Journal of Virology最新文献

Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic alphavirus. Members of the Alphavirus genus are found across the globe, transmitted by arthropod vectors, and cause significant disease burdens in humans and animals. There are currently no FDA-approved antivirals for human use against any member of the Alphavirus genus. While a vaccine exists against chikungunya virus (CHIKV), a member of the arthritogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus, and Western equine encephalitis virus. 4'-Fluorouridine (4'-FlU, EIDD-2749) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. 4'-FlU can potently inhibit VEEV-TC83 replication, with submicromolar potency in cell culture. However, the emergence of antiviral resistance represents a hurdle for antiviral drug development and the implementation of effective treatment strategies. Here, we have identified novel mutations in the VEEV nsP4 RNA-dependent RNA polymerase that reduce susceptibility to 4'-FlU, including P187A, Q191L, L289F, and T296I. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of these mutations on fitness and pathogenicity. In addition, we assessed the impact of mutations reducing sensitivity to 4'-FlU in a mouse model. Although mutations against 4'-FlU arise quickly in vitro, treatment can still alleviate severe disease and lethal encephalitis. Together, these data highlight the promising therapeutic potential of 4'-FlU for the treatment of alphavirus encephalitis.IMPORTANCEVenezuelan equine encephalitis virus (VEEV) is a mosquito-spread virus that can cause encephalitis in people and animals. There are no FDA-approved countermeasures to treat VEEV infections in humans. 4'-Fluorouridine (4'-FlU) is currently being developed to treat multiple viral infections, including VEEV. A major problem with antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we treated infected mice with 4'-FlU and measured how well the compound inhibited virus replication and prevented severe disease. In addition, we identified mutations in VEEV's polymerase that confer reduced susceptibility to 4'-FlU. We then assessed if viruses encoding for these mutations were still pathogenic. Although VEEV can develop mild resistance to 4'-FlU in vitro, administration of 4'-FlU still reduced severe disease and prevented lethality in the animals infected with viruses that possess mutations that decrease susceptibility to 4'-FlU. These results suggest that 4'-FlU has strong potential as a future treatment for alphavirus infections.

Antagonism of the host responses that limits viral replication is critical to the success of infection. Recently, we identified that the hypervariable region (HVR) of SARS-CoV-2 NSP3 binds to FXR1 and disrupts stress granule formation during the early stages of infection. Despite variation across the rest of the HVR, a 20-amino acid region, highly conserved in the Sarbecovirus family, is required for NSP3-FXR1 binding, but the critical residues remained unresolved. In this study, we explore the individual residues in NSP3 driving FXR1 binding and determine their impact on viral replication, pathogenesis, and stress granule formation. Our results indicate that the tyrosine at position 138 (Y138) and a phenylalanine at position 145 (F145) are required for FXR1 binding and affinity. Using reverse genetics, we showed that mutating NSP3 Y138A/F145A (YF mutant) reduced viral replication in vitro and in vivo. Importantly, we demonstrate that attenuation is not due to differential type I interferon responses but rather loss of stress granule control by the NSP3 mutant as compared to wild type. Together, our findings demonstrate the importance of Y138 and F145 within the NSP3-HVR in regulating stress granule formation at the early times post-infection.IMPORTANCEStress granules play a key role in host-antiviral defenses, and viruses have developed strategies to antagonize their activity. For SARS-CoV-2, the virus has two proteins that antagonize stress granules, with NSP3 acting early and nucleocapsid acting at late times. Here, we show that key NSP3 residues Y138 and F145, conserved across the Sarbecovirus family, are necessary to bind FXR1 and disrupt its activity in stress granule formation. Mutating these residues results in attenuation of SARS-CoV-2 replication and induces stress granule formation at early times post-infection. These results show the importance of these NSP3 residues in disrupting stress granule formation early and highlight multiple approaches SARS-CoV-2 uses to antagonize stress granule activation.

Genus beta (β) human papillomaviruses (HPVs) potentially contribute to the development of non-melanoma skin cancer. Yet, comparatively little is known about their biology. In particular, details about initial infection, i.e., host cell entry, remain mostly elusive. During initial characterization of β HPV5 pseudovirion (PsV) preparations, surprisingly large amounts of filamentous particles were found besides the prototypical icosahedral (T = 7) virions. Whether these filamentous particles actively contribute to or interfere with infectivity of the spherical viruses is unknown. Using a combination of morphological, biochemical, and virological methods, we showed that the filamentous particles are non-infectious. Moreover, they interfered with the initial step of infection, i.e., binding to cellular heparan sulfate proteoglycans (HSPGs), and served as a decoy for soluble glycosaminoglycans, thereby modulating infectivity by enhancing infectious PsV binding. This explains previous seemingly contradictory findings on HPV5 binding to HSPGs. Importantly, in HPV5 skin warts from an immunocompromised patient, no filamentous particles were observable highlighting differences in the assembly of pseudovirions and native viruses.IMPORTANCEPapillomaviruses contribute to numerous cancer incidents and significant mortality despite available vaccinations. Hence, high-risk α HPVs have been the focus of most research in the past. However, there are indications that less well-studied β HPVs may also contribute to certain malignancies. Little is known about their mode of cell invasion, and available data appear partially contradictory. Our work demonstrated that HPV5 as a model β HPVs yielded high amounts of non-infectious filamentous particles during PsV production. These acted as modulators of infection by the infectious spherical particles. Removing these filamentous particles showed that HPV5 engaged HSPGs as the primary receptor for cell binding, similar to high-risk α HPV, indicating a conserved feature not only among α, but also among β HPVs, thereby explaining previous contradictions.

Emerging and re-emerging swine viral infectious diseases impose substantial economic burdens. Additionally, swine, which frequently interact with humans, may facilitate virus evolution, posing a risk to public health security. Consequently, there is a pressing need to develop safe, effective, and rapid vaccine platforms, with vector vaccine being a viable option. In this study, we utilized the enhanced green fluorescent protein (eGFP) as an exogenous reporter to investigate the intergenic regions of the PIV5-JS17 strain for expressing exogenous proteins. These regions included N-P, P-M, M-F, F-SH, SH-HN, and HN-L. Our findings revealed that exogenous gene expression varied at different positions, with the expression cassette containing the non-coding sequence within the P-M intergenic region achieving the highest eGFP fluorescence intensity. Then, we successfully established a porcine infection model through oral administration of the recombinant virus, identified the target organs infected, and verified the safety of the viral vector in swine. Furthermore, using porcine deltacoronavirus (PDCoV)-S protein as a model antigen, it was demonstrated that the recombinant virus triggered a robust humoral and cellular immune responses. In conclusion, we have developed a novel oral gene delivery system for swine, providing insights and guidance for the design of vector vaccines based on the newly discovered porcine PIV5, the selection of appropriate exogenous gene insertion sites, and vaccine delivery strategies.IMPORTANCEThe research presented in this paper hinges on the fortunate isolation of the PIV5-JS17 strain from the intestines of pigs. Given the pressing demand for oral vaccines, the emergence of this novel PIV5 strain capable of infecting both the respiratory and intestinal tracts has sparked our interest in developing it as a vector vaccine. Utilizing eGFP as a model exogenous gene, our findings reveal that the P/M intergenic region serves as the optimal site for the insertion of exogenous genes. Using the PDCoV-S protein as a model antigen, the study shows that this novel porcine-derived PIV5 virus vector presents innovative prevention methods and gene delivery strategies for addressing porcine infectious diseases.

Norovirus is a leading cause of acute gastroenteritis worldwide, imposing a major burden on public health and healthcare systems. Despite its significant medical impact, no licensed vaccines or specific antiviral therapies are currently available. Norovirus vaccine development is complicated by several factors, including the extreme genetic and antigenic diversity. In particular, the predominant genotype GII.4 exhibits a continuous emergence of novel variants that can evade immune responses acquired from previous infections. In this manuscript, we will summarize the characteristics and current knowledge of the B cell responses elicited by conserved and variable GII.4 epitopes and discuss how these findings inform our understanding of responses to other pandemic norovirus genotypes. We also highlight how ongoing research in this area may provide critical insights for the development of broadly protective norovirus vaccines.

Viruses are metabolic engineers of host cells. As obligate intracellular pathogens, they rely on host cell metabolism for efficient viral replication. The manipulation of host metabolic processes is a strategy shared among diverse virus families to secure the necessary resources for replicating new genomes, building more virus particles, and supporting cell growth and proliferation. Key metabolic pathways targeted by viruses for disruption and manipulation are glycolysis, glutaminolysis, and lipid metabolism. However, the mechanisms behind virus-induced metabolic reprogramming and the viral proteins mediating it remain poorly understood. This review explores how specific viral proteins reshape the metabolic milieu of host cells during viral infections. We also highlight common themes and outline gaps in knowledge to stimulate further investigations into how viral proteins manipulate host metabolism. Such mechanistic insights will deepen our understanding of virus-host interactions and may reveal novel therapeutic targets.

To counteract host antiviral responses, influenza A virus triggers a global reduction of cellular gene expression, a process termed "host shutoff." A key effector of influenza A virus host shutoff is the viral endoribonuclease PA-X, which degrades host mRNAs. While many of the molecular determinants of PA-X activity remain unknown, a previous study found that N-terminal acetylation of PA-X is required for its host shutoff activity upon ectopic expression. However, it remains unclear how this co-translational modification promotes PA-X activity. Here, we report that PA-X N-terminal acetylation has two functions-it promotes nuclear localization but is also needed directly for host shutoff. Moreover, these two functions can be separated based on whether acetylation occurs on the first amino acid, the initiator methionine, or the second amino acid following initiator methionine excision. Modification at either site is sufficient to ensure PA-X localization to the nucleus, whereas N-terminal acetylation of the initiator methionine is specifically required for normal PA-X host shutoff activity. We also demonstrate that PA-X N-terminal acetylation is needed for its activity during infection. Our studies thus uncover a multifaceted role for PA-X N-terminal acetylation in the regulation of this important immunomodulatory factor.IMPORTANCEInfluenza A viruses pose a significant threat to human health through seasonal epidemics and recurrent pandemics. Our immune and inflammatory responses have a key role in disease outcome. They clear the virus but can also cause lung damage. Influenza A viruses encode factors that modulate these responses, including PA-X, which destroys cellular mRNAs to control immune responses (a phenomenon called "host shutoff"). PA-X is modified with an acetylation at its N-terminus. This modification is needed for its activity, but it has remained unclear why. We show that PA-X N-terminal acetylation ensures that PA-X goes to the nucleus but also separately contributes to host shutoff activity. For host shutoff activity, the specific location of the modification matters, whereas for entry into the nucleus, it does not. These findings uncover how influenza A viruses exploit a widespread protein modification to support the activity of one of their important immunomodulatory proteins.

Porcine epidemic diarrhea virus (PEDV) causes acute intestinal disease in pigs and remains a major threat to the global swine industry due to its high morbidity and mortality in neonatal piglets. To investigate host metabolic alterations upon PEDV infection, we performed untargeted metabolomic profiling in LLC-PK1 and Vero E6 cells. Pathway enrichment analysis revealed significant changes in nucleotide metabolism, cofactor biosynthesis, amino acid biosynthesis, and purine metabolism. Notably, PEDV infection led to divergent regulation of purine metabolism in the two cell types-upregulation in Vero E6 cells and downregulation in LLC-PK1 cells at 18 h post-infection. We further identified inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide biosynthesis, as a critical host factor for PEDV replication. Both genetic knockdown of IMPDH2 and pharmacological inhibition using merimepodib (VX-497, MMPD) significantly reduced viral RNA levels and impaired replication. These treatments also suppressed host nucleotide biosynthetic activity. Together, our findings demonstrate that PEDV hijacks the IMPDH-dependent guanosine biosynthesis pathway to support its replication and identify IMPDH as a promising host-directed antiviral target against PEDV.

Importance: PEDV poses a major global threat to swine health. This study uncovers a key mechanism of pathogenesis: PEDV exploits host nucleotide metabolism, inducing significant reprogramming with emphasis on purine biosynthesis. Comparative infection of porcine (LLC-PK1) and primate (Vero E6) cells revealed cell-specific metabolic adaptations. Crucially, we identify inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme for guanosine biosynthesis, as an essential host dependency factor for PEDV replication. Inhibiting IMPDH genetically or pharmacologically significantly reduced viral titers, validating it as a critical vulnerability. These findings reveal a novel mechanism by which PEDV hijacks host metabolism and establishes IMPDH as a promising host-directed therapeutic target for combating this economically devastating virus.

Copy-back viral genomes (cbVGs) are generated during the replication of negative-sense RNA viruses when the polymerase drops off from the genome and reattaches to the nascent strand. cbVGs have strong immunostimulatory properties and impact infection outcomes. Despite their importance, the composition and mechanisms of de novo cbVG generation and accumulation remain unclear due to challenges in obtaining cbVG-free virus stocks (clean stocks). Here, we obtained several clean stocks by independently rescuing recombinant Sendai virus (SeV) six times and verified their cleanliness through PCR, RNA sequencing, and absence of immunostimulatory activity. High multiplicity-of-infection (MOI) passaging of clean stocks produced six high-MOI passaged stocks, each with distinct cbVG populations. Among them, polymerase drop-off (break) positions occurred throughout the genome, while polymerase reattachment (rejoin) positions preferentially occurred near the trailer end. Few common breaks were observed between stocks, while there was a hot rejoin region near the trailer end. In each stock, a few cbVG species dominated and remained stable across passages, all conforming to the "rule of six," regardless of length. Low-abundance cbVGs were variable across passages, indicating the continuous generation of new cbVGs, despite the stabilization of a subset of species. Intriguingly, cbVG species that originated from polymerase drop-off at or close to nucleotide 1 were present in all stocks, suggesting that cbVG species originating at the 3' end of the genome are conserved products of SeV replication.IMPORTANCECopy-back viral genomes (cbVGs) are generated during infection when the polymerase drops off from the template and reattaches to the nascent strand, and they are major drivers of antiviral immunity. However, natural isolates contain pre-existing cbVGs, limiting our ability to understand how cbVGs are generated and accumulated. Here, we used cbVG clean stocks obtained from cDNA to address these questions. Comparative analysis of six parallel cbVG-high stocks showed that polymerase drop-off sites are broadly distributed across the genome, with a recurrent origin near nucleotide 1, while polymerase reattaches near the trailer end. Longitudinal analysis revealed that dominant cbVG species remain stable across passages of the same stock, whereas some cbVGs are dynamic. cbVG accumulation was independent of cbVG length but strictly followed the rule of six. These findings reveal conserved and variable features of cbVG generation from clean stocks and shed light on how cbVGs accumulate during infection.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, infects the small intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED), which is particularly severe in young piglets. Owing to its strong immunogenicity, the S1 subunit of the PEDV spike (S) protein mediates viral invasion by recognizing host cell receptors and inducing neutralizing antibodies. However, effective antiviral drugs against PEDV are lacking, and current control measures are limited. In this study, the truncated S1 protein was expressed and used in surface plasmon resonance screening of 416 natural compounds. Salvianolic acid A (SalA) exhibited the strongest antiviral activity against PEDV, with a dissociation constant KD of 4.54 × 10⁻⁷ M. Molecular docking revealed multiple hydrogen bonds between SalA and key amino acid residues of the S1 protein. In vitro assays demonstrated that SalA significantly reduced viral RNA copy number, titer, and N protein expression in a dose-dependent manner. SalA inhibited viral adsorption, replication, and release and showed direct virucidal activity. In a piglet challenge model, SalA treatment improved survival, alleviated clinical symptoms and intestinal lesions, and reduced viral loads in blood, feces, and tissues. Overall, SalA was identified as a potent natural compound that targets the PEDV S1 protein and exhibits strong antiviral effects both in vitro and in vivo, highlighting its promise as a therapeutic candidate for PEDV infection.

Importance: Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in piglets, resulting in substantial economic losses to the global swine industry. However, effective antiviral therapeutics are still lacking. In this study, salvianolic acid A (SalA), a natural polyphenolic compound derived from Salvia miltiorrhiza, was identified as a potent inhibitor of PEDV through direct targeting of its spike (S1) protein. SalA efficiently suppressed viral replication, release, and infectivity in vitro and markedly alleviated intestinal damage, viral load, and clinical symptoms in infected piglets. Molecular docking and dynamic simulations further confirmed the stable binding between SalA and the S1 protein. Overall, this study provides the first comprehensive experimental evidence that SalA exhibits both prophylactic and therapeutic antiviral activities against PEDV, clearly highlighting its potential as a promising lead compound for the development of effective antiviral drugs to control PEDV and related coronavirus infections in livestock.