|JVI Current Issue|
The present study employed mass sequencing of small RNA libraries to identify the repertoire of small noncoding RNAs expressed in normal CD4+ T cells compared to cells transformed with human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma (ATLL). The results revealed distinct patterns of microRNA expression in HTLV-1-infected CD4+ T-cell lines with respect to their normal counterparts. In addition, a search for virus-encoded microRNAs yielded 2 sequences that originated from the plus strand of the HTLV-1 genome. Several sequences derived from tRNAs were expressed at substantial levels in both uninfected and infected cells. One of the most abundant tRNA fragments (tRF-3019) was derived from the 3' end of tRNA-proline. tRF-3019 exhibited perfect sequence complementarity to the primer binding site of HTLV-1. The results of an in vitro reverse transcriptase assay verified that tRF-3019 was capable of priming HTLV-1 reverse transcriptase. Both tRNA-proline and tRF-3019 were detected in virus particles isolated from HTLV-1-infected cells. These findings suggest that tRF-3019 may play an important role in priming HTLV-1 reverse transcription and could thus represent a novel target to control HTLV-1 infection.
IMPORTANCE Small noncoding RNAs, a growing family of regulatory RNAs that includes microRNAs and tRNA fragments, have recently emerged as key players in many biological processes, including viral infection and cancer. In the present study, we employed mass sequencing to identify the repertoire of small noncoding RNAs in normal T cells compared to T cells transformed with human T-cell leukemia virus type 1 (HTLV-1), a retrovirus that causes adult T-cell leukemia/lymphoma. The results revealed a distinct pattern of microRNA expression in HTLV-1-infected cells and a tRNA fragment (tRF-3019) that was packaged into virions and capable of priming HTLV-1 reverse transcription, a key event in the retroviral life cycle. These findings indicate tRF-3019 could represent a novel target for therapies aimed at controlling HTLV-1 infection.
The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo.
IMPORTANCE The study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There were several relevant findings, which could be translated into human trials. (i) Prime (systemic) and boost (mucosal) immunization is the best protocol to induce anti-self antibodies with the expected properties. (ii) Aluminum is the best adjuvant in mice and thus can be easily used in nonhuman primates (NHP) and humans. (iii) The Flock House virus (FHV) system represents a valid delivery system, as the structure is well known and is not pathogenic for humans, and it is possible to introduce constrained regions able to elicit antibodies that recognize conformational epitopes. (iv) The best CCR5 vaccine candidate should include either extracellular loop 1 or 2 (ECL1 or ECL2), but not N terminus domains.
Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways.
IMPORTANCE Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.
Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis.
IMPORTANCE This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.
Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is a pivotal intracellular mediator of signaling pathways downstream of TNFR1 and -2 with known pro- and antiviral effects. We investigated its role in the replication of the prototype poxvirus vaccinia virus (VACV). Loss of TRAF2 expression, either through small interfering RNA treatment of HeLa cells or through genetic knockout in murine embryonic fibroblasts (MEFs), led to significant reductions in VACV growth following low-multiplicity infection. In single-cycle infections, there was delayed production of both early and late VACV proteins as well as accelerated virus-induced alterations to cell morphology, indicating that TRAF2 influences early stages of virus replication. Consistent with an early role, uncoating assays showed normal virus attachment but delayed virus entry in the absence of TRAF2. Although alterations to c-Jun N-terminal kinase (JNK) signaling were apparent in VACV-infected TRAF2nndash;/nndash; MEFs, treatment of wild-type cells with a JNK inhibitor did not affect virus entry. Instead, treatment with an inhibitor of endosomal acidification greatly reduced virus entry into TRAF2nndash;/nndash; MEFs, suggesting that VACV is reliant on the endosomal route of entry in the absence of TRAF2. Thus, TRAF2 is a proviral factor for VACV that plays a role in promoting efficient viral entry, most likely via the plasma membrane.
IMPORTANCE Tumor necrosis factor receptor-associated factors (TRAFs) are key facilitators of intracellular signaling with roles in innate and adaptive immunity and stress responses. We have discovered that TRAF2 is a proviral factor in vaccinia virus replication in both HeLa cells and mouse embryonic fibroblasts and that its influence is exercised through promotion of efficient virus entry.
Persistent infection is a key feature of hepatitis C virus (HCV). However, chimpanzee infections with cell culture-derived viruses (JFH1 or related chimeric viruses that replicate efficiently in cell culture) have been limited to acute-transient infections with no pathogenicity. Here, we report persistent infection with chronic hepatitis in a chimpanzee challenged with cell culture-derived genotype 1a virus (H77S.2) containing 6 cell culture-adaptive mutations. Following acute-transient infection with a chimeric H77/JFH1 virus (HJ3-5), intravenous (i.v.) challenge with 106 FFU H77S.2 virus resulted in immediate seroconversion and, following an unusual 4- to 6-week delay, persistent viremia accompanied by alanine aminotransferase (ALT) elevation, intrahepatic innate immune responses, and diffuse hepatopathy. This first persistent infection with cell culture-produced HCV provided a unique opportunity to assess evolution of cell culture-adapted virus in vivo. Synonymous and nonsynonymous nucleotide substitution rates were greatest during the first 8 weeks of infection. Of 6 cell culture-adaptive mutations in H77S.2, Q1067R (NS3) had reverted to Q1067 and S2204I (NS5A) was replaced by T2204 within 8 weeks of infection. By 62 weeks, 4 of 6 mutations had reverted to the wild-type sequence, and all reverted to the wild-type sequence by 194 weeks. The data suggest H77S.2 virus has greater potential for persistence and pathogenicity than JFH1 and demonstrate both the capacity of a nonfit virus to persist for weeks in the liver in the absence of detectable viremia as well as strong selective pressure against cell culture-adaptive mutations in vivo.
IMPORTANCE This study shows that mutations promoting the production of infectious genotype 1a HCV in cell culture have the opposite effect and attenuate replication in the liver of the only fully permissive animal species other than humans. It provides the only example to date of persistent infection in a chimpanzee challenged with cell culture-produced virus and provides novel insight into the forces shaping molecular evolution of that virus during 5 years of persistent infection. It demonstrates that a poorly fit virus can replicate for weeks within the liver in the absence of detectable viremia, an observation that expands current concepts of HCV pathogenesis and that is relevant to relapses observed with direct-acting antiviral therapies.
Serious permanent neurological or psychiatric dysfunction may result from virus infections in the central nervous system (CNS). Olfactory sensory neurons are in direct contact with the external environment, making them susceptible to infection by viruses that can enter the brain via the olfactory nerve. The rarity of full brain viral infections raises the important question of whether unique immune defense mechanisms protect the brain. Here we show that both RNA (vesicular stomatitis virus [VSV]) and DNA (cytomegalovirus [CMV]) virus inoculations of the nasal mucosa leading to olfactory bulb (OB) infection activate long-distance signaling that upregulates antiviral interferon (IFN)-stimulated gene (ISG) expression in uninfected remote regions of the brain. This signaling mechanism is dependent on IFN-aalpha;/bbeta; receptors deep within the brain, leading to the activation of a distant antiviral state that prevents infection of the caudal brain. In normal mice, VSV replication is limited to the OB, and these animals typically survive the infection. In contrast, mice lacking the IFN-aalpha;/bbeta; receptor succumbed to the infection, with VSV spreading throughout the brain. Chemical destruction of the olfactory sensory neurons blocked both virus trafficking into the OB and the IFN response in the caudal brain, indicating a direct signaling within the brain after intranasal infection. Most signaling within the brain occurs across the 20-nm synaptic cleft. The unique long-distance IFN signaling described here occurs across many millimeters within the brain and is critical for survival and normal brain function.
IMPORTANCE The olfactory mucosa can serve as a conduit for a number of viruses to enter the brain. Yet infections in the CNS rarely occur. The mechanism responsible for protecting the brain from viruses that successfully invade the OB, the first site of infection subsequent to infection of the nasal mucosa, remains elusive. Here we demonstrate that the protection is mediated by a long-distance interferon signaling, particularly IFN-bbeta; released by infected neurons in the OB. Strikingly, in the absence of neurotropic virus infection, ISGs are induced in the posterior regions of the brain, activating an antiviral state and preventing further virus invasion.
Barrier dysfunction of airway epithelium may increase the risk for acquiring secondary infections or allergen sensitization. Both rhinovirus (RV) and polyinosinic-polycytidilic acid [poly(Immiddot;C)], a double-stranded RNA (dsRNA) mimetic, cause airway epithelial barrier dysfunction, which is reactive oxygen species (ROS) dependent, implying that dsRNA generated during RV replication is sufficient for disrupting barrier function. We also demonstrated that RV or poly(Immiddot;C)-stimulated NADPH oxidase 1 (NOX-1) partially accounts for RV-induced ROS generation. In this study, we identified a dsRNA receptor(s) contributing to RV-induced maximal ROS generation and thus barrier disruption. We demonstrate that genetic silencing of the newly discovered dsRNA receptor Nod-like receptor X-1 (NLRX-1), but not other previously described dsRNA receptors, abrogated RV-induced ROS generation and reduction of transepithelial resistance (RT) in polarized airway epithelial cells. In addition, both RV and poly(Immiddot;C) stimulated mitochondrial ROS, the generation of which was dependent on NLRX-1. Treatment with Mito-Tempo, an antioxidant targeted to mitochondria, abolished RV-induced mitochondrial ROS generation, reduction in RT, and bacterial transmigration. Furthermore, RV infection increased NLRX-1 localization to the mitochondria. Additionally, NLRX-1 interacts with RV RNA and poly(Immiddot;C) in polarized airway epithelial cells. Finally, we show that NLRX-1 is also required for RV-stimulated NOX-1 expression. These findings suggest a novel mechanism by which RV stimulates generation of ROS, which is required for disruption of airway epithelial barrier function.
IMPORTANCE Rhinovirus (RV), a virus responsible for a majority of common colds, disrupts the barrier function of the airway epithelium by increasing reactive oxygen species (ROS). Poly(Immiddot;C), a double-stranded RNA (dsRNA) mimetic, also causes ROS-dependent barrier disruption, implying that the dsRNA intermediate generated during RV replication is sufficient for this process. Here, we demonstrate that both RV RNA and poly(Immiddot;C) interact with NLRX-1 (a newly discovered dsRNA receptor) and stimulate mitochondrial ROS. We show for the first time that NLRX-1 is primarily expressed in the cytoplasm and at the apical surface rather than in the mitochondria and that NLRX-1 translocates to mitochondria following RV infection. Together, our results suggest a novel mechanism for RV-induced barrier disruption involving NLRX-1 and mitochondrial ROS. Although ROS is necessary for optimal viral clearance, if not neutralized efficiently, it may increase susceptibility to secondary infections and alter innate immune responses to subsequently inhaled pathogens, allergens, and other environmental factors.
Ross River virus (RRV) is one of a group of mosquito-transmitted alphaviruses that cause debilitating, and often chronic, musculoskeletal disease in humans. Previously, we reported that replacement of the nonstructural protein 1 (nsP1) gene of the mouse-virulent RRV strain T48 with that from the mouse-avirulent strain DC5692 generated a virus that was attenuated in a mouse model of disease. Here we find that the six nsP1 nonsynonymous nucleotide differences between strains T48 and DC5692 are determinants of RRV virulence, and we identify two nonsynonymous nucleotide changes as sufficient for the attenuated phenotype. RRV T48 carrying the six nonsynonymous DC5692 nucleotide differences (RRV-T48-nsP16M) was attenuated in both wild-type and Rag1nndash;/nndash; mice. Despite the attenuated phenotype, RRV T48 and RRV-T48-nsP16M loads in tissues of wild-type and Rag1nndash;/nndash; mice were indistinguishable from 1 to 3 days postinoculation. RRV-T48-nsP16M loads in skeletal muscle tissue, but not in other tissues, decreased dramatically by 5 days postinoculation in both wild-type and Rag1nndash;/nndash; mice, suggesting that the RRV-T48-nsP16M mutant is more sensitive to innate antiviral effectors than RRV T48 in a tissue-specific manner. In vitro, we found that the attenuating mutations in nsP1 conferred enhanced sensitivity to type I interferon. In agreement with these findings, RRV T48 and RRV-T48-nsP16M loads were similar in mice deficient in the type I interferon receptor. Our findings suggest that the type I IFN response controls RRV infection in a tissue-specific manner and that specific amino acid changes in nsP1 are determinants of RRV virulence by regulating the sensitivity of RRV to interferon.
IMPORTANCE Arthritogenic alphaviruses, including Ross River virus (RRV), infect humans and cause debilitating pain and inflammation of the musculoskeletal system. In this study, we identified coding changes in the RRV nsP1 gene that control the virulence of RRV and its sensitivity to the antiviral type I interferon response, a major component of antiviral defense in mammals. Furthermore, our studies revealed that the effects of these attenuating mutations are tissue specific. These findings suggest that these mutations in nsP1 influence the sensitivity of RRV to type I interferon only in specific host tissues. The new knowledge gained from these studies contributes to our understanding of host responses that control alphavirus infection and viral determinants that counteract these responses.
Hepatitis C virus (HCV)-mediated liver diseases are one of the major health issues in the United States and worldwide. HCV infection has been reported to modulate microRNAs (miRNAs) that control various cell surface receptors and gene-regulatory complexes involved in hepatic inflammation and liver diseases. We report here that specific downregulation of miRNA-107 and miRNA-449a following HCV infection in patients with HCV-mediated liver diseases modulates expression of CCL2, an inflammatory chemokine upregulated in patients with chronic liver diseases, by targeting components of the interleukin-6 receptor (IL-6R) complex. Computational analysis for DNA-bound transcription factors in the CCL2 promoter identified adjacent binding sites for CCAAT/CEBPaalpha;, spleen focus-forming virus, proviral integration oncogene (SPI1/PU.1), and STAT3. We demonstrate that CEBPaalpha;, PU.1, and STAT3 interacted with each other physically to cooperatively bind to the promoter and activate CCL2 expression. Analysis of IL-6R and JAK1 expression in HCV patients by quantitative PCR showed significant upregulation when there was impaired miRNA-107 and miRNA-449a expression, along with upregulation of PU.1 and STAT3, but not CEBPaalpha;. miRNA-449a and miRNA-107 target expression of IL-6R and JAK1, respectively, in vitro and also inhibit IL-6 signaling and impair STAT3 activation in human hepatocytes. Taken together, our results demonstrate a novel gene-regulatory mechanism in which HCV-induced changes in miRNAs (miRNA-449a and miRNA-107) regulate CCL2 expression by activation of the IL-6-mediated signaling cascade, which we propose will result in HCV-mediated induction of inflammatory responses and fibrosis.
IMPORTANCE Hepatitis C virus (HCV)-induced hepatitis is a major health concern worldwide. HCV infection results in modulation of noncoding microRNAs affecting major cellular pathways, including inflammatory responses. In this study, we have identified a microRNA-regulated pathway for the chemokine CCL2 in HCV-induced hepatitis. Understanding microRNA-mediated transcriptional-regulatory pathways will result in development of noninvasive biomarkers for better disease prediction and development of effective therapeutics.
We report the diversity of latent membrane protein 1 (LMP1) gene founder sequences and the level of Epstein-Barr virus (EBV) genome variability over time and across anatomic compartments by using virus genomes amplified directly from oropharyngeal wash specimens and peripheral blood B cells during acute infection and convalescence. The intrahost nucleotide variability of the founder virus was 0.02% across the region sequences, and diversity increased significantly over time in the oropharyngeal compartment (P = 0.004). The LMP1 region showing the greatest level of variability in both compartments, and over time, was concentrated within the functional carboxyl-terminal activating regions 2 and 3 (CTAR2 and CTAR3). Interestingly, a deletion in a proline-rich repeat region (amino acids 274 to 289) of EBV commonly reported in EBV sequenced from cancer specimens was not observed in acute infectious mononucleosis (AIM) patients. Taken together, these data highlight the diversity in circulating EBV genomes and its potential importance in disease pathogenesis and vaccine design.
IMPORTANCE This study is among the first to leverage an improved high-throughput deep-sequencing methodology to investigate directly from patient samples the degree of diversity in Epstein-Barr virus (EBV) populations and the extent to which viral genome diversity develops over time in the infected host. Significant variability of circulating EBV latent membrane protein 1 (LMP1) gene sequences was observed between cellular and oral wash samples, and this variability increased over time in oral wash samples. The significance of EBV genetic diversity in transmission and disease pathogenesis are discussed.
Foreskin is the principal site of heterosexual HIV-1 infection in men. However, little is known about HIV-1-specific immune responses or inflammation in foreskin. To the best of our knowledge, no previous studies have assessed immune responses to candidate HIV-1 vaccines in foreskin. Using the rhesus monkey model, we show that intramuscular immunization with adenovirus serotype 26 and 35 vectors expressing SIV antigens elicited durable SIV Gag-specific CD4+ and CD8+ T cell responses in foreskin that were detectable for more than 1 year following vaccination. Gag-specific CD4+ and CD8+ T cells were also detectable in foreskin of SIV- and SHIV-infected animals and were at least comparable in magnitude to those in peripheral blood. However, unlike peripheral blood T cells, the majority of foreskin T cells exhibited transitional memory or effector memory phenotype and expressed higher levels of the activation markers CD69, HLA-DR, and CCR5, although vaccination did not further enhance foreskin CD4+ T cell activation. These findings suggest that systemic vaccination strategies can elicit potentially important SIV-specific cellular immunity in foreskin. Further characterization of vaccine-elicited immune responses and inflammation in foreskin is warranted.
IMPORTANCE We demonstrate here the induction of SIV-specific cellular immune responses in foreskin by adenovirus serotype 26 and 35 vaccine vectors. Foreskin T cells were more activated than peripheral blood T cells, but foreskin T cells were not further activated by vaccination. These findings suggest that alternative serotype adenovirus vectors induce potentially important immune responses in foreskin.
The nucleocapsid of a negative-strand RNA virus is assembled with a single nucleocapsid protein and the viral genomic RNA. The nucleocapsid protein polymerizes along the length of the single-strand genomic RNA (viral RNA) or its cRNA. This process of encapsidation occurs concomitantly with genomic replication. Structural comparisons of several nucleocapsid-like particles show that the mechanism of RNA encapsidation in negative-strand RNA viruses has many common features. Fundamentally, there is a unifying mechanism to keep the capsid protein protomer monomeric prior to encapsidation of viral RNA. In the nucleocapsid, there is a cavity between two globular domains of the nucleocapsid protein where the viral RNA is sequestered. The viral RNA must be transiently released from the nucleocapsid in order to reveal the template RNA sequence for transcription/replication. There are cross-molecular interactions among the protein subunits linearly along the nucleocapsid to stabilize its structure. Empty capsids can form in the absence of RNA. The common characteristics of RNA encapsidation not only delineate the evolutionary relationship of negative-strand RNA viruses but also provide insights into their mechanism of replication.
IMPORTANCE What separates negative-strand RNA viruses (NSVs) from the rest of the virosphere is that the nucleocapsid of NSVs serves as the template for viral RNA synthesis. Their viral RNA-dependent RNA polymerase can induce local conformational changes in the nucleocapsid to temporarily release the RNA genome so that the viral RNA-dependent RNA polymerase can use it as the template for RNA synthesis during both transcription and replication. After RNA synthesis at the local region is completed, the viral RNA-dependent RNA polymerase processes downstream, and the RNA genome is restored in the nucleocapsid. We found that the nucleocapsid assembly of all NSVs shares three essential elements: a monomeric capsid protein protomer, parallel orientation of subunits in the linear nucleocapsid, and a (5H + 3H) motif that forms a proper cavity for sequestration of the RNA. This observation also suggests that all NSVs evolved from a common ancestor that has this unique nucleocapsid.
Epstein-Barr virus (EBV) latent antigen EBNA3C is implicated in B-cell immortalization and linked to several B-cell malignancies. Deregulation of H2AX can induce genomic instability with increased chromosomal aberrations, which ultimately leads to tumorigenesis. Here we demonstrated that EBNA3C can attenuate H2AX expression at the transcript and protein levels. A reduction of total H2AX levels was clearly observed upon infection of primary B cells with wild-type EBV but not with EBNA3C knockout recombinant EBV. H2AX also interacted with EBNA3C through its N-terminal domain (residues 1 to 100). Furthermore, H2AX mutated at Ser139 failed to interact with EBNA3C. Luciferase-based reporter assays also revealed that the binding domain of EBNA3C is sufficient for transcriptional inhibition of the H2AX promoter. EBNA3C also facilitated H2AX degradation through recruitment of components of the ubiquitin proteasome pathway. We further demonstrated that knockdown of H2AX in lymphoblastoid cell lines (LCLs) led to the upregulation of the Bub1 oncoprotein and downregulated expression of p53. Overall, our study provides additional insights into EBV-associated B-cell lymphomas, which are linked to the regulation of the DNA damage response system in infected cells. The importance of these insights are as follows: (i) EBNA3C downregulates H2AX expression at the protein and transcript levels in epithelial cells, B cells, and EBV-transformed LCLs, (ii) EBNA3C binds with wild-type H2AX but not with the Ser139 mutant of H2AX, (iii) the N terminus (residues 1 to 100) of EBNA3C is critical for binding to H2AX, (iv) localization of H2AX is predominantly nuclear in the presence of EBNA3C, and (v) H2AX knocked down in LCLs led to enhanced expression of Bub1 and downregulation of the tumor suppressor p53, which are both important for driving the oncogenic process.
Group A rotaviruses (RVs) remain a leading cause of childhood gastroenteritis worldwide. Although the G/P types of locally circulating RVs can vary from year to year and differ depending upon geographical location, those with G1P, G2P, G3P, G4P, G9P, and G12P specificities typically dominate. Little is known about the evolution and diversity of G2P RVs and the possible role that widespread vaccine use has had on their increased frequency of detection. To address these issues, we analyzed the 12 G2P RV isolates associated with a rise in RV gastroenteritis cases at Vanderbilt University Medical Center (VUMC) during the 2010-2011 winter season. Full-genome sequencing revealed that the isolates had genotype 2 constellations typical of DS-1-like viruses (G2P-I2-R2-C2-M2-A2-N2-T2-E2-H2). Phylogenetic analyses showed that the genome segments of the isolates were comprised of two or three different subgenotype alleles; this enabled recognition of three distinct clades of G2P viruses that caused disease at VUMC in the 2010-2011 season. Although the three clades cocirculated in the same community, there was no evidence of interclade reassortment. Bayesian analysis of 328 VP7 genes of G2 viruses isolated in the last 39 years indicate that existing G2 VP7 gene lineages continue to evolve and that novel lineages, as represented by the VUMC isolates, are constantly being formed. Moreover, G2 lineages are characteristically shaped by lineage turnover events that introduce new globally dominant strains every 7 years, on average. The ongoing evolution of G2 VP7 lineages may give rise to antigenic changes that undermine vaccine effectiveness in the long term.
IMPORTANCE Little is known about the diversity of cocirculating G2 rotaviruses and how their evolution may undermine the effectiveness of rotavirus vaccines. To expand our understanding of the potential genetic range exhibited by rotaviruses circulating in postvaccine communities, we analyzed part of a collection of rotaviruses recovered from pediatric patients in the United States from 2010 to 2011. Examining the genetic makeup of these viruses revealed they represented three segregated groups that did not exchange genetic material. The distinction between these three groups may be explained by three separate introductions. By comparing a specific gene, namely, VP7, of the recent rotavirus isolates to those from a collection recovered from U.S. children between 1974 and 1991 and other globally circulating rotaviruses, we were able to reconstruct the timing of events that shaped their ancestry. This analysis indicates that G2 rotaviruses are continuously evolving, accumulating changes in their genetic material as they infect new patients.
The 2009 H1N1 lineage represented the first detection of a novel, highly transmissible influenza A virus genotype: six gene segments originated from the North American triple-reassortant swine lineage, and two segments, NA and M, derived from the Eurasian avian-like swine lineage. As neither parental lineage transmits efficiently between humans, the adaptations and mechanisms underlying the pandemic spread of the swine-origin 2009 strain are not clear. To help identify determinants of transmission, we used reverse genetics to introduce gene segments of an early pandemic isolate, A/Netherlands/602/2009 [H1N1] (NL602), into the background of A/Puerto Rico/8/1934 [H1N1] (PR8) and evaluated the resultant viruses in a guinea pig transmission model. Whereas the NL602 virus spread efficiently, the PR8 virus did not transmit. Swapping of the HA, NA, and M segments of NL602 into the PR8 background yielded a virus with indistinguishable contact transmissibility to the wild-type pandemic strain. Consistent with earlier reports, the pandemic M segment alone accounted for much of the improvement in transmission. To aid in understanding how the M segment might affect transmission, we evaluated neuraminidase activity and virion morphology of reassortant viruses. Transmission was found to correlate with higher neuraminidase activity and a more filamentous morphology. Importantly, we found that introduction of the pandemic M segment alone resulted in an increase in the neuraminidase activity of two pairs of otherwise isogenic PR8-based viruses. Thus, our data demonstrate the surprising result that functions encoded by the influenza A virus M segment impact neuraminidase activity and, perhaps through this mechanism, have a potent effect on transmissibility.
IMPORTANCE Our work uncovers a previously unappreciated mechanism through which the influenza A virus M segment can alter the receptor-destroying activity of an influenza virus. Concomitant with changes to neuraminidase activity, the M segment impacts the morphology of the influenza A virion and transmissibility of the virus in the guinea pig model. We suggest that changes in NA activity underlie the ability of the influenza M segment to influence virus transmissibility. Furthermore, we show that coadapted M, NA, and HA segments are required to provide optimal transmissibility to an influenza virus. The M-NA functional interaction we describe appears to underlie the prominent role of the 2009 pandemic M segment in supporting efficient transmission and may be a highly important means by which influenza A viruses restore HA/NA balance following reassortment or transfer to new host environments.
pUL34 and pUL31 of herpes simplex virus (HSV) comprise the nuclear egress complex (NEC) and are required for budding at the inner nuclear membrane. pUL31 also associates with capsids, suggesting it bridges the capsid and pUL34 in the nuclear membrane to initiate budding. Previous studies showed that capsid association of pUL31 was precluded in the absence of the C terminus of pUL25, which along with pUL17 comprises the capsid vertex-specific complex, or CVSC. The present studies show that the final 20 amino acids of pUL25 are required for pUL31 capsid association. Unexpectedly, in the complete absence of pUL25, or when pUL25 capsid binding was precluded by deletion of its first 50 amino acids, pUL31 still associated with capsids. Under these conditions, pUL31 was shown to coimmunoprecipitate weakly with pUL17. Based on these data, we hypothesize that the final 20 amino acids of pUL25 are required for pUL31 to associate with capsids. In the absence of pUL25 from the capsid, regions of capsid-associated pUL17 are bound by pUL31. Immunogold electron microscopy revealed that pUL31 could associate with multiple sites on a single capsid in the nucleus of infected cells. Electron tomography revealed that immunogold particles specific to pUL31 protein bind to densities at the vertices of the capsid, a location consistent with that of the CVSC. These data suggest that pUL31 loads onto CVSCs in the nucleus to eventually bind pUL34 located within the nuclear membrane to initiate capsid budding.
IMPORTANCE This study is important because it localizes pUL31, a component previously known to be required for HSV capsids to bud through the inner nuclear membrane, to the vertex-specific complex of HSV capsids, which comprises the unique long region 25 (UL25) and UL17 gene products. It also shows this interaction is dependent on the C terminus of UL25. This information is vital for understanding how capsids bud through the inner nuclear membrane.
The strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity.
IMPORTANCE Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.
HIV undergoes high rates of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we used a system of silent marker mutations in HIV and a single round of infection in primary T lymphocytes combined with a high-throughput sequencing and mathematical modeling approach to directly estimate the viral recombination and mutation rates. From ggt;7 million nucleotides (nt) of sequences from HIV infection, we observed 4,801 recombination events and 859 substitution mutations (1.51 and 0.12 events per 1,000 nt, respectively). We used experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We found that the single-cycle virus-induced mutation rate is 4.6 x 10nndash;5 mutations per nt after correction. By sorting of our data into recombined and nonrecombined sequences, we found a significantly higher mutation rate in recombined regions (P = 0.003 by Fisher's exact test). We used a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination and is not simply colocated in the genome. By comparing mutation rates in recombined and nonrecombined regions, we found that recombination-associated mutations account for 15 to 20% of all mutations occurring during reverse transcription.
Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (Vif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferasenndash;mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3' ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious Vif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5' end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation.
IMPORTANCE One way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells.
After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection, a leading cause of central nervous system birth defects. We previously reported long-term HCMV infection in the T98G glioblastoma cell line (
IMPORTANCE Our previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus trafficked to the nucleus, and yet only a proportion of cells stained positive for viral antigens, thus allowing continual subculturing and passaging. The cells eventually transitioned to a state where viral genomes were maintained without viral antigen expression or virion production. Here we report that during long-term T98G infection, large numbers of genomes were maintained within all of the cells' nuclei for the first several passages (through passage 4 [P4]), even in the presence of continual cellular division. Surprisingly, genomes were maintained, albeit at a lower level, through day 41. This is decidedly longer than in any other latency model system that has been described to date. We believe that this system offers a useful model to aid in unraveling the cellular components involved in viral genome maintenance (and presumably replication) in cells carrying long-term latent genomes in a neural context.
We examined the antiviral response promoted by type I interferons (IFN) in primary mouse neurons. IFN treatment of neuron cultures strongly upregulated the transcription of IFN-stimulated genes but conferred a surprisingly low resistance to infection by neurotropic viruses such as Theiler's murine encephalomyelitis virus (TMEV) or vesicular stomatitis virus (VSV). Response of primary mouse neurons to IFN treatment was heterogeneous, as many neurons failed to express the typical IFN response marker Mx1 after IFN treatment. This heterogeneous response of primary neurons correlated with a low level of basal expression of IFN-stimulated genes, such as Stat1, that are involved in signal transduction of the IFN response. In addition, transcriptomic analysis identified 15 IFN-responsive genes whose expression was low in IFN-treated primary neurons compared to that of primary fibroblasts derived from the same mice (Dhx58, Gvin1, Sp100, Ifi203 isoforms 1 and 2, Irgm2, Lgals3bp, Ifi205, Apol9b, Ifi204, Ifi202b, Tor3a, Slfn2, Ifi35, Lgals9). Among these genes, the gene coding for apolipoprotein L9b (Apol9b) displayed antiviral activity against Theiler's virus when overexpressed in L929 cells or in primary neurons. Accordingly, knocking down Apol9b expression in L929 cells increased viral replication. Therefore, we identified a new antiviral protein induced by interferon, ApoL9b, whose lack of expression in primary neurons likely contributes to the high sensitivity of these cells to viral infection.
IMPORTANCE The type I interferon (IFN) response is an innate immune defense mechanism that is critical to contain viral infection in the host until an adaptive immune response can be mounted. Neurons are a paradigm for postmitotic, highly differentiated cells. Our data show that primary mouse neurons that are exposed to type I interferon remain surprisingly susceptible to viral infection. On one hand, the low level of basal expression of some factors in neurons might prevent a rapid response of these cells. On the other hand, some genes that are typically activated by type I interferon in other cell types are expressed at much lower levels in neurons. Among these genes is the gene encoding apolipoprotein L9, a protein that proved to have antiviral activity against the neurotropic Theiler's murine encephalomyelitis virus. Our data suggest important functional differences in the IFN response mounted by specific cell populations.
E4orf6 proteins of human adenoviruses form Cullin-based E3 ubiquitin ligase complexes that degrade cellular proteins, which impedes efficient viral replication. These complexes also include the viral E1B55K product, which is believed to recruit most substrates for ubiquitination. Heterogeneity in the composition of these ligases exists, as serotypes representing some species form Cul5-based complexes (species B2, C, D, and E), whereas others utilize Cul2 (species A and F). Adenovirus type 16 (Ad16; species B1) binds significant levels of both. In this report, we show that the Cul2 binding sequence in E4orf6 of Ad12 (species A) and Ad40 (species F) resembles the cellular consensus Cul2 box. Mutation within this Cul2 box prevents binding not only of Cul2 but also in some cases Elongin C and reduces the ability to degrade target proteins, such as Mre11 and p53. A comparable Cul2 box is not present in E4orf6 of Ad5 and other serotypes that bind Cul5; however, creation of this Cul2 box sequence in Ad5 E4orf6 promoted binding to Cul2 and Cul2-dependent degradation of Mre11. E4orf6 of Ad16 also binds Cul2; however, unlike Ad40, it does not contain an Ad12-like Cul2 box, suggesting that Ad16 binds Cul2 in a unique but perhaps nonfunctional manner, as only Cul5 binding complexes appeared able to degrade Mre11. Our extensive analyses have thus far failed to identify a consensus Cul5 binding sequence, suggesting that association occurs via a novel and perhaps complex pattern of protein-protein interactions. Nevertheless, the identification of the Cul2 box may allow prediction of Cullin specificity for all E4orf6-containing Adenoviridae.
IMPORTANCE The work described in this paper is a continuation of our in-depth studies on the Cullin-based E3 ligase complexes formed by the viral E4orf6 and E1B55K proteins of all human adenoviruses. This complex induces the degradation of a growing series of cellular proteins that impede efficient viral replication. Some human adenovirus species utilize Cul5, whereas others bind Cul2. In this paper, we are the first to identify the E4orf6 Cul2 binding site, which conforms in sequence to a classic cellular Cul2 box. Ours is the first detailed biochemical and genetic analysis of a Cul2-based adenovirus ligase and provides insights into both the cooperative interactions in forming Cullin-based ligases as well as the universality of formation of all adenovirus ligase complexes. Our work now permits future analysis of the evolutionary significance of the ligase complex, work that is currently in progress in our lab.
H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus.
The outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections and diseases represents a potential threat for worldwide spread and requires development of effective therapeutic strategies. In this study, we revealed a novel positive function of an exchange protein directly activated by cyclic AMP 1 (cAMP-1; Epac-1) on MERS-CoV replication. Specifically, we have shown that Epac-specific inhibitor treatment or silencing Epac-1 gene expression rendered cells resistant to viral infection. We believe Epac-1 inhibitors deserve further study as potential therapeutic agents for MERS-CoV infection.
|JVI Accepts: Articles Published Ahead of Print|
Replication of plus-strand RNA viruses of plants is a relatively simple process that involves complementary (-)RNA synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded (ds)RNA. Using ribonuclease digestion, DNAzyme, and RNA mobility-shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reaction, where the newly synthesized (+)strand replaces the original (+)strand RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD-box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate (-) and (+)-strand synthesis in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny.
Importance Positive-stranded RNA viruses of plants use their RNAs as templates for replication. First, (-)-strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new (+)-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, the authors performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as template for TBSV replication. During the production of the new (+)-strands, the viral replicase displaces the old (+)-strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with a model that the dsRNA is present in a nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.
Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV lifecycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (HCVcc), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific aalpha;-fetoprotein by using a cDNA array database, and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA, but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expressions of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for a complete propagation of HCV.
IMPORTANCE Previous studies have shown that liver-specific host factors are required for efficient replication of HCV RNA and formation of infectious particles. In this study, we screened human cancer cell lines for expression of the liver-specific aalpha;-fetoprotein by using a cDNA array database, and identified novel permissive cell lines for a complete propagation of HCVcc without any artificial manipulation. In particular, gastric cancer-derived FU97 cells exhibited a much higher susceptibility to HCVcc/JFH-2 infection than was observed in Huh7 cells, suggesting that FU97 cells would be useful for further investigation of the HCV lifecycle, as well as the development of therapeutic agents for chronic hepatitis C.
Infections with high-risk human papillomaviruses (hrHPV) contribute to cervical carcinoma. The cdk inhibitor and tumor suppressor p16INK4A is consistently upregulated in cervical carcinoma cells for reasons that are poorly understood. We report here that downregulation of p16INK4Agene expression in three different cervical carcinoma cell lines reduced expression of the E7 oncogene, suggesting a positive feedback loop involving E7 and p16INK4A. p16INK4A depletion induced cellular senescence in HeLa but not CaSki and MS-751 cervical carcinoma cells.
Importance: This study demonstrates that the cdk inhibitor p16INK4A, frequently used as surrogate marker for transforming infections by human papillomaviruses of the high-risk group, is required for high level expression of the E7 oncoproteins of HPV-16, HPV-18 and HPV-45 in cervical carcinoma cells. It is also demonstrated that depletion of p16INK4A induces senescence in HeLa but not CaSki or MS-751 cervical carcinoma cells.
Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon in that the infected cells are attached with lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A membrane viral protein VP23R binds and recruits the host nidogen-1 protein to construct a basement membrane-like structure termed virus-mock basement membrane (VMBM) on the surface of infected cells to provide attaching sites for LECs. VMBMs do not contain collagen IV protein, which is essential for maintenance of the BM integrity and functions. In this study, we identified the VP08R protein encoded by ISKNV. VP08R was predicted to be a secreted protein with a signal peptide but without a transmembrane domain. However, immunofluorescence assays demonstrated that VP08R was located on the plasma membrane of infected cells and showed a similar expression profile to that of VP23R. Co-immunoprecipitation showed that VP08R interacted with both VP23R and nidogen-1, indicating that VP08R is a component of VMBM and is present on the cell membrane by binding to VP23R. Through formation of intermolecular disulfide bonds, VP08R molecules self-organized into a multimer, which may play a role in maintenance of VMBM integrity and stability. Moreover, the VP08R multimer was easily degraded when the ISKNV-infected cells were lysed, which may be a mechanism for VMBM disassembly when necessary to free LECs and release the mature virions.
Importance Infectious spleen and kidney necrosis virus (ISKNV, genus Megalocytivirus, family Iridovirus) is most harmful to cultured fishes. In tissues, the ISKNV-infected cells are attached with lymphatic endothelial cells (LECs), which are speculated to segregate the host immune system. A viral membrane protein VP23R binds and recruits host nidogen-1 protein to construct virus-mock basement membranes (VMBMs) on the surface of infected cells to provide attaching sites for LECs. Although VMBMs lack the collagen IV network, which is an essential structural part of true BMs, VMBMs still show an intact structure. An ISKNV-encoded VP08R protein can self-assemble to a multimer and bind both VP23R and nidogen-1 to maintain the integrity and stability of VMBMs. Based on these, we redrew the putative schematic illustration of VMBM. Our study suggests a virus adopts a strategy to remodel the cellular matrix, and may provide an important reference to elucidate BM functions and lymphangiogenesis mechanisms.
The tripartite motif (TRIM) family of proteins includes the TRIM5aalpha; antiretroviral restriction factor. TRIM5aalpha; from many Old World and some New World monkeys can restrict the human immunodeficiency virus type 1 (HIV-1), while human TRIM5aalpha; restricts N-tropic Murine Leukemia Virus (N-MLV). TRIM5aalpha; forms highly dynamic cytoplasmic bodies (CBs) that associate with and translocate on microtubules. However, the functional involvement of microtubules or other cytoskeleton-associated factors in the viral restriction process had not been shown. Here, we demonstrate the dependency of TRIM5aalpha;-mediated restriction on microtubule-mediated transport. Pharmacological disruption of the microtubule network using nocodazole or disabling it using taxol decreased restriction of N-MLV and HIV-1 by human or simian alleles of TRIM5aalpha;, respectively. In addition, pharmacological inhibition of dynein motor complexes using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and siRNA-mediated depletion of the dynein heavy chain (DHC) similarly decreased TRIM5aalpha;-mediated restriction. The loss in restriction resulting from either the disassembly of microtubules or the disruption of dynein motor activity was seen for both endogenous and over-expressed TRIM5aalpha; and was not due to differences in protein stability or cell viability. Both nocodazole treatment and DHC depletion interfered with the dynamics of TRIM5aalpha; CBs, increasing their size and altering their intracellular localization. In addition, nocodazole, taxol and DHC depletion were all found to increase the stability of HIV-1 cores in infected cells, providing an alternative explanation for the decreased restriction. In conclusion, association with microtubules and the translocation activity of dynein motor complexes are required to achieve efficient restriction by TRIM5aalpha;.
Importance The primate innate cellular defenses against infection by retroviruses include a protein named TRIM5aalpha;, belonging to the family of restriction factors. TRIM5aalpha; is present in the cytoplasm where it can intercept incoming retroviruses shortly after their entry. How TRIM5aalpha; manages to be present at the appropriate subcytoplasmic location to interact with its target is unknown. We hypothesized that TRIM5aalpha;, either as a soluble protein or a high-molecular-weight complex (the cytoplasmic body) is transported within the cytoplasm by a molecular motor called the dynein complex, itself known to interact with and move along microtubules. Our results show that destructuring microtubules or crippling their function decreased the capacity of human or simian TRIM5aalpha; to restrict their retroviral targets. Inhibiting dynein motor activity, or reducing the expression of a key component of this complex, similarly affected TRIM5aalpha;-mediated restriction. Thus, we have identified specific cytoskeleton structures involved in innate antiretroviral defenses.
Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerging H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2+/+ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with gge;1,000 LD50 for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multi-basic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerging H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and thus TMPRSS2 expression is essential for IAV replication in vivo.
Despite the recent progress in the development of new anti-viral agents, hepatitis C virus (HCV) infection remains a major global health problem and there is a need for a preventive vaccine. We previously reported that adenoviral vectors expressing HCV non-structural proteins elicit protective T-cell responses in chimpanzees and were immunogenic in healthy volunteers. Furthermore, recombinant HCV E1E2 protein formulated with adjuvant MF59 induced protective antibody responses in chimpanzees and was immunogenic in humans. To develop a HCV vaccine capable of inducing both T cell and antibody responses we constructed adenoviral vectors expressing full-length and truncated E1E2 envelope glycoproteins from HCV genotype 1b. Heterologous prime-boost immunization regimes with adenovirus and recombinant E1E2 glycoprotein (genotype 1a) plus MF59 were evaluated in mice and guinea pigs. Adenovirus prime and protein boost induced broad HCV specific CD8+ and CD4+ T cell responses and functional Th1-type IgG responses. Immune sera neutralized HCVpp and a diverse panel of recombinant HCVcc strains and limited cell-to-cell HCV transmission. This study demonstrates that combining adenovirus vector with protein antigen can induce strong antibody and T cell responses that surpass immune responses achieved by either vaccine alone.
Importance Hepatitis C virus (HCV) infection is a major health problem. Despite the availability of new direct acting anti-viral agents for treating chronic infection, an affordable preventative vaccine provides the best long-term goal for controlling the global epidemic. This study describes a new anti-HCV vaccine targeting the envelope viral proteins based on adenovirus vector and protein in adjuvant. Rodents primed with the adenovirus vaccine and boosted with the adjuvanted protein developed cross-neutralizing antibodies and potent T cell responses that surpassed immune responses achieved by either vaccine component alone. If combined with the adenovirus vaccine targeting the HCV NS antigens now under clinical testing this new vaccine might lead to a stronger and broader immune response and to a more effective vaccine to prevent HCV infection. Importantly, the described approach represents a valuable strategy for other infectious diseases where both T and B cell responses are essential for protection.
During infection by human adenovirus (HAdV), the proteins encoded by the early region 1A (E1A) gene bind and appropriate components of the cellular transcriptional machinery to activate viral early genes transcription. Previously, we identified roles for the hBre1 and the hPaf1 complexes in E1A mediated transcriptional activation of HAdV early genes. Here we show that E1A binds hBre1 directly and that this complex targets the hPaf1 complex via the Rtf1 subunit. Depletion of hPaf1 reduces E1A dependent activation of transcription from the E2e, E3 and E4 viral transcription units, and this does not result from a reduced ability of RNA polymerase II to be recruited to the promoter proximal regions of these genes. In contrast, depletion of hPaf1 reduces the occupancy of RNA polymerase II across these transcription units. This is accompanied by a reduction in H3K36 trimethylation, a histone post-translational modification associated with efficient transcriptional elongation, and a reduction in full length transcripts from these genes. Together, these results indicate that E1A uses hBre1 to recruit the hPaf1 complex in order to optimally activate viral early transcription by enhancing transcriptional elongation.
IMPORTANCE This work provides the mechanism by which the hPaf1 complex contributes to E1A dependent activation of early gene transcription. The work also demonstrates that E1A induces gene expression by stimulating transcriptional elongation in addition to its better characterized effects on transcriptional initiation.
Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family that includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRP) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRP determined at 2.8 and 2.15 AAring; resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to the known RdRPs of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active site cavity in one of the crystal forms. The rearrangement affects mainly the motif A, where the conserved residue Asp240, involved in rNTP selection and its neighbor residue, Phe239, move about 10 AAring; from its expected position, within the ribose binding pocket, towards the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation- interaction established between the aromatic ring of Phe239 and the side chain of Lys56, within the fingers domain. Other contacts, involving Phe239 and different residues of motif F are also observed. The movement of motif A is connected with important conformational changes in the fingers region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N-terminus. The structures determined in this work provide essential information for studies on the Cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A.
IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRP) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRP, the EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A), containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, interfering with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting the enzyme activity and this type of modulation appears to be general to all picornaviruses.
The emergence of a highly pathogenic human coronavirus in the Middle East has sparked new interest in human coronaviruses around the world....
Epstein-Barr Virus (EBV) attachment to human CD21 on the B-cell surface initiates infection. Whether CD21 is a simple tether, or conveys vital information to the cell interior for production of host factors that promote infection of primary B-cells is controversial, as the cytoplasmic fragment of CD21 is short, though highly conserved. Ubiquity of CD21 on normal B-cells, the diversity of this population, and the well-known resistance of primary B-cells to gene transfer technologies have all impeded resolution of this question. To uncover the role(s) of the CD21 cytoplasmic domain during infection initiation, the full--length receptor (CD21=CR), a mutant lacking the entire cytoplasmic tail (CT), and the control vector (NEO) were stably expressed in two pre B-cell lines that lack endogenous receptor. Genome-wide transcriptional analysis demonstrated that stable CD21 surface expression alone (either CR or CT) produced multiple independent changes in gene expression, though both dramatically decreased class I MAGE family RNAs and upregulated genes associated with B-cell differentiation (e.g. C2TA, HLA-II, IL21R, MIC2, CD48, PTPRCAP/CD45 associated protein). Temporal analysis spanning 72 hours revealed not only CR, but also CT expressing lines initiated latency. In spite of this, the number and spectrum of transcripts altered in CR compared with CT-bearing lines at one-hour after infection further diverged. Differential modulation of immediate early cellular transcripts (e.g. c-jun, multiple histones), both novel and previously linked to CD21-initiated signaling, as well as distinct results from pathway analyses support a separate role for the cytoplasmic domain in initiation of intracellular signals.
Importance Statement: Membrane proteins that mediate virus attachment tether virus particles to the cell surface initiating infection. In addition, upon virus interaction such proteins may transmit signals to the interior of the cell that support subsequent steps in the infection process. Herein, we show that expression of the Epstein-Barr virus B-cell attachment receptor, CD21, in B-cells that lack this receptor result in significant changes in gene expression, both before and rapidly following EBV-CD21 interaction. These changes translate into major signaling pathway alterations that are predicted to support stable infection.
Purified retroviral Gag proteins can assemble in vitro to form immature virus-like particles (VLPs). By cryo-electron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N- and C-termini as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag in comparison with assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly.
Importance The structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of anti-retroviral drugs that interfere with virus assembly.
Reactivation of human cytomegalovirus (HCMV) is a significant cause of disease and death in immunocompromised patients underscoring the need to understand how latency is controlled. Here we demonstrate that HCMV has evolved to utilize cellular miRNAs in cells that promote latency to regulate expression of a viral protein critical for viral reactivation. Our data reveal that hsa-miR-200 miRNA family members target the UL122 (IE2) 3'UTR, resulting in repression of this viral protein. Utilizing recombinant viruses that mutate the miRNA-binding site results in lytic rather than latent infections compared to wild type virus in ex vivo infections of primary CD34+ cells. Cells permissive for lytic replication demonstrate low levels of these miRNAs. We propose that cellular miRNA regulation of HCMV is critical for maintenance of viral latency.
Importance Human cytomegalovirus (HCMV) is a herpesvirus that infects a majority of the population. Once acquired, individuals will harbor the virus for life, where the virus remains, for the most part, in a quiet or latent state. Under weakened immune conditions, the virus can reactivate, which can cause severe disease and often death. We have found that a family of small RNAs, termed microRNAs, encoded by human myeloid progenitor cells are capable of repressing a key viral protein, thus enabling the virus to ensure a quiet/latent state. As these progenitor cells mature further down the myeloid lineage towards cells that support active viral replication, the levels of these microRNAs decrease. Together, our data suggest that host cell microRNA regulation of HCMV is important for the quiet/latent state of this pathogen.
Varicella zoster virus (VZV) infection causes varicella, after which virus becomes latent in ganglionic neurons. In tissue culture, VZV-infected human neurons remain viable at two weeks, whereas fibroblasts develop cytopathology. Next-generation RNA sequencing was used to compare VZV transcriptomes in neurons and fibroblasts and identified only 12 differentially transcribed genes of the 70 annotated VZV ORFs, suggesting that defective virus transcription does not account for the lack of cell death in VZV-infected neurons in vitro.
Due to the essential role macrophages play in antiviral immunity, it is important to understand the intracellular and molecular processes that occur in macrophages following infection with various strains of vaccinia virus, particularly those used as vaccine vectors. Similarities as well as differences were found in macrophages infected with different poxvirus strains, particularly at the level of viral-induced apoptosis and the expression of immunomodulatory genes, as determined by microarray analyses. Interestingly, the attenuated vaccinia strain MVA was particularly efficient in triggering apoptosis, IFN-bbeta; secretion and inducing changes in the expression of genes associated with increased activation of innate immunity, setting it apart from the other five vaccinia strains tested. Taken together, these results increase our understanding of how these viruses interact with human macrophages, at the cellular and molecular level, and suggest mechanisms that may underlay their utility as recombinant vaccine vectors.
IMPORTANCE Our studies clearly demonstrate that there are substantial biological differences in the patterns of cellular gene expression between macrophages infected with different poxvirus strains and that these changes are due specifically to infection with the distinct viruses. For example, a clear induction in IFNbbeta; mRNA was observed after infection with MVA, but not with other poxviruses. Importantly, antiviral bioassays confirmed that MVA-infected macrophages secreted high level of biologically active type I IFN. Similarly, the phagocytic capacity of macrophages was also specifically increased after infection with MVA. Although the main scope of this study was not to test the vaccine potential of MVA as there are several groups in the field working extensively on this aspect, the characteristics/phenotypes we observed at the in vitro level clearly highlight the inherent advantages that MVA possess in comparison to other poxvirus strains.
Barrier-to-Autointegration Factor (BAF) is a DNA binding protein with multiple cellular functions including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAFrrsquo;s ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however the precise mechanisms are poorly understood. Here we analyze how phosphorylation impacts BAFrrsquo;s DNA-binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAFrrsquo;s mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAFrrsquo;s dimerization and DNA-binding activity. Furthermore, our studies of BAFrrsquo;s antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus both DNA-binding and localization are important determinants of BAFrrsquo;s antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well.
IMPORTANCE The Barrier to Autointegration Factor (BAF) contributes to cellular genomic integrity in multiple ways, the best-characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions; the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAFrrsquo;s DNA-binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.
Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions and cause significant morbidity and mortality worldwide. Currently, there are no vaccines or antiviral therapeutics approved for combating DENV-associated disease. In this paper, we describe a class of tricylic small molecule compoundsmmdash;dihydrobenzothiepenes (DHBTs), identified through high throughput screeningmmdash;with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as to a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle being affected revealed that an early event in the life cycle is inhibited. Due to similarity in structure of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the role of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of EGFR-related kinase (ERK) was found to negatively impact DENV infection, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds.
IMPORTANCE The dengue viruses are mosquito-borne, re-emerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue activity. Further analysis revealed that SKI-417616 acts through antagonism of the host-cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.
The Cucumber mosaic virus (CMV) 2b protein is an RNA silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays, and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nuclear/nucleolar-localized 2b protein possesses at least some ability to inhibit antiviral silencing but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence 2b-NLS engendered was unrelated to effects on microRNA- or short-interfering RNA-regulated host functions. Thus, although nuclear and nucleolar-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.
IMPORTANCE In this work, the main finding is that nuclear/nucleolar-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.
We have shown that glycoprotein K (gK) and its interacting partner UL20 protein play crucial roles in virion envelopment. Specifically, virions lacking either gK or UL20 fail to acquire an envelope, thus causing accumulation of capsids in the cytoplasm of infected cells. The HSV-1 UL37 protein has also been implicated in cytoplasmic virion envelopment. To further investigate the role of UL37 in virion envelopment, the recombinant virus DC480 was constructed by insertion of a 12-amino acid protein C epitope tag within the UL37 amino acid sequence immediately after amino acid 480. The DC480 mutant virus expressed full-size UL37 detected by the anti-ProtC antibody in western immunoblots, accumulated unenveloped capsids in the cytoplasm of infected cells, and produced very small plaques on African green monkey kidney (Vero) cells similar in size to those produced by the UL20-null and UL37-null viruses. The DC480 virus replicated nearly four logs less efficiently than the parental wild-type virus when grown on Vero cells. However, DC480 mutant virus titers increased nearly 20-fold when grown on FRT cells engineered to express the UL20 gene in comparison to Vero cells, while the UL37-null virus replicated approximately 20-fold less efficiently than the DC480 virus on FRT cells. Co-immunoprecipitation experiments and proximity ligation assays showed that gK and UL20 interact with the UL37 protein in infected cells. Collectively, these results indicate that UL37 interacts with the gK/UL20 protein complex to facilitate cytoplasmic virion envelopment.
Importance Herpes simplex viruses acquire thei final envelopes by budding into cytoplasmic membranes derived from the Trans-Golgi network (TGN). Tegument proteins UL36 and UL37 are known to be transported to the TGN sites of virus envelopment and function in virion envelopment, since mutants lacking UL37 accumulate capsids in the cytoplasm that are unable to bud into TGN membranes. Viral glycoprotein K (gK) also functions in cytoplasmic envelopment as a protein complex with the membrane associated protein UL20 (UL20mp). This work shows for the first time that the UL37 protein functionally interacts with glycoprotein K (gK) and UL20 to facilitate cytoplasmic virion envelopment. The work may lead to the design of specific drugs that can interrupt UL37 interactions with the gK/UL20 protein complex providing new ways to combat herpesviral infections.
In cells infected with herpesviruses, two capsid-associated, or inner-tegument, proteins UL37 and UL36 control cytosolic trafficking of capsids by as yet poorly understood mechanisms. Here, we report the crystal structure of the N-terminal half of UL37 from pseudorabies virus, an alphaherpesvirus closely related to herpes simplex viruses and varicella-zoster virus. The structure mmdash; the first for any alphaherpesvirus inner tegument protein mmdash; reveals an elongated molecule of a complex architecture, rich in helical bundles. To explore the function of UL37 N terminus, we used the three-dimensional framework provided by the structure in combination with evolutionary trace analysis to pinpoint several surface-exposed regions of potential functional importance and test their importance using mutagenesis. This approach identified a novel functional region important for cell-cell spread. These results suggest a novel role for UL37 in intracellular trafficking that promotes spread of viral infection, which expands the repertoire of UL37 functions in intracellular virus trafficking. Supporting this, the N terminus of UL37 shares a structural similarity with cellular multi-subunit tethering complexes (MTCs), which control vesicular trafficking in eukaryotic cells by tethering transport vesicles to their destination membranes. Our results suggest that UL37 could be the first viral MTC mimic and provide structural rationale for the importance of UL37 for viral trafficking. We propose that herpesviruses may have co-opted MTC functionality of UL37 to bring capsids to cytoplasmic budding destinations and further on to cell junctions for spread to nearby cells.
IMPORTANCE To move within an infected cell, viruses encode proteins that interact with host trafficking machinery. In cells infected with herpesviruses, two capsid-associated proteins control cytosolic movement of capsids by as yet poorly understood mechanisms. Here, we report the crystal structure for the N-terminal half of one of these proteins, UL37. Structure-based mutagenesis revealed a novel function for UL37 in virion trafficking to cell junctions for cell-cell spread. Unexpected structural similarity to components of cellular multi-subunit tethering complexes, which control vesicular traffic, suggests that UL37 could be the first viral MTC mimic and provides structural basis for the importance of UL37 for viral trafficking.
HSV-1 regulatory protein ICP0 stimulates efficient infection via its E3 ubiquitin ligase activity that causes degradation of several cellular proteins, some of which are sumoylated. Chicken adenovirus Gam1 protein also interferes with the sumoylation pathway, and both proteins disrupt PML NBs. We report that Gam1 increases the infection efficiency of ICP0-null mutant HSV-1 by approximately 100-fold, thus strengthening the hypothesis that PML-NB and sumoylation-related mechanisms are important factors in the control of HSV-1 infection.
Numerous animal and plant viruses are transmitted by arthropod vectors in a persistent, circulative manner. Tomato yellow leaf curl virus (TYLCV) is transmitted by the sweet potato whitefly Bemisia tabaci. Here we report that infection with Rickettsia spp., a facultative endosymbiont of whiteflies, altered TYLCV- B. tabaci interactions. A B. tabaci strain infected with Rickettsia acquired more TYLCV from infected plants, retained the virus longer and exhibited nearly double the transmission efficiency than a non-infected strain, with the same genetic background. Temporal and spatial antagonistic relationships were discovered between Rickettsia and TYLCV within the whitefly. Along different time course experiments, the levels of virus and Rickettsia within the insect were inversely correlated. Fluorescence in situ hybridization analysis on Rickettsia-infected midguts showed evidence for niche exclusion between Rickettsia and TYLCV. In particular, high levels of the bacterium in the midgut resulted in higher virus concentration in the filter chamber, a favored site for virus translocation along the transmission pathway, while low levels of Rickettsia in the midgut resulted in an even distribution of the virus. Taken together, these results indicate that Rickettsia, by infecting the midgut, increases TYLCV transmission efficacy, adding further insights into the complex association between persistent plant viruses, their insect vectors and microorganisms tenants that reside within these insects.
Importance Bacterial endosymbionts in arthropods are gaining great interest in influencing many aspects of their host biology in agricultural and human health systems. A recent and relevant studied example is the influence of Wolbachia on dengue virus transmission by mosquitoes. In parallel with our current studied whitefly-Rickettsia-TYLCV system, studies have now shown that dengue levels in the mosquito vector are inversely correlated with bacterial load. Our work presents evidence of unifying principles between vectors of plant and animal viruses in a role for endosymbionts in manipulating vector biology and pathogen transmission. Our results demonstrate the influence of an interesting and prominent bacterial endosymbiont in Bemisia tabaci in TYLCV transmission, a worldwide disease infecting tomatoes. Besides its agricultural importance, this system provides interesting insights into Bemisia interaction with these newly discovered endosymbionts.
Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly, and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired 4 kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3rrsquo; consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice, and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa site 37 as important for its diarrheogenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.
Importance Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in it. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly, and sequenced the 11 genes of the passaged viruses to identify mutations associated with emergence or disappearance of virulence. Sequence analysis revealed that changes in 3 genes (VP4, NSP1 and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheogenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.
STING (stimulator of interferon genes) is known to control the induction of innate immune genes in response to the recognition of cytosolic DNA species, including the genomes of viruses such as HSV1. However, while STING is essential for protection of the host against numerous DNA pathogens, sustained STING activity can lead to lethal inflammatory disease. It is known that STING utilizes interferon regulatory 3 (IRF3) and nuclear factor B (NF-B) pathways to exert its effects, although the signal transduction mechanisms remain to be fully clarified. Here, we demonstrate that in addition to the activation of these pathways, potent induction of the JNK/SAPK pathway was similarly observed in response to STING activation by dsDNA. Further, TANK-binding kinase 1 (TBK1) associated with STING, was found to facilitate dsDNA-mediated canonical activation of NF-B as well as IRF3 to promote pro-inflammatory gene transcription. The triggering of NF-B function was noted to require TRAF6 activation. Our findings detail a novel dsDNA-mediated NF-B activation pathway facilitated through a STING/TRAF6/TBK1 axis and suggest a target for therapeutic intervention to plausibly stimulate anti-viral activity or alternatively avert dsDNA-mediated inflammatory disease.
IMPORTANCE The IKK complex, which is composed of two catalytic subunits, IKKaalpha; and IKKbbeta; has been suggested to be essential for the activation of canonical NF-B signaling in response to various stimuli including cytokines (e.g. IL-1aalpha;, TNF-aalpha;), TLR ligands (e.g. LPS), and dsRNA derived from viruses or a synthetic analog. STING has been identified as a critical signaling molecule required for the detection of cytosolic dsDNA derived from pathogens and viruses. However little is known about how cytosolic dsDNA triggers NF-B signaling. In the present study, we demonstrate that TBK1, identified as an IKK-related kinase, may predominantly control the activation of NF-B in response to dsDNA signaling via STING through the IKKaalpha;bbeta; activation loop. Thus, our results establish TBK1 as a downstream kinase controlling dsDNA-mediated IRF3 and NF-B signaling dependent on STING.
Icosahedral virus assembly requires a series of concerted reactions of highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding proteinrrsquo;s coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8 we show that changing the coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein D14 is shown by crosslinking to interact with scaffolding protein residue R293, and thus is intimately involved in proper procapsid assembly. To a lesser extent, the coat protein N-arm residue E18 is also implicated in interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding proteinrrsquo;s coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions.
Importance Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, which triggers assembly of an intermediate called a procapsid. Previously, we determined there is a single amino acid in scaffolding protein required for P22 procapsid assembly, though others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving correctly shaped and sized procapsids, and that the lack of these proper protein:protein interfaces leads to aberrant structures. The present work represents an important contribution to the growing knowledge that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for anti-virals.
The success of future clinical trials with oncolytic viruses depends on the identification and the control of mechanisms that modulate their therapeutic efficacy. In particular, little is known about the role of autophagy in infection by attenuated measles virus of the Edmonston strain (MV-Edm). We investigated the interaction between autophagy, innate immune response and oncolytic activity of MV-Edm, as anti-viral immune response is a known factor limiting virotherapies. We report that MV-Edm exploits selective autophagy to mitigate the innate immune response mediated by DDX58/RIG-I like receptors (RLRs) in non-small cell lung cancer cells (NSCLC). Both RNAi and overexpression approaches demonstrate that autophagy enhances viral replication and inhibits the production of type-I interferons regulated by RLRs. We show that MV-Edm unexpectedly triggers SQSTM1/p62-mediated mitophagy resulting in decreased mitochondrion-tethered MAVS and subsequently weakening innate immune response. These results unveil a novel infectious strategy based on the usurpation of mitophagy leading to mitigation of the innate immune response. This finding provides a rationale to modulate autophagy in oncolytic virotherapy.
Importance: In vitro studies, pre-clinical experiments in vivo and clinical trials on humans all indicate that oncolytic viruses hold promise for cancer therapy. Measles virus of the Edmonston strain (MV-Edm), which is an attenuated virus derived from the common wild-type measles virus is paradigmatic for therapeutic oncolytic viruses. MV-Edm replicates preferentially in and kills cancer cells. Efficiency of MV-Edm is limited by the immune response of the host against virus. In our study, we reveal that MV-Edm usurps a homeostatic mechanism of intracellular degradation of mitochondria, coined mitophagy, to attenuate the innate immune response in cancer cells. This strategy might provide a replicative advantage for the virus against the development of anti-viral immune responses by the host. These findings are important since they may not only indicate that inducers of autophagy could enhance the efficacy of oncolytic therapies, but also provide clues for anti-viral therapy by targeting SQSTM1/p62-mediated mitophagy.
To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293-cells lines, double-positive for human Toll-like receptors (TLR) and a NF-B-inducible reporter gene. Screening a large variety of compounds and cellular extracts detected a TLR3 activating compound in a microsomal yeast extract. Fractionation of this extract identified a RNA molecule of 4.6 kb, named Nucleic Acid Band 2 (NAB2) that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to the genome of the dsRNA L-BC virus of Saccharomyces cerevisiae. A large scale production and purification process of this RNA was established based on chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin, but neither NAB2 nor Lipofectin alone, induced the secretion of IL-12(p70), IFNaalpha;, IP-10, Mip-1bbeta; and IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 signaled also via the cytoplasmic sensor for RNA recognition MDA-5. Significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding MVA and NAB2+Lipofectin. This combination of immunotherapeutics strongly increased the percentage of infiltrating Natural Killer (NK) cells and plasmacytoid dendritic cells (pDCs) at the injection sites, cell types which can modulate innate and adaptive immune responses.
IMPORTANCE Virus-based cancer vaccines offer a good therapeutic alternative to cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immune-modulatory characteristics which augments the efficacy of a MVA-based immunotherapeutic. The immune modulator consists of the purified dsRNA genome isolated from commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2+Lipofectin stimulate the immune system via TLR3 and MDA-5. Injected at the MVA-vaccination site, the immune-modulator increased survival in a preclinical tumor-model. We could demonstrate that NAB2+Lipofectin augments the MVA-induced infiltration of Natural Killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by NAB2+Lipofectin influencing innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of the immune modulator should be considered towards a rationale improvement of virus-vector-based vaccination by immune-modulators.
The herpes simplex virus (HSV) tegument protein VP1-2 contains an N-terminal nuclear localisation signal (NLS) that is critical for capsid routing to the nuclear pore. Here we analyse positionally conserved determinants in VP1-2 homologues from each of the aalpha;, bbeta; and classes of human herpesviruses. The overall architecture of the VP1-2s was similar with a conserved N-terminal ubiquitin specific protease domain separated from an internal region by a linker that was quite poorly conserved in length and sequence. Within this linker region all herpesviruses contained a conserved, highly basic motif which nevertheless exhibited distinct class-specific features. The motif in HSV functioned as a monopartite NLS while in VZV activity required an adjacent basic section defining the motif as a bipartite NLS. Neither the bbeta;- nor -herpesvirus VP1-2 motifs were identified by prediction algorithms but nevertheless functioned as efficient NLS motifs in both heterologous transfer assays and in HSV VP1-2. Furthermore though with different efficiencies, and with the exception of HHV-8, these chimeric variants rescued the replication defect of a HSV mutant lacking its NLS motif. We demonstrate that the lysine at position 428 of HSV is critical for replication with a single substitution to alanine sufficient to abrogate NLS function and virus growth. We conclude that the basic motifs of each of the VP1-2 proteins is likely to confer a similar function in capsid entry in the homologous setting and while there is flexibility in the exact type of motif employed, specific individual residues are critical for function.
IMPORTANCE To successfully infect cells all herpesviruses, along with many viruses e.g., HIV, hepatitis B virus and influenza, must navigate through the cytoplasmic environment and dock with nuclear pores for transport of their genomes into the nucleus. However we still have a limited understanding of the detailed mechanisms involved. Insight into these events are needed and could offer opportunities for therapeutic intervention. This work investigates the role of a specific determinant in the structural protein VP1-2 in herpesvirus entry. We examine this determinant in representative VP1-2s from all herpesvirus subfamilies, demonstrate NLS function, dissect key residues and show functional relevance in rescuing replication of the mutant blocked in capsid navigation to the pore. The results are important and strongly support our conclusions of the generality that these motifs are crucial for entry of all herpesviruses. They also facilitate future analysis on selective host interactions and possible routes to disrupt function.
Nelfinavir (NFV) is an HIV-1 protease inhibitor with demonstrated antiviral activity against herpes simplex virus type 1 (HSV-1) and several other herpesviruses. However, the stages of HSV-1 replication inhibited by NFV have not been explored. In this study, we investigated the effects of NFV on capsid assembly and envelopment. We confirmed the inhibitory effects of NFV on HSV-1 replication by plaque assay and found that treatment with NFV did not affect capsid assembly, activity of the HSV-1 maturational protease, or formation of DNA-containing capsids in the nucleus. Confocal and electron microscopy showed that these capsids were transported to the cytoplasm but failed to complete secondary envelopment and subsequent exit from the cell. Consistent with microscopy results, a light-scattering band corresponding to enveloped virions was not evident following sucrose gradient rate-velocity separation of lysates from drug-treated cells. Evidence of a possibly related effect of NFV on viral glycoprotein maturation was also discovered. NFV also inhibited the replication of an HSV-1 thymidine kinase mutant resistant to nucleoside analogues such as acyclovir. Given that NFV is neither a nucleoside mimic nor a known inhibitor of nucleic acid synthesis, this was expected and suggests its potential as a co-inhibitor or alternate antiviral therapeutic agent in cases of resistance.
IMPORTANCE Nelfinavir (NFV) is a clinically important antiviral drug that inhibits production of infectious human immunodeficiency virus (HIV). It was reported to inhibit herpesviruses in cell culture. Herpes simplex type 1 (HSV-1) infections are common and often associated with several diseases. The studies we describe here confirm and extend the findings by investigating how NFV interferes with HSV-1 replication. We show that early steps in virus formation appear unaffected by NFV (e.g., assembly of DNA-containing capsids in the nucleus and their movement into the cytoplasm), whereas later steps are severely restricted by the drug (e.g., final envelopment in the cytoplasm and release of infectious virus from the cell). Our findings provide the first insight into how NFV inhibits HSV-1 replication and suggest this drug may have applications in studying the herpesvirus envelopment process. Additionally, NFV may have therapeutic value alone or in combination with other antivirals in treating herpesvirus infections.
Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for MERS-CoV infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse adapted MERS-CoV for rapid assessment of therapeutics.
Human herpesvirus 6 (HHV-6) is widely spread in the human population and has been associated with several neuroinflammatory diseases, including multiple sclerosis. To develop a small animal model of HHV-6 infection, we analyzed the susceptibility of several lines of transgenic mice, expressing human CD46, identified as a receptor for HHV-6. We showed that HHV-6A (GS) infection results in the expression of viral transcripts in primary brain glial cultures from CD46-expressing mice, while HHV-6B (Z29) infection was inefficient. HHV-6A DNA persisted for up to 9 months in the brain of CD46-expressing mice but not in the non-transgenic littermates, whereas HHV-6B DNA levels decreased rapidly after infection in all mice. Persistence in the brain was observed only with infectious but not heat-inactivated HHV-6A. Immunohistological studies revealed the presence of infiltrating lymphocytes and monocytes in periventricular areas of the brain of HHV-6A-infected mice. Furthermore, HHV-6A stimulated the production of panel of proinflammatory chemokines in primary brain glial cultures, including CCL2, CCL5 and CXCL10 and induced the expression of CCL5 in the brains of HHV-6A-infected mice. HHV-6A-induced production of chemokines in the primary glial cultures was dependent on the stimulation of toll-like receptor 9 (TLR9). Finally, HHV-6A induced signaling through human TLR9 as well, extending thus observations from the murine model to human infection. Altogether, this study presents a first murine model for HHV-6A-induced brain infection and suggests a role for TLR9 in the HHV-6A- initiated production of proinflammatory chemokines in the brain, thus opening novel perspectives for the study of virus-associated neuropathology.
Importance section Human herpesvirus 6 (HHV-6) infection has been related to neuroinflammatory diseases, however, the lack of suitable small animal infection model has considerably hampered further studies of HHV-6-induced neuropathogenesis. In this study, we have characterized a new model for HHV-6 infection in mice expressing the human CD46 protein. Infection of CD46 transgenic mice with HHV-6A resulted in a long-term persistence of viral DNA in the brains of infected animals and was followed by lymphocyte infiltration and upregulation of the CCL5 chemokine, in the absence of clinical signs of disease. The secretion of a panel of chemokines was increased after infection in primary murine brain glial cultures and the HHV-6-induced chemokine expression was inhibited when TLR9 signaling was blocked. These results describe the first murine model for HHV-6A-induced brain infection and suggest the importance of TLR9 pathway in the HHV-6A-initiated neuroinflammation.
The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans based on sequence analysis and virus isolations from live bird markets, but itrrsquo;s not clear which species of birds are most likely to be infected and shedding sufficient levels of virus to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 106 EID50 of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding in quail and chickens was much higher and prolonged than in the other species. Quail effectively transmitted the virus to direct contacts but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The HA gene from samples collected from selected experimentally infected birds were sequenced and three amino acid differences were commonly observed when compared to A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for avian isolates and is associated with aalpha;;2,6 sialic acid binding. Different amino acid combinations were observed suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate that certain poultry species are reservoirs of the H7N9 influenza virus, and that the virus is highly upper respiratorytropic so testing of bird species should preferentially be conducted with oropharyngeal swabs for best sensitivity.
IMPORTANCE The recent outbreak of H7N9 in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be from poultry, but definitive data for the source of the infection is not known. This study provides experimental data to show that quail and chickens are susceptible to infection and shed large amounts of virus and are likely important in the spread of the virus to humans. Other poultry species, including Muscovy ducks, can be infected and shed virus, but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested as a possible source of virus because of isolation of virus from several pigeons in poultry markets in China, but experimental studies show they are generally resistant to infection and are unlikely to play a role in spread of the virus.
The novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires an induction of immunogenic cell death (ICD) coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study showed that in human pancreatic cancer cells (PDAC, n=4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0pplusmn;0.5 times (58pplusmn;9% of total content; up to 100 ng/ml) in all infected cultures, whether non-dying, necrotic or apoptotic. Alternative secretory route allowed H-1PV to overcome failure of gemcitabine to trigger HMGB1 release, and that without impeding cytotoxicity or other ICD-activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided but was uncoupled from an autocrine IL1b-loop. That and the pattern of the viral determinants maintained in gemcitabine-treated cells suggested an activation of inflammasome/CASP1-platform alongside with DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signalled through secretion of alarmin HMGB1, and besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD-process. A transient arrest of cells in cyclinA1-rich S-phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV which is not-pathogenic in humans into multimodal anticancer treatments.
Importance: The current therapeutic concepts targeting aggressive malignancies require induction of immunogenic cell death characterized by an exposure of CRT as well as release of ATP and HMGB1 from dying cells. In pancreatic tumor cells (PDAC) infected with oncolytic parvovirus H-1PV, only HMGB1 was released by all infected cells, whether non-dying, necrotic or succumbing to one of the programmed death pathways, including contra-productive apoptosis. Our data suggested that HMGB1 active secretion in PDAC is a sentinel reaction emerging during early stages of the viral life cycle irrespective of cell death, compatible with and complementing cytotoxic regimens. Consistent induction of HMGB1 secretion raised the possibility that this reaction might be a general llsquo;alarmingrrsquo; phenomenon characteristic for H-1PV interaction with the host cell; release of IL1b pointed to possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.
The VP24 protein plays an essential, albeit poorly understood role in the filovirus life cycle. VP24 is only 30% identical between Marburg virus and the ebolaviruses. Further, VP24 from the ebolaviruses is immunosuppressive, while that of Marburg virus is not. The crystal structure of Marburg virus VP24, presented here, reveals that although the core is similar between the viral genera, Marburg VP24 is distinguished by a projecting beta shelf and an alternate conformation of the N-terminal polypeptide.
Acute HIV-1 infection is characterized by a type I interferon response, resulting in the induction of host restriction factors. HIV-1 has evolved to counteract these factors, and one such adaptation, the ability of Vpu to counteract BST2/tetherin, is associated with the evolution of SIVcpz into the pandemic group M HIV-1. During transmission between individuals, very few or even a single virus, the "transmitted/founder (T/F) virus," gives rise to the new infection, but in the new host the selective pressure of the immune response yields the diverse "quasispecies" of chronic infection. Here we examine the functional characteristics of Vpu proteins encoded by T/F viruses compared to those from acute and chronic viruses from longitudinally sampled subjects. The studied T/F Vpu proteins showed a trend towards optimized CD4 downregulation compared to chronic Vpus but did not differ substantially in their ability to downregulate BST2 or enhance virion release, although individual clones from each group were impaired in these activities. Analysis of the functionally impaired clones identified a C-terminal residue, W76, as important specifically for Vpu enhancement of virion release. Primary Vpu clones encoding a W76G polymorphism, or site-directed mutants encoding a W76G substitution, were impaired in their ability to enhance virion release, but they were not defective for BST2 surface-downregulation. Conversely, the virion release function of impaired primary clones was restored by creating a G76W substitution. The identification of W76 as important for virion release enhancement independent of BST2 surface downregulation supports the potential to mechanistically separate these functions of Vpu.
Importance To establish infection in a host, HIV-1 must evade the hostrrsquo;s immune response, including the production of antiviral factors. HIV-1 encodes proteins that antagonize these defenses, including Vpu. Vpu counteracts the host protein BST2, which blocks the release of progeny viruses from the host cell. To determine the importance of Vpu activity to HIV-1 transmission, this study assessed the functionality of Vpu from viruses isolated soon after transmission ("transmitted/founder" viruses) compared to isolates from chronic infection. Although the anti-BST2 activity of Vpu proteins from the tested transmitted/founder viruses did not differ from the activity of the chronic Vpus, the transmitted/founder Vpus trended towards having superior activity against another host protein, CD4. Further, this study identified an amino acid near the C-terminus of Vpu that is specifically important for Vpurrsquo;s ability to enhance the release of progeny virus from the host cell, supporting a new mechanism for this function of Vpu.
Interferons (IFNs) are cytokines produced by host cells in response to the infection with pathogens. By binding to the corresponding receptors, IFNs trigger different pathways to block intracellular replication and growth of pathogens and to impede the infection of surrounding cells. Due to their key role in host defence against viral infections as well as for clinical therapies, the IFN responses and regulation mechanisms are well studied. However, studies on type I IFNs are mainly focusing on IFNaalpha; and bbeta; subtypes. Knowledge about IFN and is limited. Moreover, most of the studies are done in humans or mouse models but not in the original host of zoonotic pathogens.
Bats are important reservoirs and transmitters of zoonotic viruses like lyssaviruses. A few studies have shown an antiviral activity of IFNs in fruit bats. However, the function of type I IFNs against lyssaviruses in bats is not studied yet. Here, IFN and IFN genes from European serotine bat, Eptesicus serotinus, were cloned and functionally characterized. E. serotinus IFN and IFN genes are intron-less and well conserved between Microchiropterean species. The promoter regions of both genes contain essential regulatory elements for transcription factors. In vitro studies indicated a strong activation of IFN signalling by recombinant IFN, whereas IFN displayed weaker activation. Noticeably, both IFNs inhibit to different extents the replication of different lyssaviruses in susceptible bat cell line. The present study provides functional data on the innate host defence against lyssaviruses in endangered European bats.
Importance In this manuscript we describe for the first time the molecular and functional characterization of two type I interferons (IFN and ) from European serotine bat (Eptesicus serotinus). The importance of this study is mainly based on the fact that very limited information about the early innate immune response against bat Lyssaviruses in their natural host serotine bats is yet available. Generally, whereas the antiviral activity of other type I interferons is well studied the functional involvement of IFN and is not yet investigated.
Emerging and zoonotic pathogens pose continuing threats to human health and ongoing challenges to diagnostics. As nucleic acid tests are playing increasingly prominent roles in diagnostics, the genetic characterization of molecularly uncharacterized agents is expected to significantly enhance detection and surveillance capabilities. We report the identification of two previously unrecognized members of the family Orthomyxoviridae, which includes the influenza viruses and the tick-transmitted Thogoto and Dhori viruses. We provide morphologic, serologic and genetic evidence that Upolu virus (UPOV) from Australia and Aransas Bay virus (ABV) from North America, both previously considered potential bunyaviruses based on electron microscopy and physicochemical features, are orthomyxoviruses instead. Their genomes show up to 68% nucleotide sequence conservation to Thogoto virus (segment 2; ~74% at amino acid level) and a more distant relationship to Dhori virus, the two prototype viruses of the recognized species in the genus Thogotovirus. Despite sequence similarity, the coding potential of UPOV and ABV differed from Thogoto virus, being instead like that of Dhori virus. Our findings suggest that the tick-transmitted UPOV and ABV represent geographically distinct viruses in the genus Thogotovirus of the family Orthomyxoviridae that do not fit in the two currently recognized species of that genus.
Importance. Upolu virus (UPOV) and Aransas Bay virus (ABV) are shown to be orthomyxoviruses instead of bunyaviruses as previously thought. Genetic characterization and adequate classification of agents is paramount in this molecular age to devise appropriate surveillance and diagnostics. Although closer to Thogoto virus by sequence, UPOV and ABV differ in their coding potential by lacking a proposed pathogenicity factor. In this respect they are similar to Dhori virus, which despite this lack can cause disease. These findings enable further studies into the evolution and pathogenicity of orthomyxoviruses.
Coxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae. According to earlier studies, CVA9 binds to aalpha;Vbbeta;3 and aalpha;Vbbeta;6 integrins on the cell surface and utilizes bbeta;2-microglobulin, dynamin and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here by electron microscopy that CVA9 is found in multivesicular structures 2 h post infection (p.i.). The Neutral Red -label assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicular bodies (MVBs) is crucial for promoting infection judged by the strong inhibitory effect of the wild type form of Hrs and dominant negative form of VPS4 in the CVA9 infection. CVA9 infection is dependent on phospholipase C at the start of infection whereas Rac1 is especially important between 1 and 3 h p.i. when the virus is in endosomes. Several lines of evidence implicated that low pH does not play a role in CVA9 infection. The infection is not affected by Bafilomycin A1. In addition, CVA9 is not targeted to acidic late endosomes or lysosomes, and the MVBs accumulating CVA9 have neutral pH. Thus CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger neutral MVBs, which are important for virus infection.
IMPORTANCE We have demonstrated here that the enterovirus coxsackievirus A9 (CVA9) uses a non-clathrin and non-acidic pathway to infect cells. CVA9 is not accumulating in conventional late endosomes or lysosomes. We found out that inhibitors of phospholipase C (PLC), Rac1 and Na+/H+ exchanger decreased the CVA9 infection. PLC inhibitor acts on the early entry, Rac1 inhibitor between 1 and 3 h when the virus is in endosomes, and the Na+/H+ exchanger inhibitor during various steps along the virus life cycle. The infection depends on the formation of novel neutral multivesicular bodies (MVBs), which are accumulating CVA9 for the first hours of entry. Thus CVA9 is the second enterovirus demonstrated so far, after echovirus 1, which can trigger formation of neutral MVBs. The data show that these enteroviruses favor non-acidic conditions and complex MVBs to promote virus infection.
The lack of a vaccine against respiratory syncytial virus (RSV) is a challenging and serious gap in preventive medicine. Herein, we characterize the immunogenicity and protection following immunization with an adenoviral-based RSV vaccine encoding for the fusion protein F (Ad5.RSV-F) and assess its potential for producing enhanced disease in a cotton rat (CR) model. Animals were immunized intranasally (IN) and/or intramuscularly (IM) and subsequently challenged with RSV/A/Tracy (IN) to assess protection. Robust immune responses were seen in CRs vaccinated with Ad5.RSV-F given IM or IN, which correlated with reduced replication of virus in noses and lungs after challenge. Neutralizing antibody responses following immunization with a single dose of Ad5.RSV-F at 1x1011 viral particles (v.p.) elicited antibody titers 64- to 256- fold greater than those seen after natural infection. CRs boosted with Ad5.RSV-F IN 28 days after an IM dose also had significant increases in neutralizing antibody titers. Antibody affinity for different F protein antigenic sites revealed substantial differences between antibodies elicited by Ad5.RSV-F as compared with those seen after RSV infection; differences in antibody profiles were also seen in CRs given Ad5.RSV-F IM vs. IN. Ad5.RSV-F priming did not result in enhanced disease following live virus challenge in contrast with histopathology seen in CRs given formalin-inactivated-RSV/A/Burnett vaccine.
IMPORTANCE Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory infection in infants and young children and a serious health threat in the immunocompromised and the elderly. Infection severity increased in children in an immunization trial, hampering the over four decades-long quest for a successful RSV vaccine. In this study, we show that a genetically-engineered RSV-F encoding adenoviral vector provides protective immunity against RSV challenge without enhanced lung disease in cotton rats (CRs). CRs were vaccinated under a number of different regimens and intramuscular-intranasal prime-boost immunity induced by the recombinant adenoviral RSV vaccine may provide the best protection for young infants and children at risk of RSV infection, since this population is naiiuml;ve to adenoviral preformed immunity. Overall, this manuscript describes a potential RSV vaccine candidate that merits further evaluation in a phase I clinical study in humans.
Defective interfering (DI) RNAs are highly deleted forms of the infectious genome that are made by most families of RNA viruses. DI RNAs retain replication and packaging signals, are synthesized preferentially over infectious genomes, and are packaged as DI virus particles which can be transmitted to susceptible cells. Their ability to interfere with the replication of infectious virus in cell culture, and their potential as antivirals in the clinic has long been known. However, up to now, no realistic formulation has been described. In this review, we consider the early evidence of antiviral activity by DI viruses and, using the example of DI influenza A virus, and outline developments that have led to the production of a cloned DI RNA that is highly active in preclinical studies not only against different subtypes of influenza A virus but also against heterologous respiratory viruses. These data suggest that it is timely to reassess the potential of DI viruses as a novel class of antivirals that may have general applicability.
The brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is one of the most destructive insect pests of rice crops in Asia. Nudivirus-like sequences were identified during the whole genome sequencing of BPH. PCR examination showed that the virus sequences were present in all of the 22 BPH populations collected from East, Southeast and South Asia. Thirty-two of the 33 nudivirus core genes were identified, including 20 homologues of baculovirus core genes. In addition, several gene clusters that arranged collinearly with other nudiviruses were found in the partial virus genome. In a phylogenetic tree constructed using the supermatrix method, the original virus was grouped with other nudivirus and closely related to polydnavirus. Taken together, these data indicated that the virus sequences belong to a new member of the family Nudiviridae. More specifically, the virus sequences were integrated into the chromosome of its insect host during co-evolution. This study is the first report of a large double-stranded circular DNA virus/genome in a sap-sucking hemipteran insect.
Importance This is the first report of a large double-stranded DNA virus integrated genome in the planthopper, a plant sap-sucking hemipteran insect. It is an exciting addition to the evolutionary story of Bracoviruses (polydnaviruses), Nudiviruses and Baculoviruses. The result of the virus sequences integrated in the chromosomes of its insect host is also a successful co-evolutionary story of an invertebrate virus and a plant sap-sucking insect.
The essential immediate-early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gamma-herpesvirus 68 lacking both of the known gene 50 promoters (G50pDblKO) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero, but not in permissive murine fibroblasts following low MOI infection. However, G50pDblKo replication in permissive fibroblasts was partially rescued by high MOI infection. In addition, replication of the G50pDblKO virus was rescued by growth on MEFs isolated from IFNaalpha;/bbeta;R-/- mice, while growth on Vero cells was suppressed by the addition of IFNaalpha;. 5rrsquo; rapid amplification of cDNA ends (RACE) analyses of RNA prepared from G50pDblKo and wild-type MHV68 infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays neither of the newly identified gene 50 promoters exhibited sensitivity to IFNaalpha; treatment, although RTA levels were lower in IFNaalpha;-responsive cells infected with the G50pDblKo mutant. Infection of mice with the MHV68 G50pDblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudates cells (PECs) of C57Bl/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50pDblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50pDblKO mutant by growth on type I interferon receptor null MEFs, infection of IFNaalpha;/bbeta;R-/- mice with the G50pDblKo mutant virus demonstrated partial rescue of: (i) acute virus replication in the lungs; (ii) establishment of latency; and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlight the complex mechanisms involved in controlling expression of RTA, likely reflecting time dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression.
IMPORTANCE The viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases.
HIV-1 replication in dendritic cells (DCs) is restricted by SAMHD1. This factor is counteracted by the viral protein Vpx; Vpx is found in HIV-2 and SIVsm/SIVmac, but is absent from HIV-1. We previously observed that HIV-1 replication in immature DCs is stimulated by cocultivation with primary T/B lymphocytes, suggesting that HIV-1 restriction in DCs may be overcome in coculture conditions. Here, we aimed to decipher the mechanism of SAMHD1-mediated restriction in DC/lymphocyte coculture. We found that coculture with lymphocytes downregulated SAMHD1 expression and was associated with an increased HIV-1 replication in DCs. Moreover, in infected DC/T lymphocyte cocultures, DCs acquired maturation status and secreted IFN-aalpha;. The blockade of DC/lymphocyte crosstalk by anti-ICAM-1 antibody markedly inhibited the stimulation of HIV-1 replication and prevented the downregulation of SAMHD1 expression in cocultured DCs. These results demonstrate that, in contrast to purified DCs, crosstalk with lymphocytes downregulates SAMHD1 expression in DCs, triggering HIV-1 replication and an antiviral immune response. Therefore, HIV-1 replication and immune sensing by DCs should be investigated in more physiologically relevant models of DC/lymphocyte coculture.
IMPORTANCE SAMHD1 restricts HIV-1 replication in dendritic cells (DCs). Here, we demonstrate that, in a coculture model of DC/lymphocyte mimicking early mucosal HIV-1 infection, stimulation of HIV-1 replication in DCs is associated with a downregulation of SAMHD1 expression and an activation of innate immune sensing by DCs. We propose that DC/lymphocyte crosstalk occurring in vivo modulates host restriction factor SAMHD1, promoting HIV-1 replication in cellular reservoirs and stimulating immune sensing.
Herpes simplex virus 1 (HSV-1) infected cell protein No.0 (ICP0) is a multifunctional protein that plays a key role in overcoming numerous facets of host innate immunity. A key function of ICP0 that requires an intact RING finger domain is that of an ubiquitin E3 ligase: ICP0 interacts with at least three ubiquitin conjugating enzymes of which one, UbcH5a is required for degradation of PML and SP100. A preceding report showed that ICP0 is highly unstable at very early times after infection but becomes stable at later times. Here we report that (a) the degradation of ICP0 is not infected cells specific, (b) the degradation does not require the interaction of ICP0 with either UbcH5a, UbcH6 or UbcH9, (c) ICP0 is degraded both early and late in cells infected with a mutant lacking the UL13 protein kinase, (d) ICP0 encoded by wild-type virus or the UL13 mutant are stable in cells transfected with a plasmid encoding UL13 before infection, (e) ICP0 carrying mutations in the RING finger domain is stable both early and late in infection and finally, (f) In cells infected with both wild type and RING finger mutant only the wild-type ICP0 is rapidly degraded at early times. The results suggest that the stability of ICP0 is mediated by the UL13 protein kinase and that the target of proteolysis is a site at or near the RING domain of ICP0.
Importance ICP0, a major regulatory protein of HSV-1 turns over rapidly early in infection but becomes stable at late times. We report that stabilization requires the presence of UL13 protein kinase and that an ICP0 with mutations in RING finger is stable. In mixed infections mutant ICP0 is stable whereas the wild-type ICP0 is degraded. Our findings suggest that the life-style of HSV-1 requires an ICP0 that turns over rapidly if late proteins are absent.
Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic tick-borne flaviviruses; TBEV causes neurological disease in humans while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C-terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role of NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of tick-borne flaviviruses.
Importance Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) belong to the tick-borne encephalitis serocomplex, genus Flavivirus, family Flaviviridae. Although TBEV causes neurological disease in humans while OHFV causes a disease typically identified with hemorrhagic fever. In this study, we investigated the viral determinants responsible for the different disease phenotypes using reverse genetics technology. We identified a cluster of only four amino acids in non-structural protein 5 primarily involved in the development of neurological disease in a mouse model. Moreover, the effect of these four amino acids was independent of viral replication property, and did not affect the formation of virus-induced lesions in the brain directly. These data suggest that these amino acids may be involved in the induction of neuronal dysfunction and degeneration in virus-infected neurons, ultimately leading to the neurological disease phenotype. These findings provide new insight into the molecular mechanisms of tick-borne flavivirus pathogenesis.
Mice overexpressing the PrP sequence from various host species are widely used for measuring infectious titers in prion disease. However the impact that the transgene expression level might have on the susceptibility to infection raises some concerns about the final biological relevance of these models. Here we report that the endpoint titration of a sheep scrapie isolate in sheep and in mice overexpressing the ovine PrP result in similar estimates of the infectious titer.
The two human neurotropic alphaherpesviruses Varicella Zoster virus (VZV) and Herpes simplex virus type 1 (HSV1) both establish latency in sensory ganglia. Human trigeminal ganglia are known to frequently harbor both viruses, and there is evidence to suggest the presence of both VZV and HSV DNA in the same neuron. We ask here whether VZV and HSV can exclude themselves and each other and whether they can productively infect the same cells in human neurons and foreskin fibroblasts (HFF). Simultaneous infection (co-infection) or consecutive infection (superinfection) was assessed using cell-free HSV1 and VZV expressing fluorescent reporter proteins. Automated analysis was carried out to detect singly and dually infected cells. We demonstrate that VZV and HSV both display efficient superinfection exclusion (SE) in HFF with each virus excluding either itself or the other virus. While SE also occurred in neurons, it was with much lower efficiency. Both aalpha;-herpesviruses productively infected the same neurons, whether applied simultaneously or even consecutively, albeit at lower frequency.
Importance: Superinfection exclusion by VZV for itself or the related neurotropic alphaherpesvirus HSV has been studied here for the first time. We find that while these viruses display classic SE in fibroblasts, SE is less efficient for both HSV1 and VZV in human neurons. The ability of multiple VZV to productively infect the same neurons has important implications in terms of recombination of both wild type and vaccine strains in patients.
Acute coxsackievirus B3 (CVB3) infection is one of the most prevalent causes of acute myocarditis, a disease that frequently is identified only after the sudden death of apparently-healthy individuals. CVB3 infects cardiomyocytes, but the infection is highly focal, even in the absence of a strong adaptive immune response, suggesting that virus spread within the heart may be tightly constrained by the innate immune system. Type I interferons (T1IFN) are an obvious candidate, and T1IFN receptor knockout mice are highly susceptible to CVB3 infection, succumbing within a few days of challenge. Here, we investigated the role of T1IFNs in the heart using a mouse model in which the T1IFN receptor (T1IFNR) gene can be ablated in vivo, specifically in cardiomyocytes. We found that T1IFN signaling into cardiomyocytes contributed substantially to the suppression of viral replication and infectious virus yield in the heart; in the absence of such signaling, virus titers were markedly elevated by d3 p.i., and remained high at d12 p.i., a time point at which virus was absent from genetically-intact littermates, suggesting that the T1IFN-unresponsive cardiomyocytes may act as a safe haven for the virus. Nevertheless, in these mice the myocardial infection remained highly focal, despite the cardiomyocytes' inability to respond to T1IFN, indicating that other factors, as yet unidentified, are sufficient to prevent the more widespread dissemination of the infection throughout the heart. The absence of T1IFN signaling into cardiomyocytes also was accompanied by a profound acceleration and exacerbation of myocarditis, and by a significant increase in mortality.
Importance Acute coxsackievirus B3 (CVB3) infection is one of the most common causes of acute myocarditis, a serious, and sometimes fatal, disease. To optimize treatment, it is vital that we identify the immune factors that limit virus spread in the heart and other organs. Type 1 interferons play a key role in controlling many virus infections, but it has been suggested that they may not directly impact CVB3 infection within the heart. Here, using a novel line of transgenic mice, we show that these cytokines signal directly into cardiomyocytes, limiting viral replication, myocarditis, and death.
Here we report the results of a late boost and three additional series of simian immunodeficiency virus (SIV) challenges in seven DNA/MVA vaccinated macaques who resisted a first series of rectal challenges. During 29 additional challenges delivered over 2.3 years, all animals became infected. However, 13 blips of virus in six rhesus and anamnestic Env-specific rectal IgA responses in three of the six suggested that local control of infections was occurring during the serial challenge.
Recombination plays a critical role in virus evolution. It helps avoid genetic decline and creates novel phenotypes. This promotes survival, and genome sequencing suggests that recombination has facilitated the evolution of human pathogens including Orthopoxviruses like variola virus. Recombination can also be used to map genes, but although recombinant poxviruses are easily produced in culture, classical attempts to map the vaccinia virus (VACV) genome this way, met with little success. We have sequenced recombinants formed when VACV TianTan and Dryvax strains are crossed under different conditions. These were a single round of growth in co-infected cells, five rounds of sequential passage, or using Leporipoxvirus-mediated DNA reactivation. Our studies showed that recombinants contain a patchwork of DNA, with the number of exchanges increasing with passage. Further passage also selected for TianTan DNA and correlated with increased plaque size. The recombinants produced through a single round of co-infection contain a disproportionate number of short conversion tracks (llt;1 kbp) and exhibited 1 exchange per 12 kbp, close to the ~1 per 8 kbp in the literature. One byproduct of this study was that rare mutations were also detected, VACV replication produces ~1x10-8 mutations per nucleotide copied per cycle of replication and ~1 large (21 kbp) deletion per 70 rounds of passage. Viruses produced using DNA reactivation appeared no different from recombinants produced using ordinary methods. An attractive feature of this approach is that, when combined with selection for a particular phenotype, it provides a way of mapping and dissecting more complex virus traits.
IMPORTANCE When two closely related viruses co-infect the same cell, they can swap genetic information through a process called recombination. Recombination produces new viruses bearing different combinations of genes, and it plays an important role in virus evolution. Poxviruses are a family of viruses that includes variola (or smallpox) virus and although poxviruses are known to recombine, no one has previously mapped the patterns of DNAs exchanged between viruses. We co-infected cells with two different vaccinia poxviruses, isolated the progeny, and sequenced them. We show that poxvirus recombination is a very accurate process that assembles viruses containing DNA copied from both parents. In a single round of infection, DNA is swapped back and forth ~18 times per genome to make recombinant viruses that are a mosaic of the two parental DNAs. This mixes many different genes in complex combinations and illustrates how recombination can produce viruses with greatly altered disease potential.
Human norovirus (NoV) accounts for 95% of non-bacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Ma et al., 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice compared to rVSV-VP1. At the inoculation dose of 1.0x106 PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0x106 PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that: (i) Insertion of additional gene (HSP70 or Luc) into rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo, thus improving the safety of the vaccine candidate and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine.
SIGNIFICANCE Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is uncultivable. Thus, a live vector-based vaccine may provide an alternative vaccine strategy. In this study, we developed a vesicular stomatitis virus (VSV)-based human NoV vaccine candidate. We constructed rVSV-HSP70-VP1 that co-expressing heat shock protein (HSP70) and capsid (VP1) gene of human NoV, and rVSV-Luc-VP1 that co-expressing firefly luciferase (Luc) and VP1 genes. We found that VSVs with double gene insertion were significantly more attenuated than VSV with a single VP1 insertion (rVSV-VP1). Furthermore, we found that co-expression or co-administration of HSP70 from VSV vector significantly enhanced human NoV-specific mucosal immunity. Collectively, we developed an improved live vectored vaccine candidate for human NoV which will be useful for future clinical studies.
Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, IFN-dependent upregulation of IFN-stimulated genes (ISGs) and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterised populations, and selected mutants, of wild type viruses selected by passage through IFN non-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that non-synonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterisation induced IFN and ISGs and were unable to counteract the effects of exogenous IFN yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs, but nonetheless replicated to high titres. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results presented suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in IFN non-responsive cells allows the virus to relax from these constraints and find new genome consensus within its sequence space.
Importance In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes towards interferon evasion have not been well characterised. Here, we have examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titres had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.
Antibody capacity to recognize infectious virus is a prerequisite of many antiviral functions. We determined the infectious virion capture index (IVCI) of different antibody specificities. Whereas, broadly neutralizing antibodies (bNAbs) selectively captured infectious virions, non-neutralizing antibodies and mucosal HIV-1+ IgG captured subsets of both infectious and non-infectious virions. Infectious virion capture was additive with a mixture of antibodies, providing proof of concept for vaccine-induced antibodies that together have improved capacity to recognize infectious virions.
Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is widely expressed in EBV-associated malignancies. We demonstrate that LMP2A has transformation ability. This study shows that LMP2A-induced transformation in several human non-hematopoietic cell lines were blocked in those cells expressing Immunoreceptor tyrosine-based activation motif (ITAM) LMP2A mutant. The Syk inhibitor or Syk-specific siRNA inhibited LMP2A-induced transformation. These results indicate that the interaction of the LMP2A ITAM with Syk is a key step for LMP2A-mediated transformation.
Foot-and-mouth disease virus (FMDV) causes a highly contagious, debilitating disease in cloven-hoofed animals with devastating economic consequences. To survive in the host, FMDV has evolved to antagonize the host type I interferon (IFN) response. Previous studies have reported that Lpro and 3Cpro of FMDV are involved in the inhibition of type I IFN production. However, whether the proteins of FMDV can inhibit type I IFN signaling is less well understood. In this study, we first found that 3Cpro of FMDV functioned to interfere with the JAK-STAT signaling pathway. Expression of 3Cpro significantly reduced the transcript levels of IFN-stimulated genes (ISGs) and IFN-stimulated response element (ISRE) promoter activity. The protein level, tyrosine phosphorylation of STAT1 and STAT2 and their heterodimerization were not affected. However, the nuclear translocation of STAT1/STAT2 was blocked by the 3Cpro protein. Further mechanistic studies demonstrated that 3Cpro induced proteasome- and caspase-independent protein degradation of karyopherin aalpha;1 (KPNA1), the nuclear localization signal receptor for tyrosine-phosphorylated STAT1, but not karyopherin aalpha;2, aalpha;3, or aalpha;4. Finally, we showed that the protease activity of 3Cpro contributed to the degradation of KPNA1 and thus blocked STAT1/STAT2 nuclear translocation. Taken together, results of our experiments described for the first time a novel mechanism by which FMDV evolves to inhibit IFN signaling and counteract host innate antiviral responses.
IMPORTANCE We show that 3Cpro of FMDV antagonizes the JAK-STAT signaling pathway by blocking STAT1/STAT2 nuclear translocation. Furthermore, 3Cpro induces KPNA1 degradation, which is independent of proteasome and caspase pathways. The protease activity of 3Cpro contributes to the degradation of KPNA1 and governs the ability of 3Cpro to inhibit the JAK-STAT signaling pathway. This study uncovers a novel mechanism evolved by FMDV to antagonize host innate immune responses.
T-cell functional avidity is a crucial determinant for efficient pathogen clearance. Although recombinant DNA priming coupled with a vaccinia vectored vaccine (VACV) boost has been widely used to mount robust CD8+ T cell responses, how VACV boost shapes the properties of memory CD8+ T cells remains poorly defined. Herein, we characterize the memory CD8+ T cells boosted by VACV and demonstrate that the intrinsic expression of MyD88 is critical for their high functional avidity. Independent of selection of clones with high-affinity TCR or of enhanced proximal TCR signaling, the VACV boost significantly increased T-cell functional avidity through a decrease in the activation threshold. VACV-induced inflammatory milieu is not sufficient for this improvement as simultaneous administration of the DNA vaccine and mock VACV had no effects on the functional avidity of memory CD8+ T cells. Furthermore, reciprocal adoptive transfer models revealed that the intrinsic MyD88 pathway is required for instructing the functional avidity of CD8+ T cells boosted by VACV. Taken together, the intrinsic MyD88 pathway is required for the high functional avidity of VACV-boosted CD8+ T cells independent on TCR selection or the VACV infection-induced MyD88-mediated inflammatory milieu.
Importance paragraph The functional avidity is one of the crucial determinants of T-cell functionality. Interestingly, although it's demonstrated that DNA prime-VACV boost regimen elicits high levels of T-cell functional avidity, how VACV tunes the low avidity of CD8+ T cells primed by DNA into higher ones in vivo is less defined. Herein, we proved that the enhancement of CD8+ T cell avidity induced by VACV boost is mediated by the intrinsic MyD88 pathway but not MyD88-mediated inflammatory milieu, which might provide prompts in vaccine design.
The Epstein-Barr virus (EBV) establishes a lifelong latent infection in humans. EBV infection of primary B-cells causes cell activation and proliferation, a process driven by the viral latency III gene expression program which includes EBV nuclear proteins (EBNAs), latent membrane proteins and untranslated RNAs including microRNAs. Some latently infected cells enter the long-lived memory B-cell compartment and express only EBNA1 transiently (Lat I) or no EBV protein at all (Lat 0). Targeting the molecular machinery that controls B-cell fate decisions, including the Bcl-2 family of apoptosis-regulating proteins, is crucial to the EBV cycle of infection. Here, we show that BIK (also known as NBK), which encodes a pro-apoptotic llsquo;sensitiser' protein, is repressed by the EBNA2-driven Lat III program but not the Lat I program. BIK repression occurred soon after infection of primary B-cells by EBV but not by a recombinant EBV in which the EBNA2 gene had been knocked out. Ectopic BIK induced apoptosis in Lat III cells by a mechanism dependent on its BH3 domain and the activation of caspases. We show that EBNA2 represses BIK in EBV-negative B lymphoma-derived cell lines and that this host-virus interaction can inhibit the pro-apoptotic effect of TGF-bbeta;1, a key physiological mediator of B-cell homeostasis. Reduced levels of TGF-bbeta;1-associated regulatory SMAD proteins were bound to the BIK promoter in response to EBV Lat III or ectopic EBNA2. This data is evidence of an additional mechanism used by EBV to promote B-cell survival, namely the transcriptional repression of the BH3-only sensitiser BIK.
Importance/non-technical summary Over 90% of adult humans are infected with the Epstein-Barr virus (EBV). EBV establishes a lifelong silent infection, its DNA residing in small numbers of blood B-cells that are a reservoir from which low-level virus re-activation and shedding in saliva intermittently occurs. Importantly, EBV DNA is found in some B-cell derived tumors in which viral genes play a key role in tumor cell emergence and progression. Here, we report for the first time that EBV can shut-off a B-cell gene called BIK. When activated by a molecular signal called TGF-bbeta;1, BIK plays an important role in killing unwanted B-cells, including those infected by viruses. We describe the key EBV-B-cell molecular interactions that lead to BIK shut-off. These findings further our knowledge of how EBV prevents the death of its host cell during infection. They are also relevant to certain post-transplant lymphomas where unregulated cell growth is caused by EBV genes.
Members of the genus Parvovirus are small, non-enveloped single-stranded DNA viruses that are non-pathogenic in humans but that have potential utility as cancer therapeutics. Because the innate immune response to parvoviruses has received relatively little attention, we compared parvoviruses to several other types of virus in human cells. In normal human glia, fibroblasts, or melanocytes, vesicular stomatitis virus evoked robust interferon (IFN)-bbeta; responses. Cytomegalovirus, pseudorabies, and Sindbis virus all evoked a 2-log or greater upregulation of IFN-bbeta; in glia; in contrast, LuIII and MVMp parvoviruses did not evoke a detectable IFN-bbeta; or interferon stimulated gene (ISG: MX1, OAS, IFIT-1) response in these same cell types. The lack of response raised the question of whether parvoviral infection can be attenuated by IFN; interestingly, we found that IFN did not decrease parvovirus (MVMp, LuIII, and H-1) infectivity in normal human glia, fibroblasts, or melanocytes; the same was true in human cancers including glioma, sarcoma, and melanoma. Similarly, IFN failed to attenuate transduction by the dependovirus vector, AAV-2. Progeny production of parvoviruses was also unimpaired by IFN in both glioma and melanoma, whereas VSV replication was blocked. Sarcoma cells with upregulated IFN signaling that show high resistance to other viruses showed strong infection by LuIII. Unlike many other oncolytic viruses, we find no evidence that impairment of innate immunity in cancer cells plays a role in the oncoselectivity of parvoviruses in human cells. Parvoviral resistance to the effects of IFN in cancer cells may constitute an advantage in virotherapy of some tumors.
Importance. Understanding interactions between oncolytic viruses and the innate immune system will facilitate employing these viruses as therapeutic agents in cancer victims. The cancer-selective nature of some oncolytic viruses is based on an impaired innate immunity of many cancer cells. The parvoviruses H-1, LuIII, and MVM target cancer cells, however their relationship with the innate immune system is relatively uncharacterized. Surprisingly, we find that these parvoviruses do not evoke an interferon response in normal human fibroblasts, glia, or melanocytes. Furthermore, unlike most other types of viruses, we find that parvovirus infectivity is unaffected by interferon treatment of human normal or tumor cells. Finally, parvoviral replication was unimpaired by interferon in four human tumor types, including those with residual interferon functionality. We conclude that deficits in the interferon antiviral response of cancer cells do not contribute to parvoviral oncoselectivity in human cells. The interferon-resistant phenotype of parvoviruses may give them an advantage over interferon-sensitive oncolytic viruses in tumors showing residual interferon functionality.
Enveloped viruses carry highly specialized glycoproteins that catalyze membrane fusion under strict spatial and temporal control. To prevent premature activation after biosynthesis, viral class I fusion proteins adopt a locked conformation and require proteolytic cleavage to render them fusion-ready. This priming step may occur during virus exit from the infected cell, in the extracellular milieu or during entry at or in the next target cell. Proteolytic processing of coronavirus spike (S) fusion proteins during virus entry has been suggested but not yet formally demonstrated, while the nature and functionality of the resulting subunit is still unclear. We used the prototype coronavirus - mouse hepatitis virus (MHV) - to develop a conditional biotinylation assay that enables the specific identification and biochemical characterization of viral S proteins on virions that mediated membrane fusion with the target cell. We demonstrate that MHV S proteins are indeed cleaved upon virus endocytosis and we identified a novel processing product S2* with characteristics of a fusion-active subunit. The precise cleavage site and the enzymes involved remain to be elucidated.
Importance Virus entry determines the tropism and is a crucial step in the virus life cycle. We developed an approach to characterize structural components of virus particles after entering new target cells. A prototype coronavirus was used to illustrate how the virus fusion machinery can be controlled.
Genital HSV reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized.
To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2 seropositive persons. HSV was detected via PCR and sites of asymptomatic HSV shedding were biopsied within 24 hours. CD4+ and CD8+ T cells were quantified by immunofluorescence, and HSV specific CD4+ T cells were identified by intracellular cytokine cytometry.
HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range 0-22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (pllt;0.001). In biopsies of asymptomatic shedding sites, we found increased numbers of CD8+ T cells compared to control tissue (27 vs. 13 cells/mm2, p=0.03) and identified HSV specific CD4+ T cells.
HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV-specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract.
Importance to field: This detailed study of the anatomic patterns of genital HSV-2 shedding demonstrates that HSV-2 reactivation can be detected at multiple, bilateral sites in the genital tract, suggesting that HSV establishes latency throughout the sacral ganglia. In addition, genital biopsies from sites of asymptomatic HSV shedding have increased numbers of CD8+ T cells compared to control tissue, and HSV-specific CD4+ T cells are found at sites of asymptomatic shedding. These findings suggest that that widespread asymptomatic genital HSV-2 shedding is associated with a targeted host immune response and contributes to chronic inflammation throughout the genital tract.
Anti-vector immunity limits the response to homologous boosting for viral vector vaccines. Here, we describe a new, potent vaccine vector based on replication competent vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP), which we previously showed to be safe in mice. In mice, VSV and VSV-GP encoding ovalbumin (OVA) as a model antigen (VSV-OVA, VSV-GP-OVA) induced equal levels of OVA-specific humoral and cellular immune responses upon a single immunization. However, boosting with the same vector was only possible for VSV-GP-OVA as neutralizing antibodies to VSV limited the immunogenicity of the VSV-OVA boost. OVA specific CTL responses induced by VSV-GP-OVA were at least as potent as those induced by an adenoviral state-of-the-art vaccine vector and completely protected mice in a Listeria monocytogenes challenge model. VSV-GP is so far the only replication competent vaccine vector that does not lose efficacy upon repeated application.
Importance Section Although there has been great progress in treatment and prevention of infectious diseases in the past years, effective vaccines against some of the most serious infections, e.g. AIDS, malaria, hepatitis C or tuberculosis, are urgently needed. Here, several approaches based on viral vector vaccines are under development. However, for all viral vaccine vectors currently in clinical testing repeated application is limited by neutralizing antibodies to the vector itself. Here, we have exploited the potential of vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP) as a vaccine platform. VSV-GP is the first replication-competent viral vector vaccine that does not induce vector-specific humoral immunity, i.e. neutralizing antibodies, and therefore can boost immune responses against a foreign antigen by repeated applications. The vector allows introduction of various antigens and therefore can serve as a platform technology for the development of novel vaccines against a broad spectrum of diseases.
Hepatitis C virus (HCV) infects 180 million people worldwide and is a leading cause of liver disease such as fibrosis, cirrhosis and hepatocellular carcinoma. It has been shown that HCV can spread to naiiuml;ve cells using two distinct entry mechanisms, "cell-free" entry of infectious extracellular virions that have been released by infected cells and direct "cell-to-cell" transmission. Here, we examined host-cell requirements for HCV spread and found that the cholesterol uptake receptor NPC1L1, which we recently identified as being an antiviral target involved in HCV cell-free entry/spread, is also required for the cell-to-cell spread. In contrast, the VLDL pathway, which is required for the secretion of cell-free infectious virus and thus has been identified as antiviral target for blocking cell-free virus secretion/spread, is not required for cell-to-cell spread. Noting that HCV cell-free and cell-to-cell spread share some common factors but not others, we tested the therapeutic implications of these observations and demonstrate that inhibitors that target cell factors required for both forms of HCV spread exhibit synergy when used in combination with interferon (a representative inhibitor of intracellular HCV production), while inhibitors that block only cell-free spread do not. This provides insight into mechanistic basis of synergy between interferon and HCV entry inhibitors and highlights the broader, previously unappreciated impact blocking HCV cell-to-cell spread can have on the efficacy of HCV combination therapies.
Importance Statement HCV can spread to naiiuml;ve cells using distinct mechanisms, "cell-free" entry of extracellular virus and direct "cell-to-cell" transmission. Herein, we identify the host cell HCV entry factor NPC1L1 as also being required for HCV cell-to-cell spread, while showing that the VLDL pathway, which is required for the secretion of cell-free infectious virus, is not required for cell-to-cell spread. While both these host factors are considered viable antiviral targets, we demonstrate that only inhibitors that block factors required for both forms of HCV entry/spread (i.e. NPC1L1) exhibit synergy when used in combination with interferon, while inhibitors that block factors only required for cell-free spread (i.e. VLDL pathway components) do not. Thus, this study advances our understanding of HCV cell-to-cell spread, provides mechanistic insight into the basis of drug synergy and highlights inhibition of HCV spread as a previously unappreciated consideration in HCV therapy design.
PML-derived non-coding control region (NCCR) sequences permitted increased early viral gene expression compared with kidney-associated NCCR. This was driven in part by binding of the transcription factor Spi-B to unique PML-associated Spi-B binding sites. Spi-B is upregulated in developing B cells in response to natalizumab therapy, a known risk factor for PML. Naturally occurring JCV sequence variation, together with drug treatment-induced cellular changes, may synergize to create an environment leading to increased risk of PML.
Following human immunodeficiency virus (HIV-1) entry into the host cell, the viral capsid gradually disassembles in a process called uncoating. A proper rate of uncoating is important for reverse transcription of the HIV-1 genome. Host restriction factors such as TRIM5aalpha; and TRIMCyp bind retroviral capsids and cause premature disassembly, leading to blocks in reverse transcription. Other host factors, such as cyclophilin A, stabilize the HIV-1 capsid and are required for efficient infection in some cell types. Here, we show that a heat-labile factor greater than 100 kilodaltons in the cytoplasm of cells from multiple vertebrate species slows the spontaneous disassembly of HIV-1 capsid-nucleocapsid (CA-NC) complexes in vitro. We identified the PDZ-Domain-containing protein 8 (PDZD8) as a critical component of the capsid-stabilizing activity in the cytoplasmic extracts. PDZD8 has been previously reported to bind the HIV-1 Gag polyprotein and to make a positive contribution to the efficiency of HIV-1 infection (1). PDZD8 knockdown accelerated the disassembly of HIV-1 capsids in infected cells, resulting in decreased reverse transcription. The PDZD8 coiled coil domain is sufficient for HIV-1 capsid binding, but other parts of the protein, including the PDZ domain, are apparently required for stabilizing the capsid and supporting HIV-1 infection. In summary, PDZD8 interacts with and stabilizes the HIV-1 capsid and thus represents a potentially targetable host cofactor for HIV-1 infection.
HIV and SIV strains differ in their capacity to replicate in macrophages, but mechanisms underlying these differences are not fully understood. Here, we identify a highly conserved N-linked glycosylation site (N173 in SIV, corresponding to N160 in HIV) in the V2 region of the SIV envelope glycoprotein (Env) as a novel determinant of macrophage tropism and characterize mechanisms underlying this phenotype. Loss of the N173 glycosylation site in the non-macrophage-tropic SIVmac239 virus by introducing an N173Q mutation enhanced viral replication and multinucleated giant cell formation upon infection of rhesus macrophages, while addition of N173 to SIVmac251 had the opposite effect. Removal of N173 in SIVmac239 enhanced CD4-independent cell-to-cell transmission to CCR5-expressing cells. SIVmac239 with N173Q mediated CD4-independent cell-cell fusion, but could not infect CD4-negative cells in single-round infections. Thus, CD4-independent phenotypes were detected only in the context of cell-to-cell contact. Similar results were obtained in SIVmac251 with and without N173. N173 decreased neutralization sensitivity of SIVmac251, but had no effect on neutralization sensitivity of SIVmac239. N173Q mutation had no effect on SIVmac239 binding to CD4 in BIACORE, co-immunoprecipitation, and ELISA assays. These findings suggest that loss of the N173 N-linked glycosylation site increases SIVmac239 replication in macrophages by enhancing CD4-independent cell-to-cell virus transmission through CCR5-mediated fusion. This mechanism may facilitate escape of macrophage-tropic viruses from neutralizing antibodies, while promoting spreading infection by these viruses in vivo.
Importance In this study, we identify a genetic determinant in the viral envelope (N173) that increases replication and spreading infection of SIV strains in macrophages by enhancing cell-to-cell virus transmission. This effect is explained by a novel mechanism involving increased cell-to-cell fusion in the absence of CD4, the primary receptor that normally mediates virus entry. The same genetic determinant also affects sensitivity of these viruses to inhibition by neutralizing antibodies. Most macrophage-tropic HIV/SIV strains are known to be neutralization-sensitive. Together, these findings suggest that this efficient mode of virus transmission may facilitate escape of macrophage-tropic viruses from neutralizing antibodies, while promoting spreading infection by these viruses to cells expressing little or no CD4 in vivo.
Rates of spontaneous mutation determine viral fitness and adaptability. In RNA viruses, treatment with mutagenic nucleoside analogues selects for polymerase variants with increased fidelity, showing that viral mutation rates can be adjusted in response to imposed selective pressures. However, this type of resistance is not possible in viruses that do not encode their own polymerases, such as single-stranded DNA viruses. We previously showed that serial passaging of bacteriophage X174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU) favored substitutions in the lysis protein E. Here, we found that approximately one half (6/12) of the amino acid replacements in the N-terminal region of this protein led to delayed lysis, and two of these changes (V2A and D8A) also conferred partial resistance to 5-FU. By delaying lysis, the V2A and D8A substitutions allowed the virus to increase the burst size per cell in the presence of 5-FU. Furthermore, these substitutions tended to alleviate drug-induced mutagenesis by reducing the number of rounds of copying required for population growth, thus revealing a new mechanism of resistance. This form of mutation rate regulation may also be utilized by other viruses whose replication mode is similar to that of bacteriophage X174.
Importance Many viruses display high rates of spontaneous mutations due to defects in proofreading or post-replicative repair, allowing them to rapidly adapt to changing environments. Viral mutation rates may have been optimized to achieve high adaptability without incurring in an excessive genetic load. Supporting this, RNA viruses subjected to chemical mutagenesis treatments have been shown to evolve higher-fidelity polymerases. However, many viruses cannot modulate replication fidelity because they do not encode their own polymerase. Here we show a new mechanism for regulating viral mutation rates. We found that, under mutagenic conditions, the single-stranded bacteriophage X174 evolved delayed lysis, and that this allowed the virus to increase the amount of progeny produced per cell. As a result, the viral population was amplified in fewer infection cycles, therefore reducing the chances for mutation appearance.
CD4+ and CD8+ memory T cells with stem cell-like properties (TSCM) have been identified in mice, humans, and non-human primates and are being investigated for antitumor and antiviral vaccines and immunotherapies. Whether CD4+ TSCM are infected by HIV was investigated using a combination HIV reporter virus system in vitro and by direct staining for HIV p24 antigen ex vivo. A proportion of TSCM were found to express the HIV coreceptors CCR5 and CXCR4 and were infected by HIV both in vitro and in vivo. Analysis of viral outcome following fusion using the combination reporter virus system revealed that TSCM can become productively or latently infected, although the vast majority of TSCM are abortively infected. Knockdown of the HIV restriction factor SAMHD1 using Vpx-containing SIV virion-like particles enhanced the productive infection of TSCM, indicating SAMHD1 contributes to abortive infection in these cells. These results demonstrate that CD4+ TSCM are targets for HIV infection, become productively and latently infected at low levels, and that SAMHD1 expression promotes abortive infection of this important memory subset.
Importance Here, we demonstrate CD4+ memory stem cells (TSCM) are susceptible to infection by HIV in vitro and in vivo and provide an in-depth analysis of coreceptor expression, HIV infection of naiiuml;ve and memory CD4+ T cell subsets with both CCR5- and CXCR4-tropic HIV, and also perform outcome analysis to calculate the percentage of cells that are productively, latently, and abortively infected. Through these outcome studies, we determined that the vast majority of TSCM are abortively infected by HIV and demonstrate that knockdown of SAMHD1 significantly improves the infection of this CD4+ T cell subset, indicating that SAMHD1 is an active restriction factor in TSCM.
The Middle East Respiratory Syndrome coronavirus (MERS-CoV) recently spread from an animal reservoir to infect humans, causing sporadic severe, and frequently fatal respiratory disease. Appropriate public health and control measures will require discovery of the zoonotic MERS coronavirus reservoirs. The relevant animal hosts are liable to be those that offer optimal MERS virus-cell entry. Cell entry begins with virus spike (S) protein binding to DPP4 receptors. We constructed chimeric DPP4 receptors that have the virus-binding domains of indigenous Middle Eastern animals, and assessed the activities of these receptors in supporting S protein binding and virus entry. Human, camel, and horse receptors were potent and nearly equally effective MERS virus receptors, while goat and bat receptors were considerably less effective. These patterns reflected S protein affinities for the receptors. However, even the low-affinity receptors could hyper-sensitize cells to infection when S-cleaving protease(s) were present, indicating that affinity thresholds for virus entry must be considered in the context of host-cell proteolytic environments. These findings suggest that virus receptors and S protein-cleaving proteases combine in a variety of animals to offer efficient virus entry, and that several Middle Eastern animals are potential reservoirs for transmitting MERS-CoV to humans.
Importance Middle East Respiratory Syndrome (MERS) is a frequently fatal disease that is caused by a zoonotic coronavirus (CoV). The animals transmitting MERS-CoV to humans are not yet known. Infection by MERS-CoV requires receptors and proteases on host cells. We compared the receptors of humans and Middle Eastern animals, and found that human, camel and horse receptors sensitized cells to MERS-CoV infection more robustly than goat and bat receptors. Infection susceptibility correlated with affinities of the receptors for viral spike proteins. We also found that the presence of a cell-surface lung protease will greatly increase susceptibility to MERS-CoV, particularly in conjunction with low-affinity receptors. This cataloguing of human and animal host cell factors allows one to make inferences on the distribution of MERS-CoV in nature.
Hepatitis A virus (HAV) has a highly biased and deoptimized codon usage compared to the host cell and fails to inhibit host protein synthesis. It has been proposed that an optimal combination of abundant and rare codons controls the translation speed required for the correct capsid folding. The artificial shut-off of host protein synthesis results in the selection of variants containing mutations in the HAV capsid coding region critical for folding, stability, and function. Here we show that these capsid mutations resulted in changes in their antigenicity, in a reduced stability to high temperature, low pH, and biliary salts and in an increased efficacy of cell entry. In conclusion, the adaptation to cellular shut-off resulted in the selection of large plaque-producing virus populations.
Importance HAV has a naturally deoptimized codon usage with respect to that of its cell host and is unable to shut down the cellular translation. This fact contributes to the low replication rate of the virus, in addition to other factors such as the highly inefficient IRES, and explains the outstanding physical stability of this pathogen in the environment mediated by a folding-dependent highly cohesive capsid. Adaptation to artificially induced cellular transcription shut-off, resulted in a re-deoptimization of its capsid codon usage, instead of an optimization.
These genomic changes are related to an overall change of capsid folding, which in turn induces changes in the cell entry process. Remarkably, the adaptation to cellular shut-off allowed the virus to significantly increase its RNA uncoating efficiency resulting in the selection of large plaque-producing populations. However, these populations produced much debilitated virions.
Epstein-Barr virus (EBV) lytic replication involves complex processes, including DNA synthesis, DNA cleavage and packaging, and virion egress. These processes require many different lytic gene products, but the mechanisms of their actions remain unclear, especially for DNA cleavage and packaging. According to sequence homology analysis, EBV BALF3, encoded by the BamHI A fragment leftward open reading frame 3 in the viral genome, is a homologue of herpes simplex virus type 1 (HSV-1) UL28. This gene product is believed to possess the properties of a terminase, such as exhibiting a nucleolytic activity on newly synthesized viral DNA and translocating unit-length viral genomes into procapsids. In order to characterize EBV BALF3, the protein was produced and purified from recombinant baculoviruses and examined in an enzymatic reaction in vitro, determining that EBV BALF3 acts as an endonuclease, and its activity is modulated by Mg2+, Mn2+, and ATP. Moreover, in EBV-positive epithelial cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following lytic induction, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Interestingly, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. Based on these results, we suggest that EBV BALF3 is involved simultaneously in DNA synthesis and packaging and is required for the production of mature virions.
IMPORTANCE Virus lytic replication is essential to produce infectious virions, which is responsible for virus survival and spread. This work shows that an uncharacterized gene product of human herpesvirus EBV, BALF3, is expressed during the lytic cycle. In addition, BALF3 mediates an endonucleolytic reaction and is involved in viral DNA synthesis and packaging, leading to influence the production of mature virions. According to sequence homology and physical properties, the lytic gene product BALF3 is considered a terminase in EBV. These findings identify a novel viral gene with an important role in conducing more to an understanding of the EBV life.
Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immunodeficiency syndrome (AIDS)-related malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease. The interaction of human immunodeficiency virus type 1 (HIV-1) and KSHV has a central role in promoting the aggressive manifestations of AIDS-KS. We have previously shown that negative factor (Nef), a secreted HIV-1 protein, synergizes with KSHV vIL-6 to promote angiogenesis and tumorigenesis by activating the AKT pathway. Here, we further demonstrated the role of soluble and ectopic Nef in the regulation of KSHV latency. We found that both soluble Nef protein and ectopic expression of Nef by transfection suppressed the expression of KSHV viral lytic mRNA transcripts and proteins, and production of infectious viral particles. MicroRNAs (miRNAs) microarray analysis identified a number of Nef-regulated miRNAs. Bioinformatics and luciferase reporter analyses showed that one of the Nef-upregulated miRNAs, a cellular miRNA 1258 (hsa-miR-1258), directly targeted a seed sequence in the 3' untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls KSHV reactivation from latency. Ectopic expression of hsa-miR-1258 impaired RTA synthesis and enhanced Nef-mediated inhibition of KSHV replication, whereas repression of hsa-miR-1258 has the opposite effect. Mutation of the seed sequence in the RTA 3'UTR abolished down-regulation of RTA by hsa-miR-1258. Collectively, these novel findings demonstrate that, by regulating cellular miRNA, Nef may inhibit KSHV replication to promote viral latency, and thus contribute to the pathogenesis of AIDS-related malignancies.
Importance This study presented that negative factor (Nef), a secreted HIV-1 protein, suppressed KSHV lytic replication to promote KSHV latency. Mechanistic studies indicated that a Nef-upregulated cellular microRNAs (miRNAs) hsa-miR-1258 inhibits KSHV replication by directly targeting a seed sequence in the KSHV RTA 3' UTR. These results illustrate that, in addition to viral miRNAs, cellular miRNAs also play an important role in regulating the life cycle of KSHV. Overall, this is the first study to report the involvement of Nef in KSHV latency implying its likely important role in the pathogenesis of AIDS-related malignancies.
Swine influenza A virus is an endemic and economically important pathogen in pigs with the potential to infect other host species. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major component in swine influenza A vaccines. However, as a result of antigenic drift, vaccine strains must be regularly updated to reflect currently circulating strains. Characterizing the cross-reactivity between strains in pigs and seasonal influenza strains in humans is also important in assessing the relative risk of interspecies transmission of viruses from one host population to the other. Hemagglutination inhibition (HI) assay data for swine and human H3N2 viruses were used with antigenic cartography to quantify the antigenic differences among H3N2 viruses isolated from pigs in the USA from 1998-2013 and the relative cross-reactivity between these viruses and current human seasonal influenza A strains. Two primary antigenic clusters were found circulating in the pig population, but with enough diversity within and between the clusters to suggest updates in vaccine strains are needed. We identified single amino acid substitutions likely responsible for antigenic differences between the two primary antigenic clusters and between each antigenic cluster and outliers. The antigenic distance between current seasonal influenza H3 strains in humans and those endemic in swine suggests that population immunity may not prevent the introduction of human viruses into pigs and possibly vice-versa, reinforcing the need to monitor and prepare for potential incursions.
Importance Influenza A virus (IAV) is an important pathogen in pigs and humans. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major target of vaccines. However, vaccine strains must be updated to reflect current strains. Characterizing the differences between seasonal IAV in humans and swine IAV is important in assessing the relative risk of interspecies transmission of viruses. We found two primary antigenic clusters of H3N2 in the U.S. pig population with enough diversity to suggest updates in swine vaccine strains. We identified changes in the HA protein that are likely responsible for these differences that may be useful in predicting when vaccines need to be updated. The difference between human H3N2 and those in swine is enough that population immunity is unlikely to prevent new introductions of human IAV into pigs or vice-versa, reinforcing the need to monitor and prepare for potential introductions.
Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza viruses. We have analysed here the role of the serine protease TMPRSS2 activating HA in the human respiratory tract on pathogenesis in a mouse model.
Replication of the human H7N9 isolate A/Anhui/1/13 and of human H1N1 and H3N2 viruses was compared in TMPRSS2-knockout (TMPRSS2-/-) and wildtype (WT) mice. Knockout of TMPRSS2 expression inhibited H7N9 influenza virus replication in explants of murine trachea, bronchi and lung. H1N1 virus replication was also strongly suppressed in airway explants of TMPRSS2-/- mice, while H3N2 virus replication was only marginally affected. H7N9 and H1N1 viruses were apathogenic in TMPRSS2-/- mice, whereas WT mice developed severe disease with mortality rates of 100% and 20%, respectively. In contrast, all H3N2 infected TMPRSS2-/- and WT mice succumbed to lethal infection. Cleavage analysis showed that H7 and H1 are efficiently activated by TMPRSS2, whereas H3 is less susceptible to the protease.
Our data demonstrate that TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 and H1N1 influenza virus in mice. In contrast, replication of H3N2 virus appears to depend on another, not yet identified protease, supporting the concept that human influenza viruses differ in protease specificity.
Importance Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza virus, but little is known about its relevance for pathogenesis in mammals. Here, we show that knockout mice that do not express the HA-activating protease TMPRSS2 are resistant to pulmonary disease with lethal outcome when infected with influenza A viruses of subtypes H7N9 and H1N1, whereas they are not protected from lethal H3N2 virus infection. These findings demonstrate that human influenza viruses differ in protease specificity, and that expression of the appropriate protease in respiratory tissues is essential for pneumotropism and pathogenicity. Our observations also demonstrate that HA-activating proteases and in particular TMPRSS2 are promising targets for influenza therapy.
There are currently no FDA approved vaccines or therapeutics to prevent or treat Argentine Hemorrhagic Fever (AHF). The causative agent of AHF is Junin virus (JUNV); a new world arenavirus classified as an NIAID/CDC Category A priority pathogen. The PTAP Late (L) domain motif within JUNV Z protein facilitates virion egress and transmission by recruiting host Tsg101 and other ESCRT complex proteins to promote scission of the virus particle from the plasma membrane. Here we describe a novel compound (compound 0013) that blocks the JUNV Z-Tsg101 interaction and inhibits budding of virus-like particles (VLPs) driven by ectopic expression of the Z protein and live attenuated JUNV Candid-1 strain in cell culture. As inhibition of the PTAP-Tsg101 interaction inhibits JUNV egress, compound 0013 serves as a prototype therapeutic that could reduce virus dissemination and disease progression in infected individuals. Moreover, since PTAP L-domain-mediated Tsg101 recruitment is utilized by other RNA virus pathogens (e.g. Ebola virus and HIV-1), PTAP inhibitors such as compound 0013 have the potential to function as potent broad-spectrum, host-oriented antiviral drugs.
The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8+ and CD4+ T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof-of-principle that functional EBNA-1-specific T cells can be expanded by vaccination.
IMPORTANCE: EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more robust T cell responses through modified vaccines.
Ikaros is a zinc-finger DNA-binding protein that regulates chromatin remodeling and expression of genes involved in the cell cycle, apoptosis, and Notch signaling. It is a master regulator of lymphocyte differentiation and functions as a tumor suppressor in acute lymphoblastic leukemia. Nevertheless, no previous reports described effects of Ikaros on the life cycle of any human lymphotropic virus. Here, we demonstrate that full-length Ikaros (IK-1) functions as a major factor in maintenance of viral latency in EBV-positive Burkittrrsquo;s lymphoma Sal and MutuI cell lines. Either silencing of Ikaros expression by shRNA knockdown or ectopic expression of a non-DNA-binding isoform induced lytic-gene expression. These effects synergized with other lytic inducers of EBV, including TGF-bbeta; and the hypoxia mimic desferrioxamine. Data from ChIP-qPCR and ChIP-seq analyses indicated that Ikaros did not bind to either the EBV immediate-early BZLF1 or BRLF1 gene. Rather, Ikaros affected expression of Oct-2 and Bcl-6, other transcription factors that directly inhibit EBV reactivation and plasma cell differentiation, respectively. IK-1 also complexed with the EBV immediate-early R protein in co-immunoprecipitation assays and partially co-localized with R within cells. Presence of R alleviated IK-1-mediated transcriptional repression, with IK-1 then cooperating with Z and R to enhance lytic-gene expression. Thus, we conclude that Ikaros plays distinct roles at different stages of EBVrrsquo;s life cycle: it contributes to maintaining latency via indirect mechanisms; and it may also synergize with Z and R to enhance lytic replication through direct association with R and/or R-induced alterations in Ikarosrrsquo; functional activities via cellular signaling pathways.
Importance: This is the first report showing that the cellular protein Ikaros, a known master regulator of hematopoiesis and critical tumor suppressor in acute lymphoblastic leukemia, also plays important roles in the life cycle of Epstein-Barr virus in B cells.
Recent evidence identified multiple Henipavirus species in Africa distinct from those in Southeast Asia and Australia. The reported fusion glycoprotein (F) sequence of the African Gh-M74a strain (GhV-F) is likely incorrect: a single base-pair deletion near the N-terminus results in multiple aberrancies. Rectifying single nucleotide insertions result in a GhV-F that now possesses a signal peptide, is efficiently cell-surface expressed, exhibits syncytia formation when co-expressed with GhV-G protein, and mediates pseudotyped viral particle entry.
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I:C). Expression of MERS-CoV 4a protein suppressed interferon production induced by poly(I:C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5, but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner, but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5, but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1 and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA binding protein to circumvent innate antiviral response by perturbing the function of cellular double-stranded RNA binding protein PACT. PACT targeting might be a common strategy used by different viruses including Ebola virus and herpes simplex virus 1 to counteract innate immunity.
IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging and highly lethal human pathogen. Why MERS-CoV causes severe disease in human is unclear and one possibility is that MERS-CoV is particularly efficient in counteracting host immunity including the sensing of virus invasion. It will therefore be critical to clarify how MERS-CoV cripples the host proteins that sense viruses and to compare MERS-CoV with its ancestral viruses in bats in the counteraction of virus sensing. This work not only provides a new understanding of the abilities of MERS-CoV and closely related bat viruses to subvert virus sensing, but might also prove useful in revealing new strategies for the development of vaccines and antivirals.
While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here, we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus sub-family of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ~122-nt long pri-miRNA, encoded within the BFV long terminal repeat "U3" region, that is subsequently cleaved to generate two pre-miRNA that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III and the three resultant mature miRNAs were found to contribute a remarkable ~70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs using embedded proviral RNA polymerase III promoters.
Importance Foamy viruses are a ubiquitous family of non-pathogenic retroviruses that have potential as gene therapy vectors in humans. Here, we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ~122-nt long pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo.
We compared the kinetics and magnitude of HBV infection in HCV-naiiuml;ve and chronically HCV-infected chimpanzees in whose livers type I interferon stimulated gene (ISG) expression is strongly induced. HBV infection was delayed and attenuated in the HCV-infected animals and the number of HBV-infected hepatocytes was drastically reduced. These results suggest that establishment of HBV infection and its replication space is limited by the antiviral effects of type I interferon in the chronically HCV-infected liver.
Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFbbeta; was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFbbeta; remains unclear. Several studies noted reduced Vif expression in CBFbbeta; knockdown cells while others saw no significant impact of CBFbbeta; on Vif stability. Here we confirmed that CBFbbeta; increases Vif steady-state levels. CBFbbeta; neither affected expression of viral Gag nor Vpu protein indicating that CBFbbeta; regulates Vif expression post-transcriptionally. Kinetic studies revealed effects of CBFbbeta; both on metabolic stability and on the rate of Vif biosynthesis. These effects were dependent on the ability of CBFbbeta; to interact with Vif. Importantly, at comparable Vif levels, CBFbbeta; further enhanced A3G degradation suggesting that CBFbbeta; facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFbbeta; also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFbbeta; had no detectable effect on RUNX1 metabolic stability. We propose that CBFbbeta; acts like a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G.
Importance In this study we show that CBFbbeta; has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFbbeta;. Kinetic studies revealed that CBFbbeta; increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFbbeta; were non-responsive to CBFbbeta;. Our data suggest that CBFbbeta; exerts a chaperone-like activity to (i) minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.
We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicity in mice. We found that four amino acid mutations (PA-P224S, PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. Particularly, PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced its 50% mouse lethal dose (LD50) by almost 1,000-fold.
Infection with laboratory-attenuated rabies virus (RABV) enhances Blood-brain Barrier (BBB) permeability, which has been demonstrated to be an important factor for host survival since it allows immune effectors to enter into the CNS to clear RABV. To probe the mechanism by which RABV infection enhances BBB permeability, the expression of tight junction (TJ) proteins in the CNS was investigated following intracranial inoculation with laboratory-attenuated or wt RABV. BBB permeability was significantly enhanced in mice infected with laboratory-attenuated, but not wt, RABV. The expression levels of TJ proteins (claudin-5, occludin, and Zonula Occludens-1) were decreased in mice infected with laboratory-attenuated, but not wt, RABV, suggesting that enhancement of BBB permeability is associated with reduction of TJ protein expression in RABV infection. RABV neither infects the brain microvascular endothelial cells (BMECs) nor modulates the expression of TJ proteins in BMECs. However, brain extracts prepared from mice infected with laboratory-attenuated, but not wt, RABV reduced TJ protein expressions in BMECs. It was found that brain extracts from mice infected with laboratory-attenuated RABV contained significantly higher levels of inflammatory chemokines/cytokines than those from mice infected with wt RABV. Pathway analysis indicates that IFN- is located in the center of the cytokine network in RABV-infected mouse brain and neutralization of IFN- ameliorated both disruption of BBB permeability in vivo and down-regulation of TJ protein expression in vitro. These findings indicate that enhancement of BBB permeability and reduction of TJ protein expressions are not due to RABV infection per se, but due to virus-induced inflammatory chemokines/cytokines.
Importance Previous studies have shown that infection with only laboratory-attenuated, not wild-type, rabies virus (RABV) enhances Blood-brain Barrier (BBB) permeability, which allows immune effectors to enter into the central nervous system (CNS) and clear RABV from the CNS. This study investigated the mechanism by which RABV infection enhances BBB permeability. It was found that RABV infection enhances BBB permeability by down-regulation of tight junction (TJ) protein expression in the brain microvasculature. It was further found that it is not RABV infection per se, but the chemokines/cytokines induced by RABV infection that down-regulate the expression of TJ proteins and enhance the BBB permeability. Blocking some of the cytokines such as IFN- ameliorated both disruption of BBB permeability and down-regulation of TJ protein expression. These studies may provide a foundation for developing therapeutics for clinical rabies such as medication that could be used to enhance the BBB permeability.
MicroRNAs (miRNAs) are single stranded small RNA molecules that regulate various cellular processes. miR-155 regulates various aspects of innate and adaptive immune response and plays a key role in various viral infections and resulting neuroinflammation. In the present study, evaluated the involvement of miR-155 in modulating Japanese Encephalitis Virus (JEV)- induced neuroinflammation. We observed that miR-155 expression was up regulated during JEV infection of mouse primary microglia, BV-2 microglia cell line, and in both mouse and human brain. In vitro and in vivo knockdown of miR-155 minimized JEV-induced inflammatory responses. In the present study, we confirmed targeting of SHIP1 3rrsquo;UTR by miR-155 in the context of JEV infection. Inhibition of SHIP1 by miR-155 resulted in higher interferon beta (IFN-bbeta;) and pro-inflammatory cytokine production through activation of TANK-binding kinase 1 (TBK-1). Based on these observations we conclude that miR-155 modulates neuroinflammatory response during JEV infection via negative regulation of SHIP1 expression. Thus, modulation of miR-155 could be a novel strategy to regulate JEV induced neuroinflammation.
Importance Japanese Encephalitis Virus, a member of flaviviridae family that causes Japanese encephalitis (JE), is the most common mosquito-born encephalitis in the Asia-Pacific region. The disease is feared as currently there are no specific antiviral drugs available. JE Virus targets central nervous system, and leading to high mortality and neurological and psychiatric sequeale in some of those who survive. The level of inflammation correlates well with the clinical outcome in patients. Recently, microRNA (miRNA), a single stranded non-coding RNA have been implicated in various brain disorders. The present study investigates the role of miRNA in JEV induced neuroinflammation. Our results showed that miR-155 targets SHIP1 protein and promotes inflammation by regulating NFB pathway, increasing expression of various pro-inflammatory cytokines and anti-viral response. Thus, miR-155 is a potential therapeutic target to develop anti viral in JE and other brain disorders where inflammation plays significant role in diseases progression.
The NFB signaling pathway plays a critical role in inflammation and innate immunity. Consequently, many viruses have evolved strategies to inhibit NFB in order to facilitate replication and evasion of the host immune response. Recently, we determined that ectromelia virus, the causative agent of mousepox, contains a family of four BTB/kelch proteins that interact with cullin-3-based ubiquitin ligases. Here we demonstrate that expression of EVM150, one of the four BTB/kelch proteins, inhibited NFB activation induced by TNFllt; and IL-1rreg;. Although EVM150 inhibited NFB p65 nuclear translocation, IBaalpha; degradation was observed, indicating that EVM150 functioned downstream of IBaalpha; degradation. Significantly, expression of the BTB-only domain of EVM150 blocked NFB activation, demonstrating that EVM150 functioned independently of the kelch domain and its role as an adapter for cullin-3-based ubiquitin ligases. Furthermore, cullin-3 knock down by siRNA demonstrated that cullin-3-based ubiquitin ligases are dispensable for TNFllt;-induced NFB activation. Interestingly, nuclear translocation of IRF3 and STAT1 still occurred in the presence of EVM150, indicating that EVM150 prevented NFB nuclear translocation specifically. In addition to identifying EVM150 as an inhibitor of the NFB pathway, this study provides new insights into the role of BTB/kelch proteins during virus infection.
Importance Paragraph: With the exception of virulence studies, little work has been done to determine the role of poxviral BTB/kelch proteins during infection. This study, for the first time, has identified a mechanism for the ectromelia virus BTB/kelch protein EVM150. Here, we show that EVM150 is a novel inhibitor of the cellular NFB pathway, an important component of the antiviral response. This study adds EVM150 to the growing list of NFB inhibitors in poxviruses, and provides new insights into the role of BTB/kelch proteins during virus infection.
The non-covalent interactions that mediate trimerization of the influenza hemagglutinin (HA) are important determinants of its biological activities. Recent studies have demonstrated that mutations in the HA trimer interface affect the thermal and pH sensitivities of HA, suggesting a possible impact on vaccine stability (Farnsworth et al. 2011. Vaccine 29:1529-1533). We used size exclusion chromatography analysis of recombinant HA ectodomain to compare the differences among recombinant trimeric HA proteins from early 2009 pandemic H1N1 viruses, which dissociate to monomers, with those of more recent virus HAs that can be expressed as trimers. We analyzed differences amongst the HA sequences and identified inter-molecular interactions mediated by the residue at position 374 (HA0 numbering) of the HA2 sub-domain as critical for HA trimer stability. Crystallographic analyses of HA from the recent H1N1 virus A/Washington/5/2011 highlight the structural basis for this observed phenotype. It remains to be seen whether more recent viruses with this mutation will yield more stable vaccines in the future.
Importance Hemagglutinins from the early 2009 H1N1 pandemic viruses are unable to maintain a trimeric complex when expressed in a recombinant system. However HAs from 2010 and 2011 strains are more stable and our work highlights the improvement in stability can be attributed to an E47K substitution in the HA2 subunit of the stalk that emerged naturally in the circulating viruses.
Estradiol (E2) treatment limits the pathology associated with pulmonary diseases caused by pathogens, allergens, and asthma, partly by reducing the production of proinflammatory cytokines and chemokines. To test the hypothesis that E2 protects against influenza A virus (IAV) infection by altering the recruitment and activity of innate immune cells and T cells, chemokine concentrations were measured and innate and adaptive immune cells were enumerated from the lungs of E2- and placebo-treated ovariectomized female C57BL/6 mice following infection. Females treated with E2 experienced less morbidity, but had similar lung virus titers as placebo-treated females. Females treated with E2 had lower induction of CCL2, but higher CCL3 and CXCL1 responses, in their lungs than placebo-treated females. Pulmonary recruitment of neutrophils, NK cells, macrophages, and dendritic cells was increased following infection, but only neutrophil numbers were greater in E2-treated than placebo-treated females. Neutrophils enhance the responses of influenza-specific CD8 T cells to promote virus clearance and improve the outcome of infection. Total numbers of virus-specific CD8 T cells were not altered by treatment with E2, but the proportion of IFN- and TNF-aalpha; producing, virus-specific CD8 T cells was increased. Neutrophil depletion in E2-treated females increased morbidity, reduced pulmonary production of chemoattractants for neutrophils, and reduced IFN- production by virus-specific CD8 T cells. Neutrophils mediate both inflammation and tissue repair during IAV infection, and are regulated by E2 to improve the outcome of influenza in females.
Importance Severe influenza is associated with excessive inflammation that leads to tissue damage. We demonstrate that estradiol (E2) is a potent anti-inflammatory hormone that reduces the severity of influenza A virus infection in females. Treatment of female C57BL/6 mice with E2 does not affect virus replication, but rather alters the production of chemokines, pulmonary recruitment of neutrophils, and the cytokine responses of virus-specific CD8 T cells to protect females against severe influenza.
The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, full length (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activity. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir.
IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth and high level resistance to maribavir when compared to the predominant full length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo.
The extent to which HIV-1 clade B strains exhibit population-specific adaptations to host HLA alleles remains incompletely known, in part due to incomplete characterization of HLA-associated HIV-1 polymorphisms (HLA-APs) in different global populations. Moreover, it remains unknown to what extent the same HLA alleles may drive significantly different escape pathways across populations. As the Japanese population exhibits distinctive HLA class I allele distributions, comparative analysis of HLA-APs between HIV-1 clade B-infected Japanese and non-Asian cohorts could shed light on these questions. However HLA-APs remain incompletely mapped in Japan. In a cohort of 430 treatment-naiiuml;ve Japanese with chronic HIV-1 clade B infection, we identified 284 HLA-APs in Gag, Pol and Nef using phylogenetically-corrected methods. The number of HLA-associated substitutions in Pol, notably those restricted by HLA-B*52:01, was weakly inversely correlated with plasma viral load (pVL), suggesting that the transmission and persistence of B*52:01-driven Pol mutations could modulate pVL. Differential selection of HLA-APs between HLA subtype members, including those differing only with respect to substitutions outside the peptide-binding groove, was observed, meriting further investigation as to their mechanisms of selection. Notably, two-thirds of HLA-AP identified in Japan had not been reported in previous studies of predominantly Caucasian cohorts, and were attributable to HLA alleles unique to, or enriched in, Japan. We also identified 71 cases where the same HLA allele drove significantly different escape pathways between Japan versus predominantly Caucasian cohorts. Our results underscore the distinct global evolution of HIV-1 clade B as a result of host population-specific cellular immune pressures.
Importance section CTL escape mutations in HIV-1 are broadly predictable based on the HLA class I alleles expressed by the host. Because HLA allele distributions differ among worldwide populations, the pattern and diversity of HLA-associated escape mutations are likely to be somewhat distinct to each race and region. HLA-associated polymorphisms (HLA-APs) in HIV-1 have previously been identified at the population level in European, North American, Australian and African cohorts, however, large-scale analyses of HIV-1 clade B-specific HLA-APs in Asians are lacking. Differential intra-clade HIV-1 adaptation to global populations can be investigated via comparative analyses of HLA-associated polymorphisms across ethnic groups, but such studies are rare. Here, we identify HLA-APs in a large Japanese HIV-1 clade B cohort using phylogeneticaly-informed methods and observe that the majority of them had not been previously characterized in predominantly Caucasian populations. Results highlight HIVrrsquo;s unique adaptation to cellular immune pressures imposed by different global populations.
Herpesviruses have evolved a unique mechanism for nuclear egress of nascent progeny nucleocapsids: the nucleocapsids bud through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes (primary envelopment) and enveloped nucleocapsids then fuse with the outer nuclear membrane to release nucleocapsids into the cytoplasm (de-envelopment). We have shown that the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (or VP13/VP14) is a novel regulator for HSV-1 nuclear egress. In particular: (i) UL47 formed a complex(es) with HSV-1 proteins UL34, UL31, and/or Us3, which have all been reported to be critical for viral nuclear egress, and these viral proteins co-localized at the nuclear membrane in HSV-1-infected cells; (ii) the UL47-null mutation considerably reduced primary enveloped virions in the perinuclear space, although capsids accumulated in the nucleus; and (iii) UL47 was detected in primary enveloped virions in the perinuclear space by immunoelectron microscopy. These results suggested that UL47 promoted HSV-1 primary envelopment, probably, by interacting with the critical HSV-1 regulators for viral nuclear egress and by modulating their functions.
Importance Like other herpesviruses, herpes simplex virus 1 (HSV-1) has evolved a vesicle-mediated nucleocytoplasmic transport mechanism for nuclear egress of nascent progeny nucleocapsids. Although previous reports identified and characterized several HSV-1 and cellular proteins involved in viral nuclear egress, complete details of HSV-1 nuclear egress remain to be elucidated. In this study, we have presented data suggesting that (i) the major HSV-1 virion structural protein UL47 (or VP13/14) formed a complex with known viral regulatory proteins critical for viral nuclear egress, and (ii) UL47 played a regulatory role in HSV-1 primary envelopment. Thus, we identified UL47 as a novel regulator for HSV-1 nuclear egress.
During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but the temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences with mammalian cells.
Novel influenza A viruses of the H7N9 subtype [A(H7N9)] emerged in the spring of 2013 in China and have infected 163 people as of January 10, 2014; fifty of them died of the severe respiratory infection caused by these viruses. Phylogenetic studies have indicated that the novel A(H7N9) viruses emerged from reassortment of H7, N9 and H9N2 viruses. Inspections of protein sequences from A(H7N9) virusesand their immediate predecessorsrevealed several amino acid changes in A(H7N9) viruses that may have facilitated transmission and replication in the novel host.Since mutations that occurred more ancestrally may also have contributed to the genesis of A(H7N9) viruses,weinferred historical evolutionary events leading to the novel viruses. We identified a number ofamino acid changes on the evolutionary path to A(H7N9) viruses, including substitutions that may be associated with host range, replicative ability, and/or host responses to infection. The biological significance of these amino acid changes can be tested in future studies.
IMPORTANCE The novel influenza A viruses of the H7N9 subtype [A(H7N9)], which first emerged in the spring of 2013, cause severe respiratory infections in humans. Here, we performed a comprehensive evolutionary analysis of the progenitors of A(H7N9) viruses to identify amino acid changes thatmay have been critical for the emergence of A(H7N9) viruses and their ability to infect humans. We provide a list of potentially important amino acid changes which can be tested for their significance in influenza virus host range, replicative ability, and/or host responses to infection.
Human papillomavirus (HPV) causes a number of neoplastic diseases in humans. Here we show a complex normal HPV community in a cohort of 103 healthy human subjects, by metagenomics analysis of the shotgun sequencing data generated from the NIH Human Microbiome Project. The overall HPV prevalence was 68.9%, highest in the skin (61.3%), followed by vagina (41.5%), mouth (30%), and gut (17.3%). Of 109 HPV types as well as unclassified types detected, most were undetectable by the widely used commercial kits targeting the vaginal/cervical HPV types. These HPVs likely represent true HPV infections rather than transitory exposure because of strong organ tropism and persistence of same HPV types in repeat samples. Co-existence of multiple HPV types was found in 48.1% HPV-positive samples. Networking between HPV types, co-occurrence or exclusion, was detected in vaginal and skin samples. Large contigs assembled from short HPV reads were obtained from several samples, confirming their genuine HPV origin. This first large scale survey of HPV using shotgun sequencing approach yielded a comprehensive map of HPV infections among different human body sites of healthy human subjects.
IMPORTANCE This non-biased survey indicates that HPV community in healthy humans is much more complex than previously defined by widely used kits selective to target only a few high and low risk HPV types for cervical cancer. The importance of non-oncogenic viruses in a mixed HPV infection could be stimulating or inhibiting a co-existing oncogenic virus via viral interference or immune cross-reaction. Knowledge gained from this study will be helpful to guide designing of epidemiological and clinical studies in future to study the impact of non-oncogenic HPV types on the outcome of HPV infections.
Background: The mechanisms of increased memory CD4+ T cell cycling in HIV disease are incompletely understood but have been linked to antigen stimulation, to homeostatic signals or exposure to microbial products and the inflammatory cytokines they induce.
Methods: We examined phenotype and Vbbeta; family distribution in cycling memory CD4+ T cells among 52 healthy and 59 HIV+ donors.
Results: Cycling memory CD4+ T cells were proportionally more frequent in HIV infection than in controls, more often expressed CD38 and PD-1, and less frequently expressed OX40 and intracellular CD40L. OX40 expression on memory CD4+ T cells was induced in vitro by CD3, IL-2, IL-7 or IL-15, but not by TLR ligands. In HIV+ donors, memory CD4+ T cell cycling was related directly to plasma LPS levels, to plasma HIV RNA levels, to memory CD8+ T cell cycling and inversely to peripheral blood CD4+ T cell counts, but not to levels of IL-2, IL-7 or IL-15 while in HIV- donors, memory CD4+ T cell cycling was related to IL-7 levels and negatively to plasma levels of LPS. In both controls and HIV+ donors, cycling of memory CD4+ T cells had broad distribution of Vbbeta; families comparable to that of non-cycling cells.
Conclusions: Increased memory CD4+ T cell cycling in HIV disease is reflective of generalized immune activation and not driven primarily by cognate peptide stimulation or exposure to common gamma chain cytokines. This cycling may be a consequence of exposure to microbial products, to plasma viremia or otherwise through proinflammatory cytokines.
Importance: This work provides evidence that the increased memory CD4+ T cell cycling in HIV infection is not a result of cognate peptide recognition but rather is more likely related to the inflammatory environment of HIV infection.
Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection with the African green monkey (AGM) appearing to most faithfully reproduce human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment glycoprotein (G) in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing alhydrogel; four AGMs received the sGHeV with alhydrogel and CpG; and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived challenge. All four controls became severely ill with symptoms consistent with HeV infection and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use.
Importance A Hendra virus attachment G glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection by Hendra virus. It was found that all vaccinated subjects where completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates now provides critical data in support of its further development for future human use.
The coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was substituted by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging.
IMPORTANCE This work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective packaging of genomic RNA into assembled virions.
Tegument proteins pp150 and pUL96 function at a late step in cytomegalovirus (CMV) maturation. Here, we show that pp150 interacts directly with pUL96; however, the N-terminal region of pp150 and the C-terminal region of pUL96 that are critical for these proteins to function are not required for this interaction. Moreover, the largely dispensable C-terminal region of pp150 is critical for pp150-pUL96 interaction. To further study the role of pUL96, several point and clustered mutations were engineered into the cytomegalovirus Towne-BAC genome replacing the conserved negative charged C-terminal residues of pUL96. Although individual point mutants (E122A, D124A, and D125A) reduced virus growth slightly, the clustered mutations of 122EVDDAV127 significantly reduced virus growth, produced small syncytial plaque phenotypes and impacted a late stage of virus maturation. On serially passaging the UL96 C-terminal alanine conversion mutant (B6-BAC) virus in cell culture, it gained a plaque size comparable to Towne-BAC, displayed an altered restriction fragment length pattern, and replicated with an increased growth kinetics. Whole genome sequencing of this passaged virus (UL96P10) and the similarly passaged Towne-BAC virus revealed major differences only in RNA4.9 and UL96 regions. When one of the mutations in the UL96 coding region was engineered into the B6-BAC, it significantly increased the plaque size and rescued virus growth rate. Thus, accumulation of compensatory mutations only in UL96 in this revertant and the specific involvement of functionally dispensable regions of pp150 in pUL96-pp150 interaction points towards a role of pUL96 in virus maturation that does not depend upon pp150.
IMPORTANCE: Human cytomegalovirus (CMV) causes significant medical problems in newborns as well as in people with low immunity. In this study, we investigated the functions of two essential virus proteins pp150 and pUL96 and determined the impact of their mutual interaction on virus replication. These studies provide valuable information that is critical for the development of targeted antiviral therapies.
New human noroviruses strains emerge every 2-3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (mAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A'B' and E'F' loops. Six mutations in the E'F' loop, V378F, A382K, A382P, A382R, D385G, and L386F, mediated escape from mAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudo-atomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, llsquo;Arrsquo;, and llsquo;Brrsquo;, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the llsquo;Arrsquo; conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes towards the less preferred llsquo;Brrsquo; conformation of the P domain. The shift in the structural equilibrium of the P domain towards the conformation with poor structural complementarity to the antibody strongly support a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding.
Importance Human noroviruses cause the majority of all non-bacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid, which mediate escape from antibody neutralization, and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the virion/antibody complex identifies two conformations of the antibody binding domain of the viral capsid, one with a superior and the other with an inferior fit to the antibody. These data suggest a unique mode of antibody neutralization. In contrast to other viruses that largely escape antibody neutralization through direct disruption of the antibody/virus interface, we identify mutations that acted indirectly by limiting the conformation of the antibody binding loop in the viral capsid and drive the antibody binding domain into the conformation unable to be bound by the antibody.
Measles virus (MV) is being considered for global eradication, which would likely reduce compliance with MV vaccination. As a result children will grow up without MV-specific immunity, creating a potential niche for closely related animal morbilliviruses such as canine distemper virus (CDV). Natural CDV infection causing clinical signs has never been reported in humans, but recent outbreaks in captive macaques have shown that CDV can cause disease in primates. We studied virulence and tropism of recombinant CDV expressing enhanced green fluorescent protein in naive and measles-vaccinated cynomolgus macaques. In naive animals CDV caused viremia and fever and predominantly infected CD150+ lymphocytes and dendritic cells. Virus was re-isolated from the upper and lower respiratory tract, but infection of epithelial or neuronal cells was not detectable at the time points examined and the infections were self-limiting. This demonstrates that CDV readily infects non-human primates, but suggests that additional mutations are necessary to achieve full virulence in non-natural hosts. Partial protection against CDV was observed in measles-vaccinated macaques, as demonstrated by accelerated control of virus replication and limited shedding from the upper respiratory tract. While neither CDV infection nor MV vaccination induced detectable cross-reactive neutralizing antibodies, MV-specific neutralizing antibody levels of MV-vaccinated macaques were boosted by CDV challenge infection, suggesting that cross-reactive VN epitopes exist. Rapid increases in white blood cell counts in MV-vaccinated macaques following CDV challenge suggested that cross-reactive cellular immune responses were also present. This study demonstrates that zoonotic morbillivirus infections can be controlled by measles vaccination.
Importance Paragraph Throughout history viral zoonoses have had a substantial impact on human health. Given the drive towards global eradication of measles it is essential to understand the zoonotic potential of animal morbilliviruses. Morbilliviruses are thought to have evolved from a common ancestral virus that jumped species and adapted to new hosts. Recently canine distemper virus (CDV), a morbillivirus normally restricted to carnivores, caused disease outbreaks in non-human primates. Here we report that experimental CDV infection of monkeys resulted in fever and leukopenia. The virus replicated to high levels in lymphocytes, but did not spread to epithelial cells or the central nervous system. Importantly, like measles virus in macaques, the infections were self-limiting. In measles-vaccinated macaques CDV was cleared more rapidly resulting in limited virus shedding from the upper respiratory tract. These studies demonstrate that although CDV can readily infect primates, measles immunity is protective and CDV infection is self-limiting.
Mumps virus (MuV), a paramyxovirus containing a negative sense non-segmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel anti-viral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced, and mutating residue T at 101 to A (T101A) significantly enhanced, activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis.
IMPORTANCE Mumps virus (MuV) is a re-emerging paramyxovirus that caused large outbreaks in the U.S. where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. The work leads to a better understanding of viral RNA synthesis as well as to potential novel strategies to control mumps.
Background: Human papillomavirus (HPV) type 45 is a member of the HPV18-related alpha-7 species and accounts for approximately 5% of all cervical cancer cases worldwide.
Methods: This study evaluated the genetic diversity of HPV45 and the association of HPV45 variants with the risk of cervical cancer by sequencing the entire E6 and E7 open reading frames of 300 HPV45-positive cervical samples from 36 countries.
Results: A total of 43 HPV45 sequence variants were identified that formed 5 phylogenetic sub-lineages: A1, A2, A3, B1, and B2, the distribution of which varied by geographical region. Among 192 cases of cervical cancer and 101 controls, the B2 sub-lineage was significantly overrepresented in cervical cancer, both overall, and in Africa and Europe separately.
Conclusion: We show that the sequence analysis of E6 and E7 allows the classification of HPV45 variants and that the risk of cervical cancer may differ by HPV45 variant sub-lineage.
Importance: This manuscript describes the largest study to date of HPV45-positive cervical samples and provides a comprehensive reference for phylogenetic classification for use in epidemiological studies of the carcinogenicity of HPV45 genetic variants, particularly as our findings suggest that the B2 sublineage of HPV45 is associated with a higher risk of cervical cancer.
Depletion of central memory CD4+ T-cells (T
Importance Statement Comparing the immunologic effects of simian immunodeficiency virus (SIV) infection on rhesus macaques (RMs), a species characterized by progression to AIDS, and natural host sooty mangabeys (SMs), a species which remains AIDS-free, has become a useful tool for identifying mechanisms of HIV disease progression. One such distinguishing feature is that central memory CD4+ T-cells (T
Lymphocyte colonization by gamma-herpesviruses (HVs) is an important target for cancer prevention. However how it works is not clear. Epstein-Barr virus drives autonomous B cell proliferation in vitro, but in vivo may more subtly exploit the proliferative pathways provided by lymphoid germinal centers (GCs). Murid Herpesvirus-4 (MuHV-4), which realistically infects inbred mice, provides a useful tool with which to understand further how a HV colonizes B cells in vivo. Not all HVs necessarily behave the same, but common events can with MuHV-4 be assigned an importance for host colonization, and so a potential as therapeutic targets. MuHV-4-driven B cell proliferation depends quantitatively on CD4+ T cell help. Here we show that it also depends on T cell-independent survival signals provided by the BAFF receptor (BAFF-R). B cells could be infected in BAFF-R-/- mice, but virus loads remained low. This corresponded to a BAFF-R-dependent defect in GC colonization. The close parallels between normal, antigen-driven B cell responses and virus-infected B cell proliferation argue that in vivo, HVs mostly induce infected B cells into normal GC reactions, rather than generating large numbers of autonomously proliferating blasts.
Importance HVs cause cancers by driving the proliferation of infected cells. B cells are a particular target. Thus, we need to know how virus-driven B cell proliferation works. Controversy exists as to whether viral genes drive it directly, or less directly orchestrate the engagement of normal, host-driven pathways. Here we show that the B cell proliferation driven by a murid HV requires BAFF-R. This supports the idea that HVs exploit host proliferation pathways, and suggests that interfering with BAFF-R could more generally reduce HV-associated B cell proliferation.
Rotaviruses (RV) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the ESCRT complex, and the formation of endosomal intralumenal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking-down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UKxRRV reassortant viruses we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention.
RELEVANCE OF THE WORK Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EE, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EE to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated to the virus spike protein VP4. These findings illustrate the elaborated pathway of RV entry that could be used for drug intervention.
N-acetyl- and N-glycolylneuraminic acids (Sia) and aalpha;2bbeta;1 integrin are frequently used by rotaviruses as cellular receptors, through recognition by virion spike protein VP4. The VP4 subunit VP8* derived from Wa rotavirus binds the internal N-acetylneuraminic acid on ganglioside G
IMPORTANCE Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the G
An understanding of the antigen-specific B-cell response to the influenza hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly, because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind non-specifically to B cells, and this mutation has no effect on binding to broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 can elicit B cells expressing stem mAbs. Although these mAbs mostly originated from the IGHV1-69 germline, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem reactive IGHV1-69 alleles.
IMPORTANCE Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the blood to produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza than previously thought.
Previously, we reported on the in vitro antiviral activity of single domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in vitro setting, and showed EC50 values in the nanomolar range. In the present study, we further investigated the mechanism of action of these VHHs. All five VHHs interfere at multiple levels of the viral replication cycle, as they interfere both with attachment of the virus to cells and with viral uncoating. The latter effect is consistent with their ability to stabilize the poliovirus capsid, as observed in a thermofluor thermal shift assay, where the virus is gradually heated and the temperature causing 50% of the RNA to be released from the capsid was defined, either in the presence or absence of the VHHs. The VHH-capsid interactions were also seen to induce aggregation of the virus-VHH complexes. However, this observation cannot yet be linked to their mechanism of action. Cryo-electron microscopy (EM) reconstructions of two VHHs in complex with poliovirus type 1 show no conformational changes of the capsid to explain this aggregation. On the other hand, these reconstructions do show that the binding sites of VHHs PVSP6A and PVSP29F overlap with the binding site for the poliovirus receptor (CD155/PVR) and span interfaces that are altered during receptor-induced conformational changes associated with cell entry. This may explain the interference at the level of cell attachment of the virus as well as their effect on uncoating.
Importance paragraph: The study describes the mechanism of neutralization and the capsid stabilizing activity of five single domain antibody fragments (VHHs) that have an in vitro neutralizing activity against poliovirus type 1. The results show that the VHHs interfere at multiple levels of the viral replication cycle (cell-attachment and viral uncoating). These mechanisms are possibly shared by some conventional antibodies and may therefore provide some insight in the natural immune responses. Since the binding sites of two VHHs studied by cryo-EM are very similar to that of the receptor, the VHHs can be used as probes to study the authentic virus-cell interaction. The structures and conclusions in this study are original, and raise interesting findings regarding virus-receptor interactions and the order of key events early in infection.
Periodontal pathogens such as Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) produce five different short chain fatty acids (SCFAs) as metabolic by-products. We detect significantly higher levels of SCFAs in the saliva of patients with severe periodontal disease. The different SCFAs stimulate lytic gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) dose-dependently and synergistically. SCFAs inhibit class-1/2 histone deacetylases (HDACs) and down regulate expression of silent information regulator-1 (SIRT1). SCFAs also down regulate expression of enhancer of zeste homolog2 (EZH2) and suppressor of variegation 3-9 homolog1 (SUV39H1), which are two histone N-lysine methyltransferases (HLMTs). By suppressing the different components of host epigenetic regulatory machinery, SCFAs increase histone acetylation and decrease repressive histone tri-methylations to transactivate the viral chromatin. These new findings provide mechanistic support that SCFAs from periodontal pathogens stimulate KSHV replication and infection in the oral cavity and are potential risk factors for development of oral Kaposi's sarcoma (KS).
IMPORTANCE OF THE STUDY About 20% of HIV patients develop first KS lesions in the oral cavity while other patients never develop oral KS. It is not known if the oral microenvironment plays a role in oral KS tumor development. In this work, we demonstrate that a group of metabolic by-products, namely short chain fatty acids, from bacteria that cause periodontal disease promote lytic replication of KSHV, the etiological agent associated with KS. These new findings provide mechanistic support that periodontal pathogens create a unique microenvironment in the oral cavity that contributes to KSHV replication and development of oral KS.
HLA-B*57:01 and HLA-B*57:03, the most prevalent HLA-B*57 subtypes in Caucasian and African populations, respectively, are the HLA alleles most protective against HIV disease progression. Understanding the mechanisms underlying this immune control is of critical importance and yet remains unclear. Unexplained differences are observed in the impact of the dominant CTL response restricted by HLA-B*57:01 and HLA-B*57:03 in chronic infection towards the Gag epitope KAFSPEVIPMF (llsquo;KF11rrsquo; ,Gag162-172). We previously showed that the HLA-B*57:03-KF11 response is associated with a ggt;1 log lower viral setpoint in C-clade infection and that this response selects escape mutants within the epitope. We first examined the relationship of KF11 responses in B-clade infected subjects with HLA-B*57:01 to immune control and observed that a detectable KF11 response was associated with a ggt;1 log higher viral load (p=0.02). No evidence of HLA-B*57:01-KF11 associated selection pressure was identified in previous comprehensive analyses of ggt;1800 B-clade infected subjects infected. We then studied a B-clade infected cohort in Barbados where HLA-B*57:03 is highly prevalent. In contrast to B-clade infected subjects expressing HLA-B*57:01, we observed strong selection pressure driven by the HLA-B*57:03-KF11 response for the escape mutation S173T. This mutation reduces recognition of virus-infected cells by HLA-B*57:03-KF11 CTL, and is associated with a ggt;1 log increase in viral load in HLA-B*57:03-positive subjects (p=0.009). We demonstrate functional constraints imposed by HIV clade relating to the residue at Gag-173 that explain the differential clade-specific escape patterns in HLA-B*57:03 subjects. Further studies are needed to evaluate the role of the KF11 response in HLA-B*57:01-associated HIV disease protection.
IMPORTANCE SECTION HLA-B*57 is the HLA class I molecule that affords the greatest protection against disease progression in HIV infection. Understanding the key mechanism(s) underlying immune suppression of HIV is of importance in guiding therapeutic and vaccine-related approaches to improve the levels of HIV control occurring in nature. Numerous mechanisms have been proposed to explain the HLA associations with differential HIV disease outcome but no consensus exists. These studies focus on two subtypes of HLA-B*57, prevalent in Caucasian and African populations, HLA-B*57:01 and HLA-B*57:03, respectively. These alleles appear equally protective against HIV disease progression. The CTL epitopes presented are in many cases identical, and the dominant response in chronic infection in each case is to the Gag epitope KF11. However, there the similarity ends. This paper seeks to better understand the reasons for these differences and what this teaches us about which immune responses are contributing to immune control of HIV infection.
Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in a turkey in Massachusetts, USA, in 1965. Since 1997, H6 viruses with different NA subtypes have been detected frequently in the live poultry markets of southern China. Although sequence information has been gathered over the last few years, the H6 viruses have not been fully biologically characterized. To investigate the potential risk posed by H6 viruses to humans, here, we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China from 2008nndash;2011. Among the 257 H6 strains tested, 87 viruses recognized the human-type receptor. Genome sequence analysis of 38 H6 representative viruses revealed 30 different genotypes, indicating that these viruses are actively circulating and reassorting in nature. Thirty-seven of 38 viruses tested in mice replicated efficiently in the lungs and some caused mild disease; none, however, were lethal. We also tested the direct contact transmission of 10 H6 viruses in guinea pigs, and found that five viruses did not transmit to the contact animals, three viruses transmitted to one of the three contact animals, and two viruses transmitted to all three contact animals. Our study demonstrates that the H6 avian influenza viruses pose a clear threat to human health and emphasizes the need for the continued surveillance and evaluation of the H6 influenza viruses circulating in nature.
Importance Avian influenza viruses continue to present a challenge to human health. Research and pandemic preparedness have largely focused on the H5 and H7 subtype influenza viruses in recent years. Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in USA, in 1965. Since 1997, H6 viruses have been detected frequently in the live poultry markets of southern China, however, the biological characterization of these viruses is very limited. Here, we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China, and found 34% of the viruses are able to bind human-type receptors and some of them are able to transmit efficiently to contact animals. Our study demonstrates that the H6 viruses pose a clear threat to human health.
Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low pathogenic (LPAI) progenitor viruses is currently poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultra-deep sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high and low pathogenic viruses undertaken to date, and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties.
Importance The Italian H7 avian influenza (AI) epidemic of 1999-2001 was one of the most important AI outbreaks described in Europe. H7 viruses have the ability to evolve into HP (highly pathogenic) forms from LP (low pathogenic) precursors, although the mechanisms underlying this evolutionary transition are only poorly understood. We combined epidemiological information, whole-genome sequence data and ultra-deep sequencing approaches to provide the most complete characterization of the evolution of HPAI from LPAI viruses undertaken to date. Our analysis revealed that the LPAI viruses were the direct ancestors of the HPAI strains and identified low frequency minority variants with HPAI mutations that were present in the LPAI samples. Spatial analysis provided key information for the design of effective control strategies for AI at both local and global scales. Overall, this work highlights the importance of implementing rapid eradication measures to prevent the emergence of novel influenza viruses with severe pathogenic properties.
Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Vaccinia virus (VACV) is a member of the Poxviridae family and its use as a vaccine enabled the eradication of variola virus, the etiologic agent of smallpox. A global metabolic screen of VACV-infected primary human foreskin fibroblasts suggested glutamine metabolism is altered during infection. Glutamine, along with glucose, represent the two main carbon sources for mammalian cells. Depriving VACV-infected cells of exogenous glutamine led to a substantial decrease in infectious virus production, whereas starving infected cells of exogenous glucose had no significant impact on replication. Viral yield in glutamine-deprived cells or in cells treated with an inhibitor of glutaminolysis, the pathway of glutamine catabolism, could be rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Thus, VACV infection induces a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle. VACV protein synthesis, but not viral transcription, was decreased in glutamine-deprived cells, which corresponded with a dramatic reduction in all VACV morphogenetic intermediates. This study reveals the unique carbon utilization program implemented during poxvirus infection and provides a potential metabolic pathway to target viral replication.
Importance Viruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for critical processes, including genome replication, viral protein synthesis, and membrane production. This study investigates how vaccinia virus (VACV) infection alters global cellular metabolism, providing the first metabolomic analysis for a member of the poxvirus family. Unlike most viruses examined to date, VACV does not activate glycolysis and exogenous glucose is not required for maximal virus production. Instead, VACV requires exogenous glutamine for efficient replication and inhibition of glutamine metabolism effectively blocks VACV protein synthesis. This study defines a major metabolic perturbation essential for replication of a poxvirus and may lead to the discovery of novel antiviral therapies based on metabolic inhibitors.
Severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus, Hendra and Nipah viruses are members of different viral families and are known causative agents of fatal viral diseases. These viruses depend on cathepsin L for entry into their target cells. The viral glycoproteins need to be primed by protease cleavage rendering them active for fusion with the host cell membrane. In this study, we developed a novel high throughput screening assay based on peptides, derived from the glycoproteins of the aforementioned viruses, which contain the cathepsin L cleavage site. We screened a library of 5,000 small molecules and discovered a small molecule that can inhibit the cathepsin L cleavage of all viral peptides with minimal inhibition of cleavage of a host protein derived peptide (Pro-NPY). The small molecule inhibited the entry of all pseudotyped viruses in vitro and the cleavage of SARS-CoV spike glycoprotein in an in vitro cleavage assay. In addition, the Hendra and Nipah virus fusion glycoproteins were not cleaved in the presence of the small molecule in a cell based cleavage assay. Furthermore, we demonstrate that the small molecule is a mixed inhibitor of cathepsin L. Our broad-spectrum antiviral small molecule appears to be an ideal candidate for future optimization and development into a potent antiviral against SARS-CoV, Ebola, Hendra, and Nipah viruses.
We developed a novel high throughput screening assay to identify small molecules that can prevent cathepsin L cleavage of viral glycoproteins derived from SARS-CoV, Ebola, Hendra and Nipah viruses required for their entry into the host cell. We identified a novel broad-spectrum small molecule that could block cathepsin L mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from SARS-CoV, Ebola, Hendra or Nipah viruses. The small molecule can be further optimized and developed into a potent broad-spectrum antiviral drug.
In recent years, genotype I (GI) of Japanese encephalitis virus (JEV) has displaced genotype III (GIII) as the dominant virus genotype throughout Asia. In this study, the largest collection of GIII and GI envelope gene-derived viral sequences assembled to date were used to reconstruct the spatiotemporal chronology of the genotype displacement throughout Asia, and to determine the evolutionary and epidemiological dynamics underlying this significant event. GI consists of two clades, GI-a and GI-b with the latter associated with displacement of GIII as the dominant JEV genotype throughout Asia in the 1990s. Phylogeographic analysis indicated that GI-a diverged in Thailand or Cambodia and has remained confined to tropical Asia, whereas GI-b diverged in Vietnam and then dispersed northwards to China, where it was subsequently dispersed to Japan, Korea and Taiwan. Molecular adaptation was detected by ggt; 1 method at one site (15) and co-evolution was detected at two pairs of sites (89-360 and 129-141) within the GI E gene protein alignment. Viral multiplication and temperature sensitivity analyses in avian and mosquito cells revealed that the JE-91 GI-b isolate had significantly higher infectivity titers in mosquito cells from 24-48 hours post infection compared to the GI-a and GIII isolates. If the JE-91 isolate is indeed representative of GI-b, an increased multiplicative ability of GI-b viruses compared to GIII viruses early in mosquito infection may have resulted in a shortened extrinsic incubation period that led to an increased number of GI enzootic transmission cycles and the subsequent displacement of GIII.
IMPORTANCE Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, represents the most significant etiology of childhood viral neurological infection in Asia. Despite the existence of effective vaccines, JEV is responsible for an estimated 68,000 human cases and a reported 10,000 to 15,000 deaths annually. Phylogenetic studies divide JEV into five geographically and epidemiologically distinct genotypes (GI-V). GIII has been the source of numerous JE epidemics throughout history and was the most frequently isolated genotype throughout most of Asia from 1935 until the 1990s. In recent years, GI has displaced GIII as the most frequently isolated virus genotype. To date, the mechanism of this genotype replacement has remained unknown. In this study we have identified genetic determinants underlying the genotype displacement as it unfolded across Asia. JEV provides a paradigm for other flaviviruses, including West Nile, yellow fever and dengue viruses, and the critical role of the selective advantages in the mosquito vector.
The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm, which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to yeast retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P-bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cyclohexamide treatment, suggesting these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Live-cell imaging with fluorescence recovery after photobleaching (FRAP) analysis revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly.
Importance Cellular mRNA processing factors regulate the post-transcriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules. This report shows for the first time that mouse mammary tumor virus, a mammalian retrovirus that assembles intracytoplasmic virus particles, commandeers the cellular factor YB-1, a key regulator of translation involved in the cellular stress response. YB-1 is essential for the efficient production of MMTV particles, a process directed by the viral Gag protein. We found that Gag and YB-1 localize together in cytoplasmic granules. Functional studies of Gag/YB-1 granules suggest they may be sites where virus particles assemble. These studies provide significant insights into the interplay between mRNA processing factors and retroviruses.
Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa Fucci cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with NEK9 kinase and GEM interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP.
Importance: Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins from three different herpesviruses that contribute to this block. Several of the proteins we identified had previously unknown functions or were structural components of the virion. Subsets of these proteins from Epstein-Barr virus were studied for their effects on the cell cycle regulatory proteins p53 and p21, thereby identifying two proteins that induce p53 and one that induces p21 (BGLF2). We identified interactions of BGLF2 with two human proteins, both of which regulate p21, suggest that BGLF2 induces p21 by interfering with the functions of these two host proteins. Our study indicates that multiple herpesvirus proteins contribute to the cell proliferation block, including components of the incoming virions.
Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the Long Terminal Repeats (LTRs) of Murine Leukemia Virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (llt;15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ~60,000 tight clusters. These clusters comprise ~1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection.
Importance We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors.
Kaposi's sarcoma herpesvirus (KSHV) is a human -herpesvirus casually linked to Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL). Previously, we showed that LANA (LANA) encoded by KSHV upregulates expression of Survivin, a member of the inhibitor of apoptosis (IAP) family. This leads to an increase in the rate of cell proliferation of KSHV infected B-cells. LANA is required for tethering of the KSHV episome to the host chromosomes and efficiently segregates the viral genomes into dividing tumor cells. Here we show that LANA interacts with Aurora Kinase B (AK-B) and induces phosphorylation of Survivin at residue T34. Phosphorylation of Survivin specifically on residue T34 enhances the activity of p300 and inhibits the activity of HDAC-1 which then leads to an increase in acetylation of histone H3 on the viral genome. Phosphorylation of Survivin specifically on residue T34 upregulates the activities of histone acetyltransferases and deacetylases which then leads to an increase in viral copy number in KSHV infected B-cells. This results in a boost of KSHV replication in latently infected B-lymphoma cells. The studies showed that LANA can also function to regulate viral replication prior to mitosis of the latently infected cells, suggesting that LANA possesses a novel role in regulating KSHV replication in infected B-cells.
Importance This work represents a report of KSHV latent protein LANA and its interactions with Aurora Kinase B (AK-B) leading to induction of phosphorylation of the oncoprotein Survivin at residue T34. Phosphorylation of Survivin specifically on residue T34 upregulates the activities of histone acetyltransferases and deacetylases. This leads to an increase in viral copy number in KSHV infected B-cells. These studies support a role for LANA in regulating KSHV replication through post-translation modification in KSHV infected B-cells.
NS1 of influenza A virus is a potent antagonist of host antiviral interferon responses. This multifunctional protein with two distinctive domains, a RNA binding domain (RBD) and an effector domain (ED) separated by a linker region (LR), is implicated in replication, pathogenesis and host range. Although the structures of individual domains of NS1 from different strains of influenza viruses have been reported, the only structure of full-length NS1 available to date is from a H5N1 strain (A/Vietnam/1203/2004). By carrying out crystallographic analyses of full-length H6N6-NS1 (A/blue-winged teal/MN/993/1980) and a LR deletion mutant, combined with mutational analysis, we show here that these full-length NS1 structures provide an exquisite structural sampling of various conformational states of NS1 that based on the orientation of the ED with respect to RBD can be summarized as llsquo;openrrsquo;, llsquo;semi-openrrsquo; and llsquo;closedrrsquo; conformations. Our studies show that preference for these states is clearly dictated by determinants such as linker length, residue composition at position 71, and a mechanical hinge, providing a structural basis for strain-dependent functional variations in NS1. Because of the flexibility inherent in the LR, any particular NS1 could sample the conformational space around these states to engage ED in different quaternary interactions so that it may participate in specific protein-protein or protein-RNA interactions to allow for the known multi-functionality of NS1. We propose that such conformational plasticity provides a mechanism for auto-regulating NS1 functions, depending on its temporal distribution, post-translational modifications, and nuclear or cellular localization, during the course of virus infection.
IMPORTANCE NS1 of influenza A virus is a multifunctional protein associated with numerous strain-specific regulatory functions during viral infection, including conferring resistance to antiviral interferon induction, replication, pathogenesis, virulence and host range. NS1 has two domains, a RNA binding domain and an effector domain separated by a linker. To date, the only full-length NS1 structure available is that from a H5N1 strain (A/Vietnam/1203/2004). Here, we determined crystal structures of wildtype and linker region mutant of the H6N6 NS1 (A/blue-winged teal/MN/993/1980), which together with the previously determined H5N1 NS1 structure show that NS1 exhibits significant strain-dependent structural polymorphism due to variations in linker length, residue composition at position 71, and a mechanical hinge. Such a structural polymorphism may be the basis for strain-specific functions associated with NS1.
Influenza virus infection is a public health problem that is generally more severe in individuals over 65 years of age (elderly). Immunosenescence enhances the susceptibility to viral infections and renders vaccination less effective. Understanding age-related changes in the immune system is crucial in order to design prophylactic and immunomodulatory strategies to reduce morbidity and mortality in the elderly.
Here, we propose different mathematical models to provide a quantitative understanding of the immune strategies in the course of influenza virus infection using experimental data from young and aged mice. Simulation results suggest a central role of CD8+ T cells for adequate viral clearance kinetics in young and aged mice. Adding the removal of infected cells by natural killer cells does not improve the fitting either in young or aged animals. We examine the infection resistant state of cells promoted by the cytokines IFN-aalpha;/bbeta;, IFN- and TNF-aalpha; separately. The combination of activated CD8+ T cells with either of the cytokines provided the best fit in young and aged animals. During the first 3 days after infection the reproductive number for aged mice is 1.5 fold lower than in young mice (p-valuellt;0.05).
IMPORTANCE Fits of our models to experimental data suggest that the increased levels of IFN-aalpha;/bbeta;, IFN- and TNF-aalpha; ("inflammaging") promote a slower viral growth in aged mice, which consequently limits the stimulation of immune cells and contributes to the reported impaired responses in the elderly. A quantitative understanding of influenza pathogenesis and its shift in the elderly is the key contribution of this work.
Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infection in young children, and the leading cause of infant hospitalization worldwide. Uncontrolled response to RSV is mediated by a toll-like receptor (TLR)-mediated immune response. Resveratrol possesses anti-RSV activity and is an inhibitor of the TRIF/TBK1/IRF-3 complex. We hypothesize that resveratrol inhibits the TRIF-dependent pathway through up-regulation of SARM post-RSV infection. BALB/c mice were infected with RSV and were injected with resveratrol one hour post-inoculation. SARM siRNA was administrated to RSV-infected and resveratrol-treated mice. Lung function was measured by whole-body plethysmography, lung histopathology was examined, and lymphocytes in bronchoalveolar lavage fluid were quantified. SARM and TRIF protein expression were detected in lung by western blot analyses. The expression of interferon- in BALF was evaluated by ELISA. SARM expression was reduced and TRIF expression was increased after infection with RSV. Resveratrol increased SARM expression and decreased TRIF expression after RSV infection. SARM knockdown in resveratrol-treated mice enhanced interferon- production, RSV-induced airway inflammation, and AHR. Resveratrol decreased TRIF expression and prevented the RSV-mediated reduction of SARM expression. Resveratrol-mediated inhibition of the TRIF-dependent pathway may be dependent on SARM expression.
IMPORTANCE Our study provides insights into the regulation of innate immunity in response to RSV infection. The results suggest that resveratrol-mediated alterations in SARM may have therapeutic potential against RSV immunopathology caused by deregulation of the TLR-mediated immune response. Ultimately, improved insight into the complex interplay between TLR adaptor proteins and the occurrence of severe RSV infection might lead to novel therapeutic treatment strategies such as TLR adjuvants.
Syncytin-1, a fusogenic protein encoded by a human endogenous retrovirus (HERV-W) element (ERVWE1), is expressed in the syncytiotrophoblast layer of the placenta. This locus is transcriptionally repressed in adult tissues through promoter CpG methylation and suppressive histone modifications. Whereas syncytin-1 appears crucial for the development and functioning of the human placenta, its ectopic expression has been associated with pathological conditions such as multiple sclerosis and schizophrenia. We previously reported on the transactivation of HERV-W elements, including ERVWE1, during influenza A/WSN/33 virus infection in a range of human cell-lines. We here report qPCR analyses of transcripts encoding syncytin-1 in both cell-lines and primary fibroblast cells. We observed that spliced ERVWE1 transcripts and those encoding the transcription factor glial cells missing 1 (GCM1), acting as an enhancer element upstream of ERVWE1, are prominently up-regulated in response to influenza A/WSN/33 virus infection in non-placental cells. Knock-down of GCM1 by siRNA, followed by infection suppressed the transactivation of ERVWE1. While the infection had no influence on CpG methylation in the ERVWE1 promoter, chromatin immunoprecipitation assays detected decreased H3K9 trimethylation (H3K9me3) and histone methytransferase SETDB1 levels along with viral proteins associated with ERVWE1 and other HERV-W loci in infected CCF-STTG1 cells. The present findings suggest that an exogenous influenza virus infection can transactivate ERVWE1 by increasing transcription of GCM1 and reducing H3K9me3 in this region and in other regions harboring HERV-W elements.
Importance Syncytin-1, a protein encoded by the env gene in the HERV-W locus ERVWE1 appears crucial for the development and functioning of the human placenta and is transcriptionally repressed in non-placental tissues. Nevertheless, its ectopic expression has been associated with pathological conditions such as multiple sclerosis and schizophrenia. In the present paper we report findings suggesting that an exogenous influenza A virus infection can transactivate ERVWE1 by increasing transcription of GCM1 and reducing the repressive histone mark H3K9me3 in this region and in other regions harboring HERV-W elements. These observations have implications of potential relevance for viral pathogenesis and for conditions associated with aberrant transcription of HERV-W loci.
The antiviral role of TRIM E3 ligases in vivo is not fully understood. To test the hypothesis that TRIM5aalpha; and TRIM22 have differential transcriptional regulation and distinct anti-HIV roles according to infection phase and compartment, we measured TRIM5aalpha;, TRIM22 and type 1 interferon (IFN-1)-inducible MxA levels in peripheral blood mononuclear cells (PBMCs) during primary and chronic HIV-1 infection, and in matched PBMCs and central nervous system (CNS)-derived cells. Associations with biomarkers of disease progression were explored. The impact of IFN-1, select pro-inflammatory cytokines and HIV on TRIM E3 ligase-specific expression was investigated. PBMCs from individuals with primary and chronic HIV-1 infection had significantly higher levels of MxA and TRIM22 compared to HIV-1 negative PBMCs (P llt; 0.05, all comparisons). PBMCs from chronic infection had lower levels of TRIM5aalpha; compared to primary infection or HIV-1 uninfected (both P = 0.0001). In matched CNS-derived samples and PBMCs, higher levels of MxA (P = 0.001) and TRIM5aalpha; (P = 0.0001) were noted in the CNS. There was negative correlation between TRIM22 levels in PBMC and plasma viral load (r = nndash;0.40, P = 0.04). In vitro, IFN-1 and rarely pro-inflammatory cytokines induced TRIM5aalpha; and TRIM22 in cell type-dependent manner and knockdown of either protein in CD4+ lymphocytes resulted in increased HIV-1 infection. These data suggest that there are infection-phase specific and anatomically compartmentalized differences in TRIM5aalpha; and TRIM22 regulation involving primarily IFN-1 and specific cell types, and indicate subtle differences in the antiviral role and transcriptional regulation of TRIM E3 ligases in vivo.
Importance Interferon type I-inducible TRIM E3 ligases are a family of intracellular proteins with potent antiviral activities mediated through diverse mechanisms. However, little is known about the contribution of these proteins to antiviral immunity in vivo and how their expression is regulated. We show here that TRIM5aalpha; and TRIM22, two prominent members of the family, have different expression patterns in vivo and that expression pattern depends on HIV-1 infection status and phase. Furthermore, expression differs in peripheral blood versus central nervous system anatomical sites of infection. Only TRIM22 expression correlates negatively with HIV-1 viral load but gene silencing of both proteins enhances HIV-1 infection of target cells. We report on subtle differences in TRIM5aalpha; and TRIM22 gene induction by IFN-1 and pro-inflammatory cytokines in CD4+ lymphocytes, monocytes and neuronal cells. This study enhances our understanding of antiviral immunity by intrinsic antiviral factors and how their expression is determined.
Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/Tap in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named CAE (cytoplasmic accumulation element). The CAE enhanced cytoplasmic accumulation of viral RNA transcripts and expression of viral proteins without significantly affecting the stability, splicing or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated the CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in a NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and non-spliced of viral RNA transcripts.
IMPORTANCE Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology since the notion of leukemogenesis in 1950s. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs into the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found; (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) cellular factor NXF1 is involved in the nuclear export of both spliced and unspliced viral RNAs, and lastly, (iii) depletion of NXF1 results in nuclear retention or degradation of viral RNAs. Our study provides novel insight into the host-virus interaction within the nuclear export of a viral life cycle.
The sudden emergence of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) in 2002 and, more recently, Middle Eastern Respiratory Syndrome-CoV (MERS-CoV) underscores the importance of understanding critical aspects of CoV infection and pathogenesis. Despite significant insights into CoV cross-species transmission, replication, and virus-host interactions, successful therapeutic options for CoVs do not yet exist. Recent identification of SARS-CoV NSP16 as a viral 2rrsquo;O methyl-transferase (Mtase) led to the possibility of utilizing this pathway to both attenuate SARS-CoV infection as well as develop novel therapeutic treatment options. Mutations were introduced into SARS-CoV NSP16 within the conserved KDKE motif and effectively attenuated the resulting SARS-CoV mutant viruses both in vitro and in vivo. While viruses lacking 2rrsquo;O Mtase activity had enhanced sensitivity to type I IFN, they were not completely restored in their absence in vivo. However, the absence of either MDA5 or IFIT1, IFN responsive genes that recognize unmethylated 2rrsquo;O RNA, resulted in restored replication and virulence of the dNSP16 mutant virus. Finally, using the mutant as a live-attenuated vaccine showed significant promise for possible therapeutic development against SARS-CoV. Together, the data underscores the necessity of 2rrsquo;O MTase activity for SARS-CoV pathogenesis and identifies host immune pathways that mediate this attenuation. In addition, the work provides novel treatment avenues that exploit this pathway and could potentially be used against a diverse range of viral pathogens that utilize 2rrsquo;O MTase activity to subvert the immune system.
IMPORTANCE Preventing recognition by the host immune response represents a critical aspect necessary for successful viral infection. Several viruses, including SARS-CoV, utilize virally encoded 2rrsquo;O methyltransferases to camouflage and obscure their viral RNA from host cell sensing machinery, thus preventing recognition and activation of cell intrinsic defense pathways. For SARS-CoV, the absence of this 2rrsquo;O methyl-transferase (Mtase) activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice. The results indicate that both MDA5, a recognition molecule, and the IFIT family play an important role in mediating this attenuation with restored virulence observed in their absence. Understanding this viral-host interaction provided an opportunity to design a successful live-attenuated vaccine for SARS-CoV and opens avenues for treatment and prevention of emerging CoVs and other RNA virus infections.
Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G). It has been suggested that hMPV F protein utilizes integrin aalpha;vbbeta;1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif (329RGD331). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that aalpha;5bbeta;1 and aalpha;v integrin are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the 329RGD331 motif are essential for cell-cell fusion whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV-R329K and D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, cotton rats inoculated with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) aalpha;5bbeta;1 and aalpha;v integrins are essential for cell-cell fusion and viral replication; (ii) the first two residues in the RGD motif are essential for fusion activity; and (iii) inhibition of the interaction of integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines.
A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (Bernstein et al., Pediatr Infect Dis J 2012;31:109-114; Yang et al., Vaccine 2013;31:2822-2827). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the 1st (pre-N), 2nd (N-P), 3rd (P-M), and 6th (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the 1st to the 6th position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts (URT, LRT) of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the 2nd, 3rd, and 6th positions conferred increased temperature-sensitivity: this was greatest for the 3rd position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN ORF, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity.
Importance The research findings presented in this manuscript will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this knowledge has general application to the development and clinical evaluation of other mononegavirus vectors and vaccines.
Rice ragged stunt virus (RRSV), an oryzavirus in the family Reoviridae, is transmitted by the brown planthopper, Nilaparvata lugens, in a persistent-propagative manner. Here, we established a continuous cell line of brown planthopper to investigate the mechanism underlying the formation of the viroplasm, the putative site for viral replication and assembly, during infection of RRSV in its insect vector cells. Within 24 h of viral infection of cultured cells, the viroplasm had formed and contained viral nonstructural proteins Pns6 and Pns10, known to be constituents of viroplasm. Core capsid protein P3, core particles and newly synthesized viral RNAs were accumulated inside the viroplasm, while outer capsid protein P8 and virions were accumulated at the periphery of the viroplasm, confirming that the viroplasm induced by RRSV infection was the site for viral replication and assembly. Pns10 formed viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm matrix was largely composed of Pns10. Pns6 was recruited in the viroplasm by direct interaction with Pns10. Core capsid protein P3 was recruited to the viroplasm through specific association with Pns6. Knockdown of Pns6 and Pns10 expression using RNA interference inhibited viroplasm formation, virion assembly, viral proteins expression and viral dsRNAs synthesis. Thus, the present study shows that both Pns6 and Pns10 of RRSV play important roles in the early stages of viral life cycle in its insect vector cells, by recruiting or retaining components necessary for viral replication and assembly.
The brown planthopper, a commonly distributed pest of rice in Asia, is the host of numerous insect endosymbionts, and the major vector of two rice viruses (RRSV and rice grassy stunt virus). For the first time, we successfully established the continuous cell line of brown planthopper. The unique uniformity of brown planthopper cells in the monolayer can support a consistent, synchronous infection by endosymbionts or viral pathogens, improving our understanding of molecular insect-microbe interactions.
HIV-1 membranes contain gp120-gp41 trimers. Binding of gp120 to CD4 and a coreceptor (CCR5 or CXCR4) reduces constraint on metastable gp41, enabling a series of conformational changes that cause membrane fusion. An analytic difficulty occurs because these steps occur slowly and asynchronously within cohorts of adsorbed virions. We previously isolated HIV-1JRCSF variants that efficiently use CCR5 mutants severely damaged in the tyrosine sulfated amino terminus or extracellular loop two. Surprisingly, both independent adaptations included gp120 mutations S298N, F313L, and N403S, supporting other evidence that they function by weakening gp120rrsquo;s grip on gp41 rather than by altering gp120 binding to specific CCR5 sites. Although several natural HIV-1 isolates reportedly use CCR5(18) lacking the amino terminus when the soluble tyrosine sulfated peptide is present, we show that HIV-1JRCSF(Ad) with the adaptive mutations functions approximately 100-times more efficiently and that coreceptor activation is reversible, enabling synchronous efficient entry control in physiological conditions. This system revealed that three-stranded gp41 folding intermediates susceptible to the inhibitor enfuvirtide form slowly and asynchronously on cell surface virions but resolve rapidly, with virions generally forming only one target. Adsorbed virions asynchronously and transiently become competent for entry at 370C but are inactivated if the CCR5 peptide is absent during their window of opportunity. This competency is conferred by endocytosis, which results in inactivation if the peptide is absent. For both wild-type and adapted HIV-1s, early gp41 refolding steps obligatorily occur on cell surfaces, whereas final step(s) are endosomal. This system powerfully dissects HIV-1 entry and inhibitor mechanisms.
Importance statement We present a powerful means to reversibly and efficiently activate or terminate HIV-1 entry by adding or removing a tyrosine sulfated CCR5 peptide from culture medium. This system uses stable cell clones and a variant of HIV-1JRCSF with three adaptive mutations. It enabled us to show that CCR5 coreceptor activation is rapidly reversible and to dissect aspects of entry that had previously been relatively intractable. Our analyses elucidate enfuvirtide (T-20) function and suggest that HIV-1 virions form only one nonredundant membrane fusion complex on cell surfaces. Additionally, we obtained novel and conclusive evidence that HIV-1 entry occurs in an assembly line manner, with some steps obligatorily occurring on cell surfaces and with final membrane fusion occurring in endosomes. Our results were confirmed for wild-type HIV-1. Thus, our paper provides major methodological and mechanistic insights about HIV-1 infection.
We have previously shown that hepatitis C virus (HCV) infection modulates the expression of forkhead box transcription factors, including FoxO1 and FoxA2, playing key roles in gluconeogenesis and bbeta;-oxidation of fatty acid, respectively. The aim of the present study was to determine the role of forkhead box transcription factors in modulating lipid metabolism. HCV infection or core protein expression alone in transfected Huh7.5 cells increased sterol regulatory element binding protein-1c (SREBP-1c) expression and downstream target fatty acid synthase (FASN), which are key genes involved in lipid synthesis. Knockdown of FoxO1 by siRNA in HCV infected cells significantly decreased SREBP-1c and FASN. Further, HCV infection or core protein expression in Huh7.5 cells significantly decreased the expression of Medium-chain acyl-CoA dehydrogenase (MCAD) and Short-chain acyl-coenzyme A dehydrogenase (SCAD), involved in the regulation of bbeta;-oxidation of fatty acids. Ectopic expression of FoxA2 in HCV infected cells rescued the expression of MCAD and SCAD. Oil Red O and neutral lipid staining indicated that HCV infection significantly increases lipid accumulation as compared to mock infected control. This was further verified by an increased expression of perilipin-2 and decreased activity of hormone sensitive lipase (HSL) in HCV infected hepatocyte, implying increased accumulation of neutral lipids. Knockdown of FoxO1 and ectopic expression of FoxA2 significantly decreased HCV replication. Taken together, these results suggest that HCV modulates forkhead box transcription factors which together increase lipid accumulation and promote viral replication.
IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic HCV infection. Its presence is a key prognostic indicator associated with the progression to hepatic fibrosis and hepatocellular carcinoma. Several mechanisms have been proposed to account for the development of steatosis and fatty liver during HCV infection. We observed that HCV infection increases expression of both SREBP-1c and FASN. Further investigation suggested that the expression of SREBP-1c and FASN are controlled by the transcription factor FoxO1 during HCV infection. In addition, HCV infection significantly decreased both MCAD and SCAD expression, which are controlled by FoxA2. HCV infection also increased LD accumulation, increased perilipin-2 expression and decreased HSL activity. Thus knockdown of FoxO1 (decreased lipogenesis) and over expression of FoxA2 (increased bbeta;-oxidation) resulted in a significant disruption of the platform, and hence decrease in HCV genome replication. Thus, targeting FoxO1 and FoxA2 might be useful in developing therapeutic approach against HCV infection.
We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of Dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and Stabilin-1 and -2, for their abilities to facilitate viral binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors, TIM-1 and -4, also enhance viral binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8 which binds viral envelope phosphatidylserine without bridging virus to cells, but surprisingly not annexin V which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection.
Importance Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, Dengue, West Nile, and Ebola viruses utilize Axl/Gas6 to facilitate their entry, suggesting that phosphatidylserine-mediated viral entry mechanism can be shared by various envelope viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to developing novel antiviral reagents that block phosphatidylserine-mediated viral entry.
Nanomaterials have the characteristic of high surface-to-volume ratio and have been explored for their antiviral activity. Despite some success, cytotoxicity has been an issue of nanomaterial-based antiviral strategy. We previously developed a novel method to fully exfoliate montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). We further modified NSP by capping with various surfactants and found the surfactant-modified NSP (NSQ) was less cytotoxic. In this study, we tested the antiviral potential of a series of nature clay-derived nanomaterials. Among the derivatives, NSP modified with anionic sodium dodecyl sulfate (NSQc), but not the pristine clay, unmodified NSP, silver nanoparticle-NSP hybrid, NSP modified with cationic n-octadecanylamine hydrochloride salt, or NSP modified with nonionic Triton X-100, significantly suppressed the plaque-forming ability of Japanese encephalitis virus (JEV) at noncytotoxic concentrations. NSQc also blocked the infection with dengue virus (DEN) and influenza A virus. Regarding the antiviral mechanism, NSQc interfered with viral binding through electrostatic interaction, since its antiviral activity can be neutralized by polybrene, a cationic polymer. Furthermore, NSQc reduced the lethality of JEV and DEN infection in mouse challenge models. Thus, the surfactant-modified exfoliated nanoclay NSQc may be a novel nanomaterial with broad and potent antiviral activity.
Importance Nanomaterials have being investigated as antimicrobial agents, yet their antiviral potential is overshadowed by the cytotoxicity. By using a novel method, we fully exfoliate montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). Here, we show that the surfactant-modified NSP (NSQ) is less cytotoxic and NSQc (NSP modified with sodium dodecyl sulfate) could potently block infection of dengue virus (DEN), Japanese encephalitis virus (JEV) and influenza A virus at noncytotoxic concentrations. For the antiviral mechanism, we find that the electrostatic interaction between the negative-charged NSQc and the positive-charged virus particles blocks viral binding. Furthermore, we used mouse challenge models of JEV and DEN to demonstrate the in vivo antiviral potential of NSQc. Thus, NSQc may function as a potent and safe antiviral nanohybrid against several viruses and our success in synthesizing surfactant-modified NSP with antiviral activity may shed some light on future antiviral development.
Lentiviral RNA genomes present a strong bias in their nucleotide composition with extremely high frequencies of A-nucleotide in HIV-1 and simian immunodeficiency virus (SIV). Based on the observation that human optimization of RNA virus gene fragments may abolish their ability to stimulate type-I interferon (IFN-I) response, we identified the most biased sequences along SIV genome and showed that they are the most potent IFN-I stimulators. With the aim of designing an attenuated SIV genome based on a reduced capacity to activate IFN-I response, we synthesized artificial SIV genomes whose biased sequences were optimized towards macaque average nucleotide composition without altering their regulatory elements or amino acid sequences. A synthetic SIV optimized with 169 synonymous mutations in gag and pol genes showed a 100-fold decrease in replicative capacity. Interestingly, a synthetic SIV optimized with 70 synonymous mutations in pol had a normal replicative capacity. Its ability to stimulate IFN-I was reduced when infected cells were cocultured with reporter cells. IRF3 transcription factor was required for IFN-I stimulation, implicating cytosolic sensors in the detection of SIV biased RNA in infected cells. No reversion of introduced mutations was observed for both optimized viruses after 10 serial passages. In conclusion, we have designed large-scale nucleotide-modified SIVs that may display attenuated pathogenic potential.
IMPORTANCE In this study, we synthesized artificial SIV genomes in which the most hyper biased sequences were optimized to bring them closer to the nucleotide composition of the macaque SIV host. Interestingly, we generated a stable synthetic SIV optimized with 70 synonymous mutations in pol gene, which had a normal replicative capacity but a reduced ability to stimulate type-I IFN. This demonstrates the possibility to rationally change viral nucleotide composition to design replicative and genetically stable lentiviruses with attenuated pathogenic potentials.
We determined the attachment pattern of avian-origin H7N9 influenza viruses A/Anhui/1/2013 and A/Shanghai/1/2013 to the respiratory tract in ferrets, macaques, mice, pigs and guinea pigs, and compared it to that in humans. The H7N9 attachment pattern in macaques, mice, and to a lesser extent pigs and guinea pigs resembled that in humans more closely than the attachment pattern in ferrets. This knowledge contributes to our knowledge on the different animal models for influenza.
The RV144 HIV vaccine trial indicated that gp120 V2 antibodies were associated with lower risk of infection, thus the mapping of V2 epitopes can contribute to the design of an effective HIV vaccine. We solved the crystal structure of human monoclonal antibody (mAb) 2158 that targets a conformational V2 epitope overlapping the aalpha;4bbeta;7 integrin binding site, and constructed a full-length model of V1V2. Comparison of computational energy stability to experimental ELISA results identified a hydrophobic core, stabilizing the V2 region for optimal 2158 binding, and residues that directly mediate side-chain interactions with mAb 2158. These data define the binding surface recognized by mAb 2158 and offer a structural explanation for why a mismatching mutation at position 181 (I181X) in the V2 loop is associated with a vaccine efficiency of 78% in the RV144 clinical vaccine trial.
IMPORTANCE Correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the second variable region (V2) of HIV-1 gp120 was responsible for the modest protection observed in the trial. V2 is a highly variable and immunogenic region and structural information for its antigenic landscape will be important for a rational design of an effective HIV-1 vaccine. Using X-ray crystallography, computational design tools, and mutagenesis assays, we have carried out a detailed and systematic investigation of the epitope recognition of human V2 mAb 2158 and demonstrated that its epitope region overlaps the integrin binding site within V2. In addition, we proposed a structural based mechanism for mismatching of isoleucine at position 181 with increased vaccine efficacy seen in the RV144 vaccine trial.
RIG-I is a cytosolic sensor critically involved in the activation of the innate immune response to RNA virus infection. In the present study, we evaluated the inhibitory effect of a RIG-I agonist on the replication of two emerging arthropod-borne viral pathogens - dengue virus (DENV) and chikungunya virus (CHIKV) - for which no therapeutic options currently exist. We demonstrate that when a low, non-cytotoxic dose of an optimized 5rrsquo;triphosphorylated RNA (5rrsquo;pppRNA) molecule was administered, RIG-I stimulation generated a robust antiviral response against these two viruses. Strikingly, 5rrsquo;pppRNA treatment before or after challenge with DENV or CHIKV provided protection against infection. In primary human monocytes and monocyte-derived dendritic cells, the RIG-I agonist blocked both primary infection and antibody dependent-enhancement of DENV infection. The protective response against DENV and CHIKV induced by 5rrsquo;pppRNA was dependent on an intact RIG-I/MAVS/TBK1/IRF3 axis and was largely independent of the type I IFN response. Altogether, this in vitro analysis of the antiviral efficacy of 5rrsquo;pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.
IMPORTANCE Dengue virus (DENV) and chikungunya virus (CHIKV) are two re-emerging mosquito-borne viruses for which no therapeutic options currently exist. Both viruses overlap geographically in tropical regions of the world, produce similar fever-like symptoms, and are difficult to diagnose. This study investigated the inhibitory effect of a RIG-I agonist on the replication of these two viruses. RIG-I stimulation using 5rrsquo;pppRNA before or after DENV or CHIKV infection generated a protective antiviral response against both pathogens in immune and non-immune cells; interestingly, the protective response against the viruses was largely independent of the classical type I interferon response. The antiviral efficacy of 5rrsquo;pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.
We have examined the requirements for virus transcription and replication, and thus the roles of input and progeny genomes, in the generation of IFN-inducing PAMPs by influenza A viruses using inhibitors of these processes. Using IRF3 phosphorylation as a marker of activation of the IFN induction cascade that occurs upstream of the IFN-bbeta; promoter, we demonstrate strong activation of the IFN induction cascade in A549 cells infected with a range of influenza A viruses in the presence of cycloheximide or NP siRNA, which inhibit viral protein synthesis and thus cRNP and progeny vRNP synthesis. In contrast, activation of the IFN induction cascade by influenza viruses was very effectively abrogated by treatment with actinomycin D and other transcription inhibitors, which correlated with the inhibition of the synthesis of all viral RNA species. Furthermore, 5,6-dichloro-1-bbeta;-D-ribofuranosyl-benzimidazole, an inhibitor that prevents viral RNA export from the nucleus, was also a potent inhibitor of IRF3 activation; thus, both viral RNA synthesis and nuclear export are required for IFN induction by influenza A viruses. Whilst the exact nature of the viral PAMPs remains to be determined, our data suggests that in this experimental system the major influenza A virus PAMPs are distinct from incoming genomes or progeny vRNPs.
IMPORTANCE The host interferon system exerts an extremely potent antiviral response that efficiently restricts virus replication and spread; the interferon response can thus dictate the outcome of a virus infection and it is therefore important to understand how viruses induce interferon. Both input and progeny genomes have been linked to interferon induction by influenza viruses. However, our experiments in tissue culture cells show that viral RNA synthesis and nuclear export are required to activate this response. Furthermore, the interferon induction cascade is activated under conditions in which the synthesis of progeny genomes is inhibited. Therefore, in tissue culture cells, input and progeny genomes are not the predominant inducers of interferon generated by influenza A viruses; the major viral interferon inducer/s still remain to be identified.
Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptors family (RLRs) results in the activation of Type I interferon (IFN) responses. To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural NSs protein of the newly described Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the IFN-bbeta; promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of Atg7, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures co-localize with Rab5 but not with Golgi or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and points toward a novel mechanism for the suppression of IFN responses.
Importance statement: The mechanism by which the newly described SFTSV inhibits host antiviral responses has not been yet fully characterized. In this study, we describe the redistribution of RIG-I signaling components into virus-induced cytoplasmic structures in cells infected with SFTSV. This redistribution correlates with an inhibition of host antiviral responses. Further characterization of the interplay between the viral protein and components of the IFN responses could potentially provide targets for the rational development of therapeutic interventions.
Recombinant Adeno-associated viral vectors (rAAV) have garnered much promise in gene therapy applications. However, widespread clinical use has been limited by transduction efficiency. Previous studies suggest that the majority of rAAV accumulates in the perinuclear region of cells, presumably unable to traffic into the nucleus. rAAV nuclear translocation remains ill-defined; therefore, we performed microscopy, genetic, and biochemical analyses in vitro in order to understand this mechanism. Lectin blockade of the nuclear pore complex (NPC) resulted in inhibition of nuclear rAAV2. Visualization of fluorescently-labeled particles revealed that rAAV2 localized to Importin-bbeta;-dense regions of cells in late trafficking steps. Additionally, siRNA knockdown of Importin-bbeta; partially inhibited rAAV2 nuclear translocation and inhibited transduction by 50-70%. Furthermore, co-immunopreciptation (IP) analysis revealed that capsid proteins from rAAV2 could interact with Importin-bbeta; and that this interaction was sensitive to the small GTPase Ran. More importantly, mutations to key basic regions in the rAAV2 capsid severely inhibited interactions with Importin-bbeta;. We tested several other serotypes and found that the extent of Importin-bbeta; interaction varied, suggesting that different serotypes may utilize alternative import proteins for nuclear translocation. Co-IP and siRNA analyses were used to investigate the role of other karyopherins and suggested that rAAV2 may utilize multiple import proteins for nuclear entry. Taken together, our results suggest that rAAV2 interacts with Importin-bbeta; alone or in complex with other karyopherins and enters the nucleus via the NPC. These results may lend insight into the design of novel AAV vectors that have enhanced nuclear entry capability and transduction potential.
Importance: Use of recombinant Adeno-associated viral vectors for gene therapy applications is limited by relatively low transduction efficiency, in part due to cellular barriers that hinder successful subcellular trafficking to the nucleus where uncoating and subsequent gene expression occur. Nuclear translocation of rAAV has been regarded as a limiting step for successful transduction but remains ill-defined. We have explored potential nuclear entry mechanisms for rAAV2 and have shown that rAAV2 can utilize the classical nuclear import pathway, involving the nuclear pore complex, the small GTPase Ran, and cellular karyopherins. These results could lend insight into the rational design of novel rAAV vectors that can more efficiently translocate to the nucleus, which could lead to more efficient transduction.
Human papillomavirus can successfully evade the host immune response to establish a persistent infection. We show here that expression of the E7 oncoprotein in primary human keratinocytes results in increased production of IL-18 binding protein (IL-18BP). This anti-inflammatory protein is a natural antagonist of IL-18 and is necessary for skin homeostasis. We map increased IL-18BP production to the CR3 region of E7 and demonstrate that this ability is shared amongst E7 proteins from different HPV types. Furthermore, mutagenesis shows that increased IL-18BP production is mediated by a gamma activated sequence (GAS) in the IL-18BP promoter. Importantly, the increased IL-18BP levels seen in E7 expressing keratinocytes are capable of diminishing IL-18 mediated CD4 lymphocyte activation. This study provides the first evidence for a virus protein that targets IL-18BP and further validates E7 as a key component of the HPV immune evasion armor.
Importance: Infection with human papillomavirus is a leading cause of morbidity and mortality worldwide. This study demonstrates that the E7 protein increases production of the anti-inflammatory IL-18BP, a major regulator of epithelial homeostasis. A number of E7 proteins can increase IL-18BP production and a region within the CR3 of E7 is necessary for mediating the increase. A consequence of increased IL-18BP production is a reduction in CD4 positive lymphocyte activation in response to IL-18 co-stimulation. These findings may shed light on the immune evasion abilities of HPV.
We previously showed that prototype macaque-tropic HIV-1 acquired non-synonymous growth-enhancing mutations within a narrow genomic region during the adaptation process in macaque cells. These adaptive mutations were clustered in the 3rrsquo; region of pol gene encoding a small portion of the C-terminal domain of integrase (IN). Mutations in HIV-1 IN have been reported to have pleiotropic effects on both the early and late phases in viral replication. Cis-acting functions in the IN-coding sequence for viral gene expression have also been reported. We here demonstrated that the adaptive mutations promoted viral growth by increasing virion production with no positive effects on the early replication phase. Synonymous codon alterations in one of the adaptive mutations influenced virion production levels, which suggested nucleotide-dependent regulation. Indeed, when the single-nucleotide natural polymorphisms observed in the 3rrsquo; regions of 196 HIV-1/SIVcpz pol genes (nucleotides (nt) 4895-4929 for HIV-1 NL4-3) were introduced into macaque- and human-tropic HIV-1 clones, more than half exhibited altered-replication potentials. Moreover, single-nucleotide mutations caused parallel increases or decreases in the expression levels of viral late proteins and viral replication potentials. We also showed that the overall expression profiles of viral mRNAs were markedly changed by single-nucleotide mutations. These results demonstrate that the 3rrsquo; region of HIV-1 pol gene (nt 4895-4929) can alter viral replication potential by modulating the expression pattern of viral mRNAs in a nucleotide-dependent manner.
Importance: Viruses have the plasticity to adapt themselves under various constraints. HIV-1 can mutate and evolve in growth-restrictive cells by acquiring adaptive changes in its genome. We have previously identified some growth-enhancing mutations in a narrow region of the IN-coding sequence, in which a number of cis-acting elements are located. We now focus on the virological significance of this pol gene region and the mechanistic basis underlying its effects on viral replication. We have found several naturally occurring synonymous mutations within this region that alter viral replication potentials. The effects caused by these natural single-nucleotide polymorphisms are linked to the definite expression patterns of viral mRNAs. We herein show that the nucleotide sequence of the pol gene (nucleotides 4895-4929 for HIV-1 NL4-3) plays an important role in HIV-1 replication by modulating viral gene expression.
We have recently shown that a cocktail of two short synthetic shRNAs (sshRNAs) targeting the internal ribosome entry site of hepatitis C virus (HCV) formulated with lipid nanoparticles was able to suppress viral replication in chimeric mice infected with HCV GT1a by up to 2.5 log10. Viral load remained about 1 log10 below pre-treatment levels 21 days after the end of dosing. We have now sequenced the HCV viral RNA amplified from serum of treated mice after the 21-d follow-up period. Viral RNA from the HCV sshRNA-treated groups was altered in sequences complementary to the sshRNAs and nowhere else in the 500-nt sequenced region, while the viruses from the control group that received an irrelevant sshRNA had no mutations in that region. The ability of the most commonly-selected mutations to confer resistance to the sshRNAs was confirmed in vitro by introducing those mutations into HCV-luciferase reporters. The mutations most frequently selected by sshRNA treatment within the sshRNA target sequence occurred at the most polymorphic residues as identified from an analysis of available clinical isolates. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNAi mechanism of action.
Importance This study presents a detailed analysis of the impact of treating a hepatitis C virus (HCV)-infected animal with synthetic hairpin-shaped RNAs that can degrade the virusrrsquo;s RNA genome. These RNAs can reduce the viral load in these animals by over 99% after 1nndash;2 injections. The study results confirm that the viral rebound that often occurred a few weeks after treatment is due to emergence of virus whose genome is mutated in the sequences targeted by the RNAs. The use of two RNA inhibitors, which is more effective than use of either one by itself, requires that any resistant virus have mutations in the targets sites of both agents, a higher hurdle if the virus is to retain the ability to replicate efficiently. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNAi mechanism of action.
Human La protein has been known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with HCV internal ribosomal entry site (IRES) around the initiator AUG within the stem-loop IV form a bbeta;-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that HCV RNA interacting bbeta;-turn is conserved only in the human and the chimpanzee, the species primarily known to be infected by the HCV. A 7-mer peptide corresponding to HCV RNA interacting region of human La inhibits HCV translation whereas another peptide corresponding to the mouse La sequence was unable to do so. Further, IRES mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed an enhanced replication with HCV subgenomic and full length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring human La bbeta;-turn sequence in mouse cells. Taken together, our results raise a possibility of creating an immunocompetent HCV mouse model using human specific cell entry factors and a humanized form of La protein.
Importance: Hepatitis C virus is known to infect only humans and chimpanzees under natural conditions. This has prevented development of a small animal model, which is important for development of new antiviral drugs. Although a number of human specific proteins is responsible for this species selectivity and some of these proteinsmmdash;mostly entry factorsmmdash;have been identified, full multiplication of the virus in mouse cells is still not possible. In this study, we show that a turn in the human La protein that is responsible for the interaction with the viral RNA, is highly specific for the human sequence. Replacement of the corresponding mouse sequence with the human sequence, allows the mouse La to behave like the human counterpart and support viral growth in the mouse cell efficiently. This observation, in combination with previously identified cell entry factors, should open up the possibility of creating a mouse model of hepatitis C.
A new type of anti-prion compound, Gly-9, was found to inhibit abnormal prion protein formation of prion-infected neuroblastoma cells in a prion-strain independent manner when the cells were treated for more than one day. It reduced the intracellular prion protein level and significantly modified mRNA expression levels of genes of two types: interferon-stimulated genes were down-regulated after more than two days of treatment; phosphodiesterase 4D interacting protein gene, a gene involved in microtubule growth, was up-regulated after more than one day of treatment. A supplement of interferon to the cells partly restored the abnormal prion protein level but did not alter the normal prion protein level. This interferon action was independent of Janus activated kinase-signal transducer and activator of transcription signaling pathway. Therefore, the changes in interferon-stimulated genes might be a secondary effect of Gly-9 treatment. However, gene knockdown of phosphodiesterase 4D interacting protein restored or increased both the abnormal prion protein level and normal prion protein level without transcriptional alteration of prion protein gene. It also altered the localization of abnormal prion protein accumulation in the cells, indicating that phosphodiesterase 4D interacting protein might affect prion protein levels by altering the trafficking of prion protein containing structures. Interferon and phosphodiesterase 4D interacting protein had no direct mutual link, demonstrating that they regulate abnormal prion protein level independently. Although the in-vivo efficacy of Gly-9 was limited, the findings of Gly-9 provide insights into the regulation of abnormal prion protein in the cells and suggest new targets for anti-prion compounds.
IMPORTANCE This report describes our study of the efficacy and the potential mechanism underlying the anti-prion action of a new anti-prion compound having a glycoside structure in prion-infected cells, as well as the efficacy of the compound in prion-infected animals. The study revealed involvements of two factors in the mechanism of the compound action: interferon and a microtubule nucleation activator, phosphodiesterase 4D interacting protein. In particular, phosphodiesterase 4D interacting protein was suggested to be important in regulating the trafficking or fusion of prion protein-containing vesicles or structures in cells. The findings of the study are expected to be useful not only for the elucidation of cellular regulatory mechanisms of prion protein, but also for the implication of new targets for therapeutic development.
A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5rrsquo; untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10-50-fold) in viral RNA packaging and viral titer. Here we examined the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and viral titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation.
IMPORTANCE To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5rrsquo; end of the viral RNA known as packaging signal. In this study we examined whether Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or viral titer. These studies provide important information on how HIV-1 regulates its genome packaging and generate infectious viruses necessary for transmission to new hosts.
Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H or G]) mediate a concerted process of host receptor recognition followed by fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H or G and the llsquo;triggering' interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active llsquo;headless' mumps and Newcastle disease virus HN proteins provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed llsquo;stalk exposure model' to other paramyxoviruses and propose an llsquo;induced fit' hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.
IMPORTANCE STATEMENT Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H or G]) mediate a concerted process of host receptor recognition followed by fusion of viral and cellular membranes. In this paper we describe the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed llsquo;stalk exposure model' first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an llsquo;induced fit' hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.
The HSV-1 UL51 gene encodes a 244 a.a. palmitoylated protein that is conserved in all herpesviruses. The alphaherpesvirus UL51 protein has been reported to function in nuclear egress and cytoplasmic envelopment. No complete deletion has been generated because of overlap of the UL51 coding sequence 5rrsquo; end with the UL52 promoter sequences, but partial deletions generated in HSV and PrV suggest an additional function in epithelial cell-to-cell spread. Here we show partial uncoupling of the replication, release and cell-to-cell spread functions of HSV-1 pUL51 in two ways. Viruses in which a.a. 73-244 were deleted from pUL51, or in which a conserved YXX motif near the N-terminus was altered showed cell-specific defects in spread that cannot be accounted for by defects in replication and virus release. Also, a cell line that expresses a C-terminal EGFP-tagged pUL51 supported normal virus replication and release to the medium, but the formation of only small plaques. This cell line also failed to support normal localization of gE to cell junctions. gE and pUL51 partially co-localized in infected cells and these two proteins could be co-immunoprecipitated from infected cells, suggesting that they can form a complex during infection. The cell-to-cell spread defect associated with pUL51 mutation was more severe than that associated with gE null virus, suggesting that pUL51 has gE-independent functions in epithelial cell spread.
Importance Herpesviruses establish and reactivate from life-long latency in their hosts. When they reactivate, they are able to spread within their hosts despite the presence of a potent immune response that includes neutralizing antibody. This ability derives in part from a specialized mechanism for virus spread between cells. Cell-to-cell spread is a conserved property of herpesviruses that likely relies on conserved viral genes. Understanding their function may aid design of vaccines and therapeutics. Here we show that one of the conserved viral genes called UL51 has an important role in cell-to-cell spread in addition to its previously demonstrated role in virus assembly. We find that its function depends on the type of cell that is infected, and we show that it interacts with and modulates the function of another viral spread factor called gE.
Mutagenic nucleoside analogues can be used to isolate RNA virus high-fidelity RNA-dependent RNA polymerase (RdRp) variants, the majority of which are attenuated in vivo. However, attenuated foot-and-mouth disease virus (FMDV) high-fidelity RdRp variants have not been isolated, and the correlations between RdRp fidelity and virulence remain unclear. Here, the mutagen ribavirin was used to select a ribavirin-resistant population of FMDV, and 4 amino acid substitutions (D5N, A38V, M194I, and M296V) were identified in the RdRp-coding region of the population. Through single or combined mutagenesis using a reverse genetics system, we generated direct experimental evidence that the rescued mutants D5N, A38V, and DAMM but not M194I and M296V are high-fidelity RdRp variants. Mutagen-resistance assays revealed that the higher replication fidelity was associated with higher resistance to ribavirin. In addition, significantly attenuated fitness and virulence phenotypes were observed for the D5N, A38V, and DAMM mutants. Based on a systematic quantitative analysis of fidelity and virulence, we concluded that higher replication fidelity is associated with a more attenuated virus. These data suggest that the resulting restricted quasispecies diversity compromises the adaptability and virulence of an RNA virus population. The modulation of replication fidelity to attenuate virulence may represent a general strategy for the rational design of new types of live, attenuated vaccine strains.
Importance The ribavirin-isolated PV RdRp G64S variant, which polymerases were of high replication fidelity, was attenuated in vivo. Vignuzzi et al. proposed that modulating replication fidelity is a promise approach for engineering attenuated virus vaccines. The subsequently mutagen-isolated RdRp variants also expressed the high fidelity polymerase, but not all of them were attenuated. Few studies have shown the exact correlation between fidelity and virulence. The present study investigates the effect of restricted quasispecies diversity on viral virulence via the several attenuated FMDV high-fidelity RdRp variants. Our findings may aid the rational design of a new type of vaccine strains.
Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development as well as the state of differentiation of susceptible cells at the time of infection affect the severity of the disease. We used human embryonic stem (ES) cell-derived primitive pre-rosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs, infection of ES-derived pNSCs with HCMV was non-progressive. At a low multiplicity of infection, we observed only a small percentage of cells expressing immediate early (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1, and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell-associated and were mostly non-neural tropic. Finally, we found that viral genomes could persist in pNSC cultures up to a month after infection despite the absence of detectable IE expression by immunofluorescence, and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion, our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus.
Importance Human cytomegalovirus (HCMV) is a bbeta;-herpesvirus that is highly prevalent in the population. Its infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study, we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells, which are similar to those present in the developing neural tube, do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS.
Gammaherpesviruses (GHVs) are a diverse and rapidly expanding group of viruses associated with a variety of disease conditions in humans and animals. To identify felid GHVs, we screened domestic cat (Felis catus), bobcat (Lynx rufus) and puma (Puma concolor) blood cell DNA samples from California, Colorado and Florida using a degenerate pan-GHV PCR. Additional pan-GHV and long-distance PCRs were used to sequence a contiguous 3.4 kb region of each putative virus species including partial glycoprotein B and DNA polymerase genes. We identified three novel GHVs, each present predominantly in one felid species: Felis catus GHV 1 (FcaGHV1) in domestic cats, Lynx rufus GHV 1 (LruGHV1) in bobcats, and Puma concolor GHV 1 (PcoGHV1) in pumas. To estimate infection prevalence, we developed real-time quantitative PCR assays for each virus and screened additional DNA samples from all three species (n = 282). FcaGHV1 was detected in 16% of domestic cats across all study sites. LruGHV1 was detected in 47% of bobcats and 13% of pumas across all study sites, suggesting relatively common interspecific transmission. PcoGHV1 was detected in 6% of pumas, all from a specific region of Southern California. The risk of infection for each host varied with geographic location. Age was a positive risk factor for bobcat LruGHV1 infection, and age and being male were risk factors for domestic cat FcaGHV1 infection. Further characterization of these viruses may have significant health implications for domestic cats and may aid studies of free-ranging felid ecology.
Importance Gammaherpesviruses (GHVs) establish life-long infection in many animal species and can cause cancer and other diseases in humans and animals. In this study we identified DNA sequences of three GHVs present in the blood of domestic cats (Felis catus), bobcats (Lynx rufus) and pumas (Puma concolor, also known as cougars or mountain lions). We found that these viruses were closely related to, but distinct from, other known GHVs of animals and represent the first GHVs identified as native to these feline species. We developed techniques to rapidly and specifically detect the DNA of these viruses in feline blood and found that the domestic cat and bobcat viruses were widespread across the US. In contrast, puma virus was found only in a specific region of southern California. Surprisingly, the bobcat virus was also detected in some pumas, suggesting relatively common virus transmission between these species. Adult domestic cats and bobcats were at greater risk for infection than juveniles. Male domestic cats were at greater risk for infection than females. This study identifies three new viruses that are widespread in three feline species, indicates risk factors for infection that may relate to route of infection, and demonstrates cross-species transmission between bobcats and pumas. These newly identified viruses may have important effects on feline health and ecology.
Potyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.
Classical scrapie is one of the Transmissible Spongiform Encephalopathies (TSE), a group of fatal infectious diseases that affect the central nervous system (CNS). Classical scrapie can transmit laterally from ewe to lamb perinatally, or between adult animals. Here we report detection of infectivity in tissues of an unborn foetus, providing evidence that in utero transmission of classical scrapie is also possible.
The emergence of avian H7N9 viruses in humans in China has renewed concerns about influenza pandemics emerging from Asia. Vaccines are still the best countermeasure against emerging influenza virus infections, but the process from identification of vaccine seed strains to the distribution of the final product can take several months. In the case of the 2009 H1N1 pandemic, a vaccine was not available before the first pandemic wave hit and therefore came too late to reduce influenza morbidity. H7 vaccines based on divergent isolates of the Eurasian and North American lineage have been tested in clinical trials, seed strains and reagents are already available and can potentially be used initially to curtail influenza-induced disease until a more appropriately matched H7N9 vaccine is ready. In a challenge experiment in the mouse model we assessed the efficacy of both inactivated virus and recombinant hemagglutinin vaccines made from seed strains that are divergent to H7N9 from each of each of the two major H7 lineages. Furthermore, we analyzed the cross-reactive responses to H7N9 of sera from human subjects vaccinated with heterologous North American and Eurasian lineage H7 vaccines. Vaccinations with inactivated virus and recombinant hemagglutinin protein preparations from both lineages raised hemagglutination-inhibiting antibodies against H7N9 viruses and protected mice from stringent viral challenges. Similar cross-reactivity was observed in sera of human subjects from a clinical trial with a divergent H7 vaccine. Existing H7 vaccine candidates based on divergent strains could be used as a first line of defense against an H7N9 pandemic. In addition it also suggests that H7N9 vaccines that are currently under development might be stockpiled and used for divergent avian H7 strains that emerge in the future.
Importance Sporadic human infections with H7N9 viruses started being reported in China in early spring 2013. Despite a significant drop in infections during the summer months of 2013, an increased number of cases has already been reported for the 2013/14 winter season. The high case fatality rate, the ability to bind to receptors in the human upper respiratory tract in combination with several family clusters and the emergence of neuraminidase inhibitor resistant variants that show no loss of pathogenicity or ability to transmit in animal models have raised concerns about a potential pandemic and have spurred efforts to produce vaccine candidates. Here we show that antigens preparations from divergent H7 strains are able to induce protective immunity against H7N9 infection.
Induction of long-lasting immunity against viral respiratory tract infections remains an elusive goal. Using a non-human primate model of human respiratory syncytial virus (hRSV) infection, we compared mucosal and systemic immune responses induced by different DNA delivery approaches to a novel parenteral DNA prime - tonsillar adenoviral vector (AdV) booster immunization regimen. Intramuscular (i.m.) electroporation (EP) of a DNA vaccine encoding the fusion protein of hRSV induced stronger systemic immune responses than intradermal EP, tattoo immunization and conventional i.m. DNA injection. A single EP i.m. followed by two atraumatic tonsillar immunizations with the adenoviral vector elicited strong systemic immune responses, an unique persistent CD4+ and CD8+ T cell response in the lower respiratory tract, and protection from intranasal hRSV challenge. Thus, parenteral DNA priming followed by booster immunization targeted to a mucosal inductive site constitutes an effective vaccine regimen for eliciting protective immune responses at mucosal effector sites.
Importance The human respiratory syncytial virus (hRSV) is the most common cause of severe respiratory tract disease in infancy and leads to substantial morbidity and morality in the elderly. In this study, we compared the immunogenicity and efficacy of several gene-based immunization protocols in rhesus macaques. Thereby, we found that the combination of an initially parenteral delivered DNA vaccine with a subsequent atraumatic tonsillar adenoviral vector immunization results in a strong systemic immune response accompanied by an exceptional high T-cell response in the mucosa. Strikingly, these animals were protected against a RSV challenge infection controlling the viral replication indicated by a 1000-fold lower viral load in the lower respiratory tract. Since mucosal cellular responses of this strength had not been described in earlier RSV vaccine studies, this heterologous DNA-prime tonsillar boost vaccine strategy is very promising and should be pursued for further preclinical and clinical testing.
The tailed dsDNA bacteriophage 29 packages its 19.3 Kbp genome into a pre-assembled procapsid structure using a transiently assembled phage-encoded molecular motor. This process is remarkable considering that compaction of DNA to near crystalline densities within the confined space of the capsid requires that the packaging motor work against significant entropic, enthalpic and DNA-bending energies. The motor consists of three phage-encoded components: the dodecameric connector protein gp10, an oligomeric RNA molecule known as the pRNA, and the homomeric ring ATPase gp16. Although atomic resolution structures of the connector and different pRNA sub-domains have been determined, the mechanism of self-assembly and the resulting stoichiometry of the various motor components on the phage capsid have been the subject of considerable controversy. Here, a sub-nanometer asymmetric cryo-electron microscopy (cryo-EM) reconstruction of a connector-pRNA complex at a unique vertex of the procapsid conclusively demonstrates the pentameric symmetry of the pRNA and illuminates the relative arrangement of the connector and the pRNA. Additionally, a combination of biochemical and cryo-EM analyses of motor-assembly intermediates suggests a sequence of molecular events that constitute the pathway by which the motor assembles on the head, thereby reconciling conflicting data regarding pRNA assembly and stoichiometry. Taken together, these data provide new insight into the assembly, structure, and mechanism of a complex molecular machine.