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Table of Contents for this page:

  • Current Issue
  • Current Issue of Virology Journal

    Virology Journal - Latest Articles

  • Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K

  • Background: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. Methods: The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy; zymogram analysis was performed on the enzyme and selected site-directed mutants. Results: The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity. Conclusions: The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.

  • Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor

  • Background: Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O.To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells. Results: Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 [rightwards arrow] Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 [rightwards arrow] Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 [rightwards arrow] Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 [rightwards arrow] Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells.Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells.Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant encoding Gln-2080 [rightwards arrow] Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells. Conclusion: The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

  • Defining viral species: making taxonomy useful

  • Virus taxonomy at present is best characterized as a categorization of convenience, without a firm basis in the principles of evolutionary biology. Specifically, virus species definitions appear to depend more on tradition and popular opinion among virologists than on firm, quantitative biological evidence. I suggest a series of changes to underlying species concepts that would shift the field from one that simply files viruses away in taxonomic boxes to one that can learn important biological lessons from its taxonomy.

  • The CD8 Antiviral Factor (CAF) can suppress HIV-1 transcription from the Long Terminal Repeat (LTR) promoter in the absence of elements upstream of the CATATAA box

  • Background: The CD8 Antiviral Factor (CAF) suppresses viral transcription from the HIV-1 Long Terminal Repeat (LTR) promoter in a non-cytolytic manner. However, the region on the LTR upon which CAF acts is unknown. Our objective was to determine the region on the LTR upon which CAF acts to suppress HIV-1 transcription. Methods: Serial deletions of the LTR from the 5aapos; end and inactivating point mutations were made. Results: Serial deletions of the LTR from the 5aapos; end indicated the importance of a short ~120 bp segment, containing the 3 SpI sites, CATA box (used by HIV-1 instead of the TATA box) and TAR region, in the suppressive process. Introduction of deletions or inactivating point mutations in the SpI sites or deletion of the TAR region did not abolish CAF-mediated transcriptional suppression. Yet, CAF-mediated transcriptional suppression was still retained in the HIV-1 CATA-TAR segment. Conclusion: CAF is able to suppress transcription from the LTR lacking all the elements upstream of the CATA box. Our results suggest that the HIV-1 CATA box may be responsible for CAF-mediated suppression of transcription from the HIV-1 LTR.

  • Detection of circulating norovirus genotypes: hitting a moving target

  • Background: Although national surveillance programs are in place to monitor norovirus epidemiology, the emergence of new strains and the genetic diversity among genotypes can be challenging for clinical laboratories. This study evaluated the analytical and clinical performance characteristics of one real-time RT-PCR and two end-point RT-PCRs commonly used in microbiology laboratories. Methods: Lower limit of detection (LoD) was determined using 10-fold dilutions of noroviruses belonging to different genotypes. The clinical performance of the real-time and end-point RT-PCRs was assessed in parallel using nucleic acids extracted from 186 stool specimens. Results: The real-time RT-PCR was highly sensitive and specific for the detection of norovirus genotypes that are currently circulating in Canada. In contrast, the two end-point RT-PCRs displayed poor analytical sensitivity or complete failure to detect certain norovirus genotypes, which was correlated to sequence mismatches in the primer-binding sites. In an attempt to improve norovirus detection with the end-point RT-PCRs, both assays were processed concurrently and detection from either assay was considered a positive result. Concurrent testing resulted in only a modest increase in clinical sensitivity (75.0%) compared to each assay alone (62.5% and 71.9%). However, the false positivity rate increased from 1.98% and 3.36% for the assays alone to 5.47% with concurrent testing. Conclusions: This study emphasizes the benefits of a real-time method and provides support for routine surveillance to monitor norovirus epidemiology and ongoing proficiency testing to ensure detection of circulating norovirus genotypes.

  • Mutations within the conserved NS1 nuclear export signal lead to inhibition of influenza A virus replication

  • Background: The influenza A virus NS1 protein is a virulence factor and an antagonist of host cell innate immune responses. During virus infection NS1 protein has several functions both in the nucleus and in the cytoplasm and its intracellular localization is regulated by one or two nuclear localization signals (NLS) and a nuclear export signal (NES). Methods: In order to investigate the role of NS1 NES in intracellular localization, virus life cycle and host interferon responses, we generated recombinant A/Udorn/72 viruses harboring point mutations in the NES sequence. Results: NS1 NES was found to be inactivated by several of the mutations resulting in nuclear retention of NS1 at late stages of infection confirming that this sequence is a bona fide functional NES. Some of the mutant viruses showed reduced growth properties in cell culture, inability to antagonize host cell interferon production and increased p-IRF3 levels, but no clear correlation between these phenotypes and NS1 localization could be made. Impaired activation of Akt phosphorylation by the replication-deficient viruses indicates possible disruption of NS1-p85β interaction by mutations in the NES region. Conclusion: We conclude that mutations within the NS1 NES result in impairment of several NS1 functions which extends further from the NES site being only involved in regulating the nuclear-cytoplasmic trafficking of NS1.

  • Chemiluminescent microparticle immunoassay based detection and prevalence of HCV infection in district Peshawar Pakistan

  • Background: Due to the high rate of asymptomatic infections an advanced screening assay is of prompt importance to be used for the clinical diagnosis of HCV. Early detection of anti HCV is the first step in the management of chronic hepatitis and in the selection of patients needing treatments. In the current study we have first time used the advanced serological diagnostic technique i.e. Chemiluminescent Microparticle Immuno Assay (CMIA) for the detection of HCV infection in Peshawar Pakistan. Methods: A total number of 982 samples were collected among the general public belongs to the different areas of district Peshawar. The samples were centrifuged at high speed to obtain a clear supernatant serum. All the samples were run on Architect system a fully automated immuno analyzer CMIA base technology. Results: Out of 982 blood samples analyzed in this study, 160 (15.9%) were confirmed to be positive for active HCV infection. The overall prevalence was found to be 13.4%. Gender wise prevalence was recorded to be higher in male (19.1%) than female (12.7%). The age group 21-30 years was identified as the highest risk group among the studied population. Conclusion: Among the tested samples, overall prevalence of active HCV infection was found to be 13.4% in the general population of Peshawar Pakistan. The young middle aged population of this region was at higher risk of HCV ailments compared to the other age groups.

  • Reactive astrogliosis in response to hemorrhagic fever virus: microarray profile of Junin virus-infected human astrocytes

  • Background: Arenavirus Junin is the causative agent of Argentine hemorrhagic fever. Limited information is available concerning the pathogenesis of this human disease, especially the pathogenesis of acute and late neurological symptoms. Methods: In our study we present for the first time cDNA microarray profile of human astrocytes infected with the virulent strain of Junin virus. Transcriptional profiling was confirmed by quantitative real-time RT-PCR and cytokine/chemokine/growth factor assay. Results: We demonstrated the impact of virus infection on immune/inflammatory response/interferon signaling and apoptosis. Pro-apoptotic response and amplification with time of pro-inflammatory cascade of human astrocytes suggested neurodegenerative dysfunctional reactive astrogliosis in response to Junin virus infection. Conclusion: Our results suggest potential pathogenic role of astroglial cells in the development of neurological symptoms and late neurological syndrome during Argentine hemorrhagic fever.

  • The combined effects of irradiation and herpes simplex virus type 1 infection on an immortal gingival cell line

  • Background: Oral mucosa is frequently exposed to Herpes simplex virus type 1 (HSV-1) infection and irradiation due to dental radiography. During radiotherapy for oral cancer, the surrounding clinically normal tissues are also irradiated. This prompted us to study the effects of HSV-1 infection and irradiation on viability and apoptosis of oral epithelial cells. Methods: Immortal gingival keratinocyte (HMK) cells were infected with HSV-1 at a low multiplicity of infection (MOI) and irradiated with 2 Gy 24 hours post infection. The cells were then harvested at 24, 72 and 144 hours post irradiation for viability assays and qRT-PCR analyses for the apoptosis-related genes caspases 3, 8, and 9, bcl-2, NFκB1, and viral gene VP16. Mann–Whitney U-test was used for statistical calculations. Results: Irradiation improved the cell viability at 144 hours post irradiation (P = 0.05), which was further improved by HSV-1 infection at MOI of 0.00001 (P = 0.05). Simultaneously, the combined effects of infection at MOI of 0.0001 and irradiation resulted in upregulation in NFκB1 (P = 0.05). The combined effects of irradiation and HSV infection also significantly downregulated the expression of caspases 3, 8, and 9 at 144 hours (P = 0.05) whereas caspase 3 and 8 significantly upregulated in non-irradiated, HSV-infected cells as compared to uninfected controls (P = 0.05). Infection with 0.0001 MOI downregulated bcl-2 in non-irradiated cells but was upregulated by 27% after irradiation when compared to non-irradiated infected cells (P = 0.05). Irradiation had no effect on HSV-1 shedding or HSV gene expression at 144 hours. Conclusions: HSV-1 infection may improve the viability of immortal cells after irradiation. The effect might be related to inhibition of apoptosis.

  • Biphasic regulation of A20 gene expression during human cytomegalovirus infection

  • Background: The A20 ubiquitin-editing enzyme is a target of nuclear factor kappa B (NF-κB) and also plays a key role in regulating the NF-κB signaling pathway. NF-κB activity is increased during human cytomegalovirus (HCMV) infection and HCMV appears to be adapted to this change. To better understand the regulation of NF-κB signaling during HCMV infection, we investigated how A20 expression is controlled during HCMV infection. Methods: The expression level of A20 in human fibroblast cells infected with HCMV or UV-inactivated virus (UV-HCMV) was measured by immunoblot analysis, cell staining, and quantitative real-time PCR. Changes of histone modifications on the A20 promoter were determined by chromatin immunoprecipitation assays. Lentiviral vectors were used to knockdown A20 in fibroblast cells. Results: A20 expression was increased at early times after HCMV infection. This increase of the A20 protein level was promoted by viral gene expression under low viral load conditions. The viral IE1 protein, which is known to activate NF-κB, increased the A20 promoter activity through the upstream NF-κB sites in reporter assays, suggesting that IE1 is at least partly involved in A20 induction. Analysis of A20 expression with a high viral load demonstrated that the A20 regulation by HCMV was biphasic; both A20 protein and mRNA levels were increased at the early stage of infection, but decreased at the late stage. Under high viral load conditions, A20 upregulation was more profound with UV-HCMV than with HCMV, indicating a role of the viral gene product(s) in limiting A20 induction. Consistently, more histone modifications for euchromatin were found on the A20 promoter during UV-HCMV infection than with HCMV infection. A20 knockdown by shRNA reduced HCMV growth. Conclusion: These results suggest that the biphasic regulation of A20 expression may be important for productive HCMV infection.
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