Journal of Virology最新文献

Capsidless yadokariviruses (members of the order Yadokarivirales) with (+)RNA genomes divert the capsid of their partner icosahedral double-stranded RNA (dsRNA) viruses in different families of the order Ghabrivirales into the replication site. A yadokarivirus, AfSV2, has been reported from a German strain of the ascomycete fungus Aspergillus foetidus coinfected by two dsRNA viruses, a victorivirus (AfSV1, family Pseudototiviridae) and an alternavirus (AfFV, family Alternaviridae). Here, we identified AfSV1 as the partner of AfSV2 in a Japanese A. foetidus strain after showing the infectiousness of AfSV2 in three forms: virus particles (heterocapsid), transforming full-length complementary DNA (cDNA), and purified replicated form (RF) dsRNA that is believed to be inactive as a translational template. Virion transfection of virus-free A. foetidus protoplasts resulted in the generation of two strains infected either by AfSV1 alone or by both AfSV1 and AfSV2. Transformants with AfSV2 full-length cDNA launched AfSV2 infection only in the presence of AfSV1, but not those with AfSV2 RNA-directed RNA polymerase mutant cDNA. The purified fractions containing AfSV2 RF dsRNA also launched infection when transfected into protoplasts infected by AfSV1. Treatment with dsRNA-specific RNase III, but not with proteinase K, S1 nuclease, or DNase I, abolished the infectivity of AfSV2 RF dsRNA. Furthermore, we confirmed the infectiousness of gel-purified AfSV2 RF dsRNA in the presence of AfSV1. Taken together, our results show the unique infectious entity of AfSV2 and the expansion of yadokarivirus partners in the family Pseudototiviridae and provide interesting evolutionary insights.IMPORTANCEThe viral phylum Pisuviricota accommodates members with both double-stranded RNA (dsRNA) and (+)RNA genomes. Some members of the second group display peculiar virus lifestyles. These include (+)RNA yadkariviruses, which are capsidless and highjack the capsid of their partner dsRNA viruses in the order Ghabrivirales of a different phylum Duplornaviricota. We identified the partner dsRNA virus (AfSV1, a victorivirus) of a yadokarivirus (AfSV2) from the ascomycete Aspergillus foetidus. AfSV2 is infectious in the presence of AfSV1 in three forms: purified particles, transforming full-length complementary DNA, and, surprisingly, the purified replicative form dsRNA. These combined results expand yadokarivirus partner viruses to the family Pseudototiviridae and provide evidence for AfSV2 as a unique infectious entity as well as interesting evolutionary insights.

The efficacy of the G-protein is influenced by N-linked glycosylation, which serves as the sole immunogen of the rabies virus vaccine. However, achieving satisfactory immune-protection efficacy remains challenging, owing to the heterogeneous glycosylation of G-proteins. Within molecular dynamics, examining the impact of N-glycan heterogeneity on the structural characteristics of G-proteins provides insights into the relationship between antigens and the efficacy of rabies virus vaccines. Glycosylation is regulated by host cells. In rabies virus cultured in Vero cells (VRV), all N-glycosylation sites of the G-protein underwent modification. In contrast, rabies virus G-protein cultured in KMB17 cells (human diploid cell vaccine [HDCV]) was only modified by N-glycans at amino acid positions 247 and 319. Furthermore, treatment of VRV with de-glycosylation significantly improved its immune-protective efficacy, whereas de-glycosylation did not alter the immune-protective efficacy of HDCV. To support the impact of glycosylation on VRV efficacy, the structures and dynamics of G-proteins were analyzed using GROMACS. Specifically, the hydrophobicity, flexibility, and radius of gyration of the G-protein trimer in VRV were significantly altered by excessive hydrogen bonds formed by the three-branched hybrid glycan at the aa 319 site. These changes increase the instability of the G-protein trimer and may lead to a decrease in vaccine protective efficacy. Ultimately, we determined that N-glycan heterogeneity affects the immune-protection effect of antigen proteins by altering their dynamic characteristics, enhancing our understanding of the correlation between antigen structural characteristics and efficacy.

Importance: N-glycosylation of rabies virus glycoprotein dynamically regulates protein folding, stability, and antigenicity. Therefore, regulation of N-glycan modification is key to improving vaccine stability and protective efficacy. How the type and modification sites of N-glycans affect the protective efficacy of rabies vaccines remains unclear. Our research indicates that there are differences in the protective efficacy of rabies virus G-proteins modified with different N-glycans. Moreover, the modification of the three-branched hybrid glycan at the aa 319 site of G-protein significantly altered the hydrophobicity, flexibility, and radius, and increased its trimeric antigen instability through molecular dynamics demonstrations. These findings update the current understanding of the impact of glycans on vaccine antigenicity and develop a system to evaluate the stability of antigen glycoproteins based on molecular dynamics.

Pre-existing T-cell responses have been linked to reduced disease severity and better clinical outcomes during the 2009 influenza pandemic and the recent COVID-19 pandemic. We hypothesized that diversifying T-cell responses, particularly targeting conserved viral proteins such as the influenza A virus (IAV) nucleoprotein (NP), could protect against both epidemic and pandemic IAV strains. To test this, we created a mosaic nucleoprotein (MNP) by synthesizing a sequence that maximized the representation of 9-mer epitopes from 7422 NP sequences across human, swine, and avian IAVs. Notably, the MNP sequence showed high homology with the NP of the H5N1 strain affecting dairy cows in the ongoing outbreak. Mucosal immunization with the adjuvanted MNP vaccine induced robust CD8 and CD4 T-cell responses against both known immunodominant and in silico predicted subdominant epitopes. MNP-vaccinated mice challenged with epidemic H1N1 and H3N2 strains, which shared immunodominant CD8 and/or CD4 T-cell epitopes, showed a significant (~4 log) reduction in lung viral load. Importantly, MNP-vaccinated mice challenged with a pandemic H1N1 strain lacking shared immunodominant CD8 or CD4 epitopes exhibited a superior reduction in lung viral load, linked to T-cell responses targeting subdominant epitopes present in both the MNP and pandemic strain NP. These results suggest that a diversified T-cell response induced by the MNP vaccine could provide broad protection against severe disease from both current and emerging IAV strains.

Importance: The World Health Organization (WHO) estimates that seasonal influenza causes 3-5 million cases of severe illness annually. The influenza virus frequently undergoes genetic changes through antigenic drift and antigenic shift, resulting in annual epidemics and occasional pandemics. Consequently, a major public health objective is to develop a universal influenza vaccine that offers broad protection against both current and pandemic influenza A strains. In this study, we designed a nucleoprotein (NP) antigen (termed mosaic NP) comprising antigenic regions found in thousands of influenza viruses, aiming to use it as a vaccine to induce broad anti-influenza T-cell responses. Our findings indicate that the mosaic NP vaccine provided significant protection against seasonal H1N1 and H3N2, as well as the pandemic H1N1 strain, demonstrating its effectiveness across various influenza subtypes. These findings suggest that the mosaic NP is a potential universal influenza vaccine antigen, capable of protecting against diverse strains of influenza viruses.

Virion formation and egress are sophisticated processes that rely on the spatial and temporal organization of host cell membranes and the manipulation of host machineries involved in protein sorting, membrane bending, fusion, and fission. These processes result in the formation of infectious virions, defective particles, and various vesicle-like structures. In herpes simplex virus 1 (HSV-1) infections, virions and capsid-less particles, known as light (L)-particles, are formed. HSV-1 infection also stimulates the release of particles that resemble extracellular vesicles (EVs). In productively infected cells, most EVs are generated through the CD63 tetraspanin biogenesis pathway and lack viral components. A smaller subset of EVs, generated through the endosomal sorting complexes required for transport (ESCRT) pathway, contains both viral and host factors. Viral mechanisms tightly regulate EV biogenesis, including the inhibition of autophagy-a process critical for increased production of CD63+ EVs during HSV-1 infection. Mutant viruses that fail to suppress autophagy instead promote microvesicle production from the plasma membrane. Additionally, the viral protein ICP0 (Infected Cell Protein 0) enhances EV biogenesis during HSV-1 infection. The different types of particles can be separated by density gradients due to their distinct biophysical properties. L-particles and ESCRT+ EVs display a pro-viral role, supporting viral replication, whereas CD63+ EVs exhibit antiviral effects. Overall, these studies highlight that HSV-1 infection yields numerous and diverse particles, with their type and composition shaped by the ability of the virus to evade host responses. These particles likely shape the infectious microenvironment and determine disease outcomes.

In a subset of SARS-CoV-2-infected individuals treated with the antiviral nirmatrelvir-ritonavir, the virus rebounds following treatment. The mechanisms driving this rebound are not well understood. We used a mathematical model to describe the longitudinal viral load dynamics of 51 individuals treated with nirmatrelvir-ritonavir, 20 of whom rebounded. Target cell preservation, either by a robust innate immune response or initiation of N-R near the time of symptom onset, coupled with incomplete viral clearance, appears to be the main factor leading to viral rebound. Moreover, the occurrence of viral rebound is likely influenced by the time of treatment initiation relative to the progression of the infection, with earlier treatments leading to a higher chance of rebound. A comparison with an untreated cohort suggests that early treatments with nirmatrelvir-ritonavir may be associated with a delay in the onset of an adaptive immune response. Nevertheless, our model demonstrates that extending the course of nirmatrelvir-ritonavir treatment to a 10-day regimen may greatly diminish the chance of rebound in people with mild-to-moderate COVID-19 and who are at high risk of progression to severe disease. Altogether, our results suggest that in some individuals, a standard 5-day course of nirmatrelvir-ritonavir starting around the time of symptom onset may not completely eliminate the virus. Thus, after treatment ends, the virus can rebound if an effective adaptive immune response has not fully developed. These findings on the role of target cell preservation and incomplete viral clearance also offer a possible explanation for viral rebounds following other antiviral treatments for SARS-CoV-2.

Importance: Nirmatrelvir-ritonavir is an effective treatment for SARS-CoV-2. In a subset of individuals treated with nirmatrelvir-ritonavir, the initial reduction in viral load is followed by viral rebound once treatment is stopped. We show that the timing of treatment initiation with nirmatrelvir-ritonavir may influence the risk of viral rebound. Nirmatrelvir-ritonavir stops viral growth and preserves target cells but may not lead to full clearance of the virus. Thus, once treatment ends, if an effective adaptive immune response has not adequately developed, the remaining virus can lead to rebound. Our results provide insights into the mechanisms of rebound and can help develop better treatment strategies to minimize this possibility.

Disulfide exchange is underexplored as a mechanism influencing HIV-1 entry. Prior studies demonstrated that redox enzyme inhibition can prevent HIV-1 infection but with limited mechanistic explanation. We hypothesize that ligand-driven rearrangement ("conformational activation") enables enzyme-mediated disulfide exchange in Env residues ("disulfide trigger") that promotes fusion transformations, enhancing virus entry. We tested soluble CD4 and CD4-binding site entry inhibitors as conformational activators and the ubiquitous redox enzyme thioredoxin-1 (Trx1) as disulfide trigger. We found that combination treatment caused fusion-like Env transformation and pseudovirus lysis, independent of cells. Notably, only compounds associated with gp120 shedding caused lysis when paired with Trx1. In each case, lysis was prevented by adding the fusion inhibitor T20, demonstrating that six-helix bundle formation is required as in virus-cell fusion. In contrast to conformationally activating ligands, neither the ground state stabilizer BMS-806 with Trx1 nor Trx1 alone caused lysis. Order of addition experiments reinforced conformational activation/disulfide trigger as a sequential process, with virus/activator preincubation transiently enhancing lysis and virus/Trx1 preincubation reducing lysis. Lastly, addition of exogenous Trx1 to typical pseudovirus infections exhibited dose-dependent enhancement of infection. Altogether, these data support conformational activation and disulfide triggering as a mechanism that can induce and enhance the fusogenic transformation of Env.IMPORTANCEHIV remains a global epidemic despite effective anti-retroviral therapies (ART) that suppress viral replication. Damage from early-stage infection and immune cell depletion lingers, as ART enables only partial immune system recovery, making prevention of initial virus entry preferable. In this study, we investigate disulfide exchange and its facilitating conformational rearrangements as underexplored, but critical, events in the HIV entry process. The HIV envelope (Env) protein effects cell entry by conformational rearrangement and pore formation upon interaction with immune cell surface proteins, but this transformation can be induced by Env's conformational activation and disulfide exchange by redox enzymes, which then integrates into established processes of HIV entry. The significance of this research is in identifying Env's conformational activation as a mechanistic requirement for initiating fusion by triggering disulfide exchange. This will aid the development of novel preventative strategies against HIV entry, particularly in the context of HIV-enhanced inflammation and comorbidities with redox mechanisms.

Seneca Valley virus (SVV), also known as Senecavirus A, a porcine pathogen that causes vesicular diseases, is prevalent in pig herds worldwide. SVV infection induces endoplasmic reticulum (ER) stress in PK-15 and BHK-21 cells, accompanied by activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) pathways, which in turn facilitates SVV replication. ER stress is associated with the regulation of Ca²⁺ homeostasis and mitochondrial apoptosis. However, the precise role of Ca²⁺ in SVV-induced apoptosis remains unclear. In this study, western blotting, flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) detection revealed that either ER stress or the PERK pathway is involved in the apoptosis of SVV-infected cells treated with specific inhibitors. Furthermore, SVV-mediated ER stress markedly contributed to the transfer of Ca²⁺ from the ER to mitochondria. The subsequent increase in mitochondrial Ca²⁺ content was accompanied by an increased number of ER membranes near the mitochondria. Finally, the inhibition of mitochondrial Ca²⁺ overload, ER stress, and the PERK pathway substantially attenuated SVV-mediated mitochondrial dysfunction, as evidenced by analyzing mitochondrial membrane potential (MMP), mitochondrial permeability transition poremPTP, reactive oxygen speciesROS, and adenosine 5'-triphosphate ATP, and the levels of mitochondrial apoptosis. These findings demonstrate that SVV induces mitochondrial apoptosis, which is dependent on ER stress-mediated transmission of Ca²⁺ from the ER to the mitochondria.

Importance: Viruses have developed multiple mechanisms to facilitate their proliferation or persistence through manipulating various organelles in cells. Seneca Valley virus (SVV), as a novel emerging pathogen associated with vesicular disease, is clinically and economically important infections that affect farm animals. Previously, we had confirmed that SVV-induced endoplasmic reticulum (ER) stress benefited for viral replication. Ca²⁺, as an intracellular signaling messenger mainly stored in the ER, is regulated by ER stress and then involved in apoptosis. However, the precise mechanism that Ca²⁺ transfer induced by SVV infection triggered apoptosis remained unclear. Here, we found that SVV infection triggered the Ca²⁺ transform from ER to mitochondria, resulting in mitochondrial dysfunction, and finally induced mitochondrial apoptosis. Our study shed light on a novel mechanism revealing how ER stress manipulates Ca²⁺ homeostasis to induce mitochondrial apoptosis and regulate viral proliferation.

Potyviruses possess one positive-sense single-stranded RNA genome, mainly dependent on polyprotein processing as the expression strategy. The resulting polyproteins are proteolytically processed by three virus-encoded proteases into 11 or 12 mature proteins. One such factor, 6 kDa peptide 1 (6K1), is an understudied viral factor. Its function in viral infection remains largely mysterious. This study is to reveal part of its roles by using pepper veinal mottle virus (PVMV) as the model. Alanine substitution screening analysis revealed that 15 of 17 conserved residues across potyviral 6K1 sequences are essential for PVMV infection. However, 6K1 protein is less accumulated in virus-infected cells, although P3-6K1 and 6K1-CI junctions are efficiently processed by NIa-Pro for its release, indicating that 6K1 undergoes a self-degradation event. Mutating the cleavage site to prevent NIa-Pro processing abolishes viral infection, suggesting that the generation of 6K1 along with its degradation might be important for viral multiplication. We corroborated that cellular autophagy is engaged in 6K1's degradation. Individual engineering of the 15 6K1 variants into PVMV allows their expression along with viral infection. Five of such variants, D30A, V32A, K34A, L36A, and L39A, significantly interfere with viral infection. The five residues are enclosed in a conserved lysine/arginine-rich motif; four of them appear crucial in engaging autophagy-mediated self-degradation. Based on these data, we envisaged a scenario which potyviral 6K1s interact with an unknown anti-viral component to be co-degraded by autophagy to promote viral infection.IMPORTANCEPotyvirus is the largest genus of plant-infecting RNA viruses, which encompasses socio-economically important virus species, such as Potato virus Y, Plum pox virus, and Soybean mosaic virus. Like all picorna-like viruses, potyviruses express their factors mainly via polyprotein processing. Theoretically, viral factors P3 through CP, including 6K1, should share an equivalent number of molecules. The 6K1 is small in size (~6 kDa) and conserved across potyviruses but less accumulated in virus-infected cells. This study demonstrates that cellular autophagy is engaged in the degradation of 6K1 to promote viral infection. In particular, we found a conserved lysine/arginine-rich motif in 6K1s across potyviruses that is engaged in this degradation event. This finding reveals one facet of a small protein that helps understand the pro-viral role of cellular autophagy in viral infection.

Influenza A viruses with fewer amino acids in the neuraminidase (NA) stalk domain are primarily isolated from chickens rather than wild ducks, indicating that a shortened NA stalk is considered an adaptation marker of avian influenza viruses (AIVs) to chickens. Experimental passages of an H7N7 nonpathogenic AIV (rgVac2-P0) in chickens resulted in a highly pathogenic variant (Vac2-P3L4) with a 34-amino-acid deletion in the NA stalk, encompassing five potential N-glycosylation sites. To investigate how amino acid truncation and deglycosylation in the NA stalk contribute to increased pathogenicity, a virus with glycosylation-deficient mutations at these sites (rgVac2-P3L4/P0NAΔGlyco) was constructed. Contrary to expectations, chickens inoculated with rgVac2-P3L4/P0NAΔGlyco exhibited variable clinical outcomes, attributed to the genetic instability of the virus. A single mutation stabilized the virus, and the mutant (rgVac2-P3L4/P0NAΔGlyco-Y65H) resulted in higher pathogenicity compared with a virus with restored glycosylation (rgVac2-P3L4/P0NA-Y65H). Glycan occupancy analysis revealed 3-4 glycans at the five potential sites. In functional analysis, glycosylation-deficient mutants, similar to the short-stalk NA virus, showed significantly reduced erythrocyte elution activity. Additionally, mutational analysis indicated variable contributions of N-glycans to elution activity across the sites. Moreover, the functionally most contributing sites of the five potential N-glycosylation motifs were consistently included in the amino acid deletions of the stalk-truncated NA in N7-subtyped field isolates, despite the varying truncation position or length. These findings suggest that the loss of glycosylation is functionally equivalent to a reduction in amino acids, and it plays a crucial role in enhancing pathogenicity in chickens and affecting NA function.IMPORTANCEAvian influenza poses significant economic challenges to the poultry industry and presents potential risks to human health. Understanding the molecular mechanisms that facilitate the emergence of chicken-adapted avian influenza viruses (AIVs) from non-pathogenic duck-origin influenza viruses is crucial for improving AIV monitoring systems in poultry and controlling this disease. Amino acid deletions in the neuraminidase (NA) stalk domain serve as one of the molecular markers for AIV adaptation to Galliformes. This study highlights the critical role of N-glycosylation in the NA stalk domain in the pathogenesis of high pathogenicity avian influenza viruses in chickens. The findings propose a novel theory that the loss of glycosylation at the NA stalk domain, rather than a reduction in stalk length, is responsible for both NA function and increased virus pathogenicity in chickens.

Zika virus (ZIKV) is associated with microcephaly in neonates and neurological disorders in adults. Chronic ZIKV infection has been identified in the testes, indicating that the virus can lead to prolonged illness, yet its pathogenesis remains poorly understood. Here, we found that ZIKV infection does not induce significant cell death in mouse macrophages despite the critical role that cell death plays in the antiviral immune response. Furthermore, we discovered that ZIKV infection impairs the activation of the NLPR3-dependent inflammasome and inhibits apoptosis. Consequently, we investigated the regulatory mechanism of the NLRP3 inflammasome and apoptosis in the context of ZIKV infection. Our results revealed significant reductions in the protein expression levels of NLRP3 and A20, attributable to post-transcriptional or translational effects during ZIKV infection. These findings suggest that ZIKV infection may disrupt cell death pathways, leading to its pathogenicity.IMPORTANCEZika virus (ZIKV), first isolated from a nonhuman primate in Africa in 1947, was relatively understudied until 2016. By then, ZIKV had already been reported in more than 20 countries and territories. The infection poses a significant risk, as it is associated with microcephaly in infants and neurological disorders in adults; however, the underlying mechanisms responsible for these severe outcomes remain unclear. In this study, we demonstrate that ZIKV infection significantly reduces the expression of NLRP3 and A20 proteins through post-transcriptional or translational processes, which leads to inhibited cell death. These findings are critical because cell death plays a vital role in the host's antiviral immune response. Our findings highlight how ZIKV infection compromises essential cell death pathways, raising serious concerns about its pathogenesis. A comprehensive understanding of this disruption is vital for developing targeted interventions to mitigate the virus' impact on public health.