|Journal of Virology Structure and Assembly|
The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain [MD]) in the large surface protein L and a narrow region (matrix binding domain [MBD]) including isoleucine 126 on the capsid surface have been mapped, in which point mutations such as core I126A specifically blocked nucleocapsid envelopment. It is possible that the two domains interact with each other during virion morphogenesis. By the systematic evolution of ligands by exponential enrichment (SELEX) method, we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications, 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50% of the clones. The main aptamer, AO-01 (13 clones, 18%), showed the lowest dissociation constant (Kd) of 180 pplusmn; 82 nM for capsid binding among the four molecules. Its Kd for I126A capsids was 1,306 pplusmn; 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47% inhibition of virion production at 3 days posttransfection, but there was no inhibition by cotransfection of an aptamer with a random sequence. The half-life of AO-01 in cells was 2 h, which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction with a low-molecular-weight substance might represent a new approach for an antiviral therapy.
IMPORTANCE Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55-base-long single-stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The results substantiate the current model for HBV morphogenesis and show that the capsid envelope interaction is a potential antiviral target.
We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals.
IMPORTANCE A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage ) as an experimental model system, we investigated the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.
The HIV-1 envelope (Env) glycoprotein mediates viral entry during both cell-free and cell-to-cell infection of CD4+ T cells. The highly conserved long cytoplasmic tail (CT) of Env is required in a cell type-dependent manner for optimal infectivity of cell-free virus. To probe the role of the CT in cell-to-cell infection, we tested a panel of mutations in the CT region that maintain or attenuate cell-free infection to investigate whether the functions of the CT are conserved during cell-free and cell-to-cell infection. The mutations tested included truncations of structural motifs in the gp41 CT and two point mutations in lentiviral lytic peptide 3 (LLP-3) previously described as disrupting the infectivity of cell-free virus. We found that small truncations of 28 to 43 amino acids (aa) or two LLP-3 point mutations, YW_SL and LL_RQ, severely impaired single-round cell-free infectivity 10-fold or more relative to wild-type full-length CT. These mutants showed a modest 2-fold reduction in cell-to-cell infection assays. Conversely, large truncations of 93 to 124 aa severely impaired cell-to-cell infectivity 20-fold or more while resulting in a 50% increase in infectivity of cell-free viral particles when produced in 293T cells. Intermediate truncations of 46 to 90 aa showed profound impairment of both modes of infection. Our results show that the abilities of Env to support cell-free and cell-to-cell infection are genetically distinct. These differences are cell type dependent for large-CT-truncation mutants. Additionally, point mutants in LLP-3 can maintain multiround propagation from cell-to-cell in primary CD4+ T cells.
IMPORTANCE The functions of HIV Env gp41 CT remain poorly understood despite being widely studied in the context of cell-free infection. We have identified domains of the gp41 CT responsible for striking selective deficiencies in either cell-free or cell-to-cell infectivity. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env or differential interaction with host or viral proteins. Our findings provide novel insight into the key regulatory potential of the gp41 CT in cell-free and cell-to-cell HIV-1 infection, particularly for short-truncation mutants of lle;43 amino acids or mutants with point mutations in the LLP-3 helical domain of the CT, which are able to propagate via cell-to-cell infection in the absence of infectious cell-free virus production. These mutants may also serve as tools to further define the contributions of cell-free and cell-to-cell infection in vitro and in vivo.
Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles.
IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.
We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.
IMPORTANCE AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.
Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28.
IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.
Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating.
IMPORTANCE Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.