Journal of Virology最新文献

Enteroviral 3C protease (3Cpro) is an essential enzyme for viral replication and is responsible for combating the host anti-viral immune response by targeting cellular proteins for cleavage. The identification and characterization of 3Cpro substrates will contribute to our understanding of viral pathogenesis. In this study, we performed a motif search for 3Cpro substrates in the human protein database using FIMO, which refers to a common cleavage sequence of 3Cpro. We identified and characterized NEDD4-binding protein 1 (N4BP1), a key negative regulator of the NF-κB pathway, as a novel 3Cpro substrate. N4BP1 is cleaved at residue Q816 by 3Cpro from several human enteroviruses, resulting in the loss of its ability to regulate tumor necrosis factor alpha-activated NF-κB signaling. In addition, we found that mouse N4BP1, which has a threonine at the P1' site, is resistant to human enteroviral 3Cpro cleavage. However, rodent enteroviral 3Cpro derived from encephalomyocarditis virus (EMCV) can cleave both human and mouse N4BP1 at a species-specific site. By combining bioinformatic, biochemical, and cell biological approaches, we identified and characterized N4BP1 as a novel substrate of enteroviral 3Cpro. These findings provide valuable insights into the interplay between 3Cpro, its substrates, and viral pathogenesis.

Importance: Targeting cellular proteins for cleavage by enteroviral 3Cpro is a conserved strategy used by enteroviruses to promote viral replication. While the cleavage of certain host proteins by 3Cpro may not affect viral replication, it is strongly associated with the pathogenesis of viral infection. In this study, we identified and characterized N4BP1, which plays such a role, using a combination of bioinformatic, biochemical, and cell biological approaches. Our data show that multiple 3Cpros cleave N4BP1 at residue Q816 and that cleavage of endogenous N4BP1 can occur during viral infection. N4BP1 has no effect on coxsackievirus B3 replication, but 3Cpro-induced N4BP1 cleavage abolishes its regulatory function in NF-κB signaling. We also show that mouse N4bp1 resists human enteroviral 3Cpro cleavage. In contrast, rodent enteroviral EMCV 3Cpro can target human and mouse N4BP1 for cleavage at different residues, which indicates that future investigations are needed to elucidate the potential mechanisms involved.

Enterovirus 71 (EV71) infection is usually accompanied by neurological damage, which is the leading cause of death in children with hand-foot-mouth disease. In this study, we demonstrated that EV71 infection can cause pathological damage in the nervous system, such as neuronal vacuolar degeneration, shrinkage of some neurons, edema of brain tissues in the hippocampus, and a decreased number of Nissl bodies in the infarction area. Also, EV71 infection caused apparent structural damage to Schwann cells, including a decreased number of cytoplasmic organelles and severe damage of rough endoplasmic reticulum and mitochondria. However, the pathological damage was alleviated with the decrease of EV71 viral load. The cell experiment in vitro showed that EV71 infection significantly reduced ATP levels and promoted Schwann cell apoptosis, thus inhibiting cell growth. The extended infection time and the decreased viral load resulted in the gradual improvement of cell growth status. Meanwhile, EV71 inhibited the expression of miR-29b and promoted the expression of PMP22 in a time-dependent manner at both mRNA and protein levels, with the most significant change at 36 h of infection. Subsequently, the expression of miR-29b and PMP22 was gradually restored with the reduction of EV71 viral load. In addition, EV71 regulated the expression of hsa_circ_0069335, which could bind and co-localize with miR-29b. Therefore, EV71 infection can cause significant damage to the nervous system and may be related to hsa_circ_0069335/miR-29b/PMP22 pathway. The present study provides a new therapeutic target for neurological damage induced by EV71 infection.IMPORTANCEEV71 can cause severe neurological damage and even death, but the mechanism remains unclear. In this study, we exhibited the pathological changes of nervous system in EV71 infection and revealed that the damage degree was consistent with the EV71 viral load. From the molecular perspective, EV71 infection up-regulated the PMP22 expression in Schwann cells, which is accompanied by apparent structural damage of Schwann cells and myelin sheaths. Furthermore, EV71 promoted the expression of PMP22 and inhibited the expression of miR-29b in a time-dependent manner, with the most significant change at 36 h of infection. Otherwise, the hsa_circ_0069335, which binds and co-localizes with miR-29b, was also regulated by EV71 infection. The hsa_circ_0069335/miR-29b/PMP22 axis may be a potential molecular mechanism involved in EV71 infection-induced fatal neuronal damage. Drug development targeting this pathway may bring clinical improvement of EV71-infected patients.

Infection by human astrovirus (HAstV), a small, positive-strand RNA virus, is a major cause of gastroenteritis and has been implicated in an increasing number of severe, sometimes fatal, neurological diseases since 2008. Currently, there are no vaccines or antiviral treatments available to treat HAstV infection. An attractive target for antiviral therapeutics is the viral protease due to its essential functions throughout infection. However, the molecular mechanisms of the HAstV protease, nonstructural protein 1a/3 (nsp1a/3), are poorly understood. In fact, the specific residues within the cleavage junctions that are targeted by nsp1a/3 during polyprotein processing have yet to be experimentally identified. To identify the junctions between viral proteins, we performed mass spectrometry and site-directed mutagenesis using epitope-tagged viral polyprotein expression plasmids. Using these strategies, we identified a consensus motif that is found throughout the polyprotein near previously proposed junctions. We found that cleavage occurs after a hydrophobic residue - X - Gln motif. Further mutagenesis of surrounding sequence identified the importance of basic residues following the motif for efficient processing. Cleavage at each junction was determined to be essential for the production of progeny virions. However, abolishing nsp1a/4-VPg cleavage allowed efficient replication, suggesting that VPg can function in an intermediate form. Overall, our results identify a conserved cleavage motif that is recognized by the nsp1a/3 protease within the viral polyprotein, and cleavage at this motif was found to be essential for the recovery of progeny virions. These findings will be instrumental in further understanding the basic functions of HAstV polyprotein processing during infection.IMPORTANCEHuman astroviruses (HAstVs) are a leading cause of non-bacterial gastroenteritis in children, elderly individuals, and immunocompromised patients. However, infection by divergent strains of HAstV is now recognized as a causative agent of severe neurological diseases, which can have fatal outcomes. Despite the global prevalence of HAstV, we currently have a limited understanding of the biology of these viruses. Translation of the viral genome leads to the production of polyproteins that are processed by viral and host proteases into functional proteins. In this study, we identified a conserved recognition sequence targeted by the viral protease for cleavage. Importantly, these findings elucidate the N- and C-termini of the nonstructural proteins within the HAstV polyprotein, offering valuable information for future studies on the function of individual viral proteins. Similar to other positive-sense RNA viruses, the necessity of proteolytic processing for the HAstV polyprotein highlights the viral protease as a promising target for antiviral development.

Pyroptosis is an inflammatory type of programmed cell death that mainly depends on the formation of plasma membrane pores by Gasdermin D (GSDMD) in mammals. However, the genetic deficiency of GSDMD in chicken renders avian pyroptosis elusive. Here, we show that infection of DF-1 cells (a chicken cell line) with infectious bursal disease virus (IBDV) induced cell death associated with chicken GSDME (chGSDME) cleavage, and so did cells with other RNA virus (VSV, AIV, or NDV) infections, indicating a broad role of chGSDME in RNA virus-induced pyroptosis in chicken. Furthermore, infection of DF-1 cells by IBDV or treatment of cells with Poly(I:C) initiated MDA5-mediated signaling pathway, followed by the activation of chCaspase-3/7 cleaving chGSDME at a specific site ₂₇₀DAVD₂₇₃. Moreover, knockdown or knockout of chGSDME expression in cells markedly reduced IBDV-induced pyroptosis and viral release. These results unravel the mechanisms of pyroptosis in chickens with RNA virus infection, providing important clues to uncover the role of GSDM proteins of different species in host response against pathogenic infection.IMPORTANCEPyroptosis is an inflammatory type of programmed cell death that mainly depends on the function of GSDMD in mammals and plays a crucial role in the pathogenesis of viral infection, whereas the mechanism of pyroptosis in chicken remains elusive. Herein, we show that IBDV and other RNA virus induced pyroptosis through the chMDA5-CASP8/9-CASP3/7-chGSDME pathway. The finding advances our understanding of GSDM proteins of different species in host response against pathogenic infection.

The occurrence of viral diseases poses a huge threat and impact on human public health safety and the development of the animal and fishery industry. Here, a strain of single-chain antibody fragment, scFv-1, was isolated from the phage antibody display library construct by immunizing New Zealand white rabbits with rhabdovirus. In vitro analysis showed that the single-chain antibody could inhibit the infection of the virus in multiple pathways, including adsorption, fusion, and release. In vivo analysis revealed scFv-1 had a preventive and protective effect against the infection of virus. In addition, we describe that transposon-based transport of neutralizing genes allows for long-term, continuous expression, avoiding the need for lifelong, repeated passive immunization for treatment. In sum, high-throughput screening of neutralization genes based on phage display technology and transposon vector-based gene transfer provides effective methods for treating and preventing diseases and avoiding repetitive passive immunotherapy. This study also provides a reference for the prevention and treatment of unknown pathogens.IMPORTANCELivestock and fisheries play an important role in economic development and food security. The frequent outbreaks of viral diseases have caused great losses to the livestock industry, while the increase in drug resistance caused by the use of antibiotics as well as the potential risks to human health have raised serious concerns. Here, we constructed a phage display antibody library by immunizing New Zealand white rabbits with purified rhabdovirus and selected a single-chain antibody, scFv-1, with good neutralizing activity, which was validated and found to be able to block multiple phases of the virus and thus play a neutralizing role. In addition, we describe that transposon-based transport of neutralizing genes allows for long-term, continuous expression, reducing the need for lifelong, repeated passive immunization for treatment. Our work not only provides methods for the prevention and treatment of viral diseases but also provides the body with long-lasting and even permanent protection against repeated passive immunization.

Podophage tails are too short to span the cell envelope during infection. Consequently, podophages initially eject the core proteins within the head for the formation of an elongated trans-envelope channel for DNA ejection. Although the core proteins of bacteriophage T7 have been resolved at near-atomic resolution, the mechanisms of core proteins and DNA ejection remain to be fully elucidated. In this study, we provided improved structures of core proteins in mature T7 and the portal-tail complex in lipopolysaccharide-induced DNA-ejected T7 to resolutions of approximately 3 Å. Using these structures, we identified three small proteins, namely gp14, gp6.7, and gp7.3, and illustrated the conformational changes in and translocation of these proteins from the mature to DNA-ejected states. Our structures indicate that gp6.7, which participates in the assembly of the core and trans-envelope channel, is a core protein, and that gp7.3 serves as a structural scaffold to assist the assembly of the nozzle into the adaptor.

Importance: Podophage T7 core proteins form an elongated trans-envelope channel for genomic DNA delivery into the host cell. The structures of the core proteins within the mature T7 and assembled in the periplasmic tunnel form in the DNA-ejected T7 have been resolved previously. Here, we resolved the structures of two new structural proteins (gp6.7 and gp7.3) within mature T7 and receptor-induced DNA-ejected T7. The gp6.7 protein participates in the assembly of the core complex within mature T7 and the trans-envelope channel during T7 infection; therefore, gp6.7 is a core protein. Before T7 infection, gp7.3 plays a role in promoting the assembly of the nozzle into the adaptor.

Viral membrane fusion is a critical process enabling viruses to invade host cells, driven by viral membrane fusion proteins (MFPs). Cholesterol plays a pivotal role in this process, which is essential for the infectivity of many enveloped viruses. The interaction between MFPs and cholesterol is often facilitated by specific amino acid motifs known as cholesterol recognition/interaction amino acid consensus (CRAC) motifs and reverse CARC motifs. In a previous study, we demonstrated that CRAC1 and CRAC2 in GP64 are required for Bombyx mori nucleopolyhedrovirus (BmNPV) infection. This study further investigates the role of CARC in the GP64 protein of BmNPV, revealing their complex interaction with cholesterol and the influence of signal peptide (SP) retention on viral infectivity. We identified six putative CARC motifs in GP64 and generated mutants to assess their function. Our findings show that CARC1, CARC2, CARC3, and CARC4 are indispensable for viral fusion and infection when the SP is retained, whereas only CARC2 and CARC3 remain essential after SP cleavage. In contrast, CARC1 and CARC4 are necessary for viral infection through a cholesterol-independent mechanism resulting from double mutations in the CRAC1 and CRAC2 motifs of GP64. These insights not only deepen our understanding of BmNPV GP64-mediated fusion but also highlight potential antiviral targets, underscoring the adaptability and resilience of viral fusion mechanisms.IMPORTANCEUnderstanding viral membrane fusion mechanisms is crucial for developing antiviral strategies. This study provides novel insights into the intricate roles of CARC and CRAC motifs in the GP64 protein of BmNPV, particularly their interaction with cholesterol and the influence of signal peptide retention. The discovery that certain CARC motifs are essential for cholesterol-dependent fusion, whereas others function in a cholesterol-independent context advances our understanding of viral fusion processes. These findings emphasize the potential of targeting CARC motifs for therapeutic interventions and underline the importance of cholesterol interactions in viral infections. This research not only deepens our understanding of BmNPV fusion mechanisms but also has broader implications for other enveloped viruses.

During viral infections, autophagy functions as a cell-intrinsic defense mechanism by facilitating the delivery of virions or viral components to the endosomal/lysosomal pathway for degradation. In this study, we report that internalized African swine fever virus (ASFV) virions enter autolysosomes during the early phase of viral infection. Autophagy selectively targets the major capsid protein p72 within the ASFV virion. The ASFV p72 protein undergoes modification through ubiquitination at the C-terminus, a process mediated by the E3 ubiquitin ligase Stub1. Subsequently, ubiquitinated p72 is recognized by the autophagy receptor SQSTM1/p62 through its ubiquitin-binding domain. Stub1 facilitates the ubiquitination and degradation of p72 in an HSPA8-dependent manner via selective autophagy. Autophagy plays a critical role in disassembling ASFV virions and further promotes the release of ASFV genomic DNA. These findings support the notion that autophagy is involved in and contributes to the capsid disassembly of ASFV, providing valuable insights into this essential viral process.IMPORTANCEAfrican swine fever (ASF), a highly contagious disease caused by the ASF virus (ASFV), affects domestic pigs and wild boars, with a mortality rate of up to 100%. The ASF epidemic poses a persistent threat to the global pig industry. Currently, no effective vaccines or antiviral drugs are available for prevention and control. In this study, we discovered that autophagy promotes the degradation of p72 and the disassembly of the capsid during the early phase of ASFV infection. Mechanically, Stub1 facilitates the polyubiquitination of ASFV p72 through the chaperone HSPA8. The polyubiquitinated p72 then interacts with the autophagy receptor SQSTM1/p62, leading to its degradation via the selective autophagy pathway. These findings reveal the mechanism of p72 degradation through autophagy and provide new insights into the capsid disassembly process of ASFV.

Swine enteric coronaviruses pose a significant challenge to the global pig industry, inflicting severe diarrhea and high mortality rates among piglets, and resulting in substantial economic losses. In our clinical practice, we observed that the addition of potassium molybdate (PM) to the feed could dramatically reduce diarrhea and diarrhea-related mortality in piglets. However, the underlying mechanisms remain elusive and merit further investigation. In this study, we revealed that PM effectively inhibited the infection of both aminopeptidase N (APN)-dependent coronaviruses, transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV), both in vitro and ex vivo. Specifically, PM was found to block TGEV and PRCV penetration by degrading the cell receptor APN through the upregulation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) expression. In addition, knockdown and knockout of PIK3C3 resulted in the attenuation of PM-induced autophagy, thereby rescuing APN expression and viral infection. Correspondingly, replenishment of PIK3C3 in PIK3C3-null ST cells restored PM-mediated APN degradation and successfully blocked viral entry. Furthermore, our findings demonstrated that PM promoted the assembly of the PIK3C3-BECN1-ATG14 complex, leading to induced autophagic degradation by upregulating PIK3C3 Ser249 phosphorylation. In vivo experiments further confirmed that PM-induced PIK3C3-mediated autophagic degradation of APN, thereby limiting the pathogenicity of TGEV. In summary, our study for the first time identified the mechanism by which PM blocked TGEV and PRCV internalization by degrading the cell receptor APN via PIK3C3-mediated autophagy. This study provides valuable insights and potential strategies for preventing APN-restricted coronavirus infection.IMPORTANCEAminopeptidase N (APN) is one of the most important host receptors of coronavirus. Modulating APN expression can represent a novel approach for controlling APN-dependent coronaviruses and their variants infection. Here we found that a chemical compound potassium molybdate (PM) negatively regulates APN expression by inducing phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3)-mediated autophagy against APN-dependent coronavirus internalization, including transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Furthermore, PM can promote PIK3C3-BECN1-ATG14 complex assembly to induce autophagic degradation of APN by upregulating PIK3C3 Ser249 phosphorylation. Lastly, results from pig experiments also confirmed that PM can trigger PIK3C3-mediated autophagic degradation of APN to restrict TGEV pathogenicity in vivo without toxicity. Our findings underscore the promising potential of PM as an effective agent against APN-dependent coronavirus and potentially emerging viral disease entry.