Journal of Virology最新文献

Polyomaviruses (PyVs) cause diverse diseases in a variety of mammalian hosts. During the life cycle, PyVs recruit nuclear host factors to viral genomes to facilitate replication and transcription. While host factors involved in DNA replication, DNA damage sensing and repair, and cell cycle regulation have been observed to bind PyV DNA, the complete set of viral and host proteins comprising the PyV replisome remains incompletely characterized. Here, the iPOND-MS technique (Isolation of Proteins on Nascent DNA coupled with Mass Spectrometry) was used to identify the proteome bound to murine PyV (MuPyV) DNA immediately following synthesis and 2 hours post-synthesis. Several novel MuPyV DNA interactors were identified on newly synthesized viral DNA (vDNA), including MCM complex members, DNA primase, DNA polymerase alpha, DNA ligase, and replication factor C. Though displaying partial overlap, the host and viral proteins bound to MuPyV DNA 2 hours post-synthesis lacked many of the replication proteins found on newly synthesized vDNA. These data help distinguish between the host factors critical for MuPyV DNA replication and those involved in downstream processing.IMPORTANCEPolyomaviruses are the causative agents of serious diseases in humans, including progressive multifocal leukoencephalopathy (PML), BK virus nephropathy, and Merkel cell carcinoma. The exact mechanisms by which the virus replicates, and which host cell proteins are required, are incompletely characterized. Identifying the host proteins necessary for efficient viral replication in the cell may reveal targets for downstream targets that may suppress viral replication in vivo.

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in non-structural protein 3. Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the glycine-isoleucine-phenylalanine motif. While we previously demonstrated the importance of the glycine residue for CoV replication and pathogenesis, the impact of the isoleucine and phenylalanine residues remains unknown. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that correlated with attenuated replication and pathogenesis of F-A mutant MERS-CoV and SARS-CoV-2 viruses in cell culture and mice. In contrast, the I-A mutant proteins had normal enzyme activity and enhanced ADP-ribose binding. Despite only demonstrating increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 viruses were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication.

Importance: The conserved coronavirus (CoV) macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases and is critical for CoV replication and pathogenesis. As such, Mac1 is a potential therapeutic target for CoV-induced disease. However, we lack a basic knowledge of how several residues in its ADP-ribose binding pocket contribute to its biochemical and virological functions. We engineered mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and viruses for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Interestingly, a Mac1 isoleucine-to-alanine mutant protein had enhanced ADP-ribose binding which proved to be detrimental for virus replication, indicating that this isoleucine controls ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.

Rabbit hemorrhagic disease virus (RHDV) poses a significant threat to rabbits, causing substantial economic losses in rabbit farming. The virus also endangers wild populations of rabbit species and the predatory animals that rely on rabbits as a food source, thereby disturbing the ecological balance. However, the structural understanding of RHDV has been limited due to the lack of high-resolution structures. Here, we present the first high-resolution cryo-EM structures of the mature virion and virus-like particles (VLPs) derived from both full-length and N-terminal arm (NTA)-truncated VP60. These structures reveal intricate structural details of the icosahedral capsid and crucial NTA-mediated interactions essential for capsid assembly. In addition, dramatic conformational differences are unexpectedly observed between the mature virion and VLP. The protruding spikes of the A-B dimers adopt a "raised" state in the mature virion and a "resting" state in the VLP. These findings enhance our understanding of the structure, assembly, and conformational dynamics of the RHDV capsid, laying the essential groundwork for further virological research and therapeutic advancements.IMPORTANCERHDV is a pathogen with significant economic and ecological impact. By presenting the first high-resolution cryo-EM structures of RHDV, we have uncovered detailed interactions among neighboring VP60 subunits of the icosahedral capsid. The NTA of VP60 is uniquely clustered around the threefold axis of the capsid, probably play a critical role in dragging the six VP60 dimers around the threefold axis during capsid assembly. Additionally, we observed dramatic conformational differences between the mature virion and VLPs. VLPs are commonly used for vaccine development, under the assumption that their structure closely resembles that of the mature virion. Our findings significantly advance the understanding of the RHDV capsid structure, which may be used for developing potential therapeutic strategies against RHDV.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (k_cat/K_m) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC₅₀) values of 247.5 µM and 3.8 µM. In both instances, the IC₅₀ obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Toxin/antitoxin (TA) systems are present in nearly every prokaryotic genome and play the important physiological roles of phage inhibition by reducing metabolism (this includes persistence for the extreme case of complete cessation of metabolism), genetic element stabilization, and biofilm formation. TA systems have also been incorporated into other cell systems, such as CRISPR-Cas and phage quorum sensing. For the simplest and best-studied case, proteinaceous toxins and antitoxins (i.e., type II), toxin activity is masked by direct binding of the antitoxin. A long-standing, unresolved question in the TA field is how toxins are activated when bound to antitoxins at nanomolar affinity. The current paradigm envisions preferential degradation of the antitoxin by a protease, but this is highly unlikely in that a protease cannot discriminate between bound toxin and bound antitoxin because both are highly structured. Strikingly, recent results from several studies show one likely mechanism for toxin activation is conformational changes in the TA complex that result in the release or activation of the toxin as a result of a protein trigger, such as that from phages, and as a result of thermally-driven refolding dynamics.

Defective viral genomes (DVGs) emerge during error-prone replication of viral genomes and contain deletions, insertions, genomic rearrangements, and hypermutations. These large-effect mutations result in the inability of DVGs to complete an infectious cycle in the absence of a helper wild-type virus. It has been shown that in vitro DVGs usually accumulate in viral populations when a virus is serially passaged in the same host at a high multiplicity of infection. To investigate the impact of host-to-host transmission on DVG formation and population dynamics in vivo, we conducted evolution experiments with tomato black ring virus (TBRV). TBRV was sequentially passaged through a combination of four distinct host species: quinoa, tobacco, lettuce, and spinach. The host was changed every fifth passage. The diversity and population dynamics of DVGs were analyzed based on the RNA-Seq data obtained through sequencing of viral RNA after 20 passages. Our findings indicate the possibility of TBRV DVGs generation when the virus was passaged through different host species. The level of DVG abundance varied across host plant combinations, with a weak indication that the host species past sequence may play a role in DVGs generation. Most abundant DVGs in the TBRV evolved populations were derived from RNA1. Deletions were the most prevalent class of DVGs, followed by insertions. The deletion DVG subpopulation exhibited substantial diversity in species composition and the richness of the deletions species was correlated with their abundance. Longer DVGs characterized by small deletions were predominant, whereas those shorter than 1,000 nucleotides constituted less than 2%.

Importance: Defective viral genomes (DVGs) have been identified in vivo and in vitro for different virus species infecting humans, animals, and plants. The ability to form DVGs during the passaging of virus in one host has been demonstrated, i.e., for tomato black ring virus (TBRV). In our research, RNA-Seq data obtained after TBRV passaging through a combination of four distinct host species were analyzed. Our results indicate that the level of DVG abundance varied across host plant combinations. Deletions were the most prevalent class of DVGs, with the domination of longer species. Additionally, the conserved junction sites in the TBRV genome were identified, resulting in the generation of identical deletions in independently evolved viral lineages. In summary, our findings provide significant insights into the origin and structure of DVGs of plant viruses. The obtained results will help in understanding viral evolution and host-virus interactions.

Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with fivefold to sixfold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a fivefold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.IMPORTANCEUpdates to COVID-19 vaccine antigens depend on assessing how much vaccine antigens differ antigenically from newer SARS-CoV-2 variants. Human sera from single variant infections are ideal for discriminating antigenic differences among variants, but such primary infection sera are now rare due to high population immunity. It remains unclear whether sera from experimentally infected animals could substitute for human sera for antigenic assessments. This report shows that neutralization titers of variant-matched human and hamster primary infection sera correlate well and recognize variants similarly, indicating that hamster sera can be a proxy for human sera for antigenic assessments. We further show that human sera following an XBB.1.5 booster vaccine broadly neutralized XBB sub-lineage variants but titers were fivefold lower against the more recent JN.1 variant. These findings support updating the current COVID-19 vaccine variant composition and developing a framework for assessing antigenic differences in future variants using hamster primary infection sera.

Cyclophilin A (CypA) binds to the HIV-1 capsid to facilitate reverse transcription and nuclear entry and counter the antiviral activity of TRIM5α. Interestingly, recent studies suggest that the capsid enters the nucleus of an infected cell and uncoats prior to integration. We have previously reported that the capsid protein regulates HIV-1 integration. Therefore, we probed whether CypA-capsid interaction also regulates this post-nuclear entry step. First, we challenged CypA-expressing (CypA^+/+) and CypA-depleted (CypA^-/-) cells with HIV-1 and quantified the levels of provirus. CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. In addition, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited proviral integration in CypA^+/+ cells but not in CypA^-/- cells. HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at the integration step in CypA^+/+ cells but not in CypA^-/- cells. Then, to understand the mechanism, we assessed the integration activity of the HIV-1 preintegration complexes (PICs) extracted from acutely infected cells. PICs from CypA^-/- cells retained lower integration activity in vitro compared to those from CypA^+/+ cells. PICs from cells depleted of both CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC was independent of TRIM5α. Finally, CypA protein specifically stimulated PIC activity, as this effect was significantly blocked by CsA. Collectively, these results provide strong evidence that CypA directly promotes HIV-1 integration, a previously unknown role of this host factor in the nucleus of an infected cell.

Importance: Interaction between the HIV-1 capsid and host cellular factors is essential for infection. However, the molecular details and functional consequences of viral-host factor interactions during HIV-1 infection are not fully understood. Over 30 years ago, Cyclophilin A (CypA) was identified as the first host protein to bind to the HIV-1 capsid. Now it is established that CypA-capsid interaction promotes reverse transcription and nuclear entry of HIV-1. In addition, CypA blocks TRIM5α-mediated restriction of HIV-1. In this report, we show that CypA promotes the post-nuclear entry step of HIV-1 integration by binding to the viral capsid. Notably, we show that CypA stimulates the viral DNA integration activity of the HIV-1 preintegration complex. Collectively, our studies identify a novel role of CypA during the early steps of HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.

Human-to-human transmission of the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) is currently inefficient. However, there is concern that the virus might mutate and thereby increase its transmissibility and thus pandemic potential. The pandemic SARS-CoV-2 depends on a highly cleavable furin motif at the S1/S2 site of the viral spike (S) protein for efficient lung cell entry, transmission, and pathogenicity. Here, by employing pseudotyped particles, we investigated whether augmented cleavage at the S1/S2 site also increases MERS-CoV entry into Calu-3 human lung cells. We report that polymorphism T746K at the S1/S2 cleavage site or optimization of the furin motif increases S protein cleavage but not lung cell entry. These findings suggest that, unlike what has been reported for SARS-CoV-2, a highly cleavable S1/S2 site might not augment MERS-CoV infectivity for human lung cells.IMPORTANCEThe highly cleavable furin motif in the spike protein is required for robust lung cell entry, transmission, and pathogenicity of SARS-CoV-2. In contrast, it is unknown whether optimization of the furin motif in the spike protein of the pre-pandemic MERS-CoV increases lung cell entry and allows for robust human-human transmission. The present study indicates that this might not be the case. Thus, neither a naturally occurring polymorphism that increased MERS-CoV spike protein cleavage nor artificial optimization of the cleavage site allowed for increased spike-protein-driven entry into Calu-3 human lung cells.