NLRP1: a jack of all trades, or a master of one?

Recent studies provide evidence that two chemically and mechanistically distinct signals activate the human NLRP1 inflammasome, challenging the concept that it-like other mammalian inflammasomes characterized thus far-evolved to detect and respond to a single danger-associated molecular pattern.

PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

Species-specific IL-1ß is an inflammatory sensor of Seneca Valley Virus 3C Protease.

Inflammasomes play pivotal roles in inflammation by processing and promoting the secretion of IL-1ß. Caspase-1 is involved in the maturation of IL-1ß and IL-18, while human caspase-4 specifically processes IL-18. Recent structural studies of caspase-4 bound to Pro-IL-18 reveal the molecular basis of Pro-IL-18 activation by caspase-4. However, the mechanism of caspase-1 processing of pro-IL-1ß and other IL-1ß-converting enzymes remains elusive. Here, we observed that swine Pro-IL-1ß (sPro-IL-1ß) exists as an oligomeric precursor unlike monomeric human Pro-IL-1ß (hPro-IL-1ß). Interestingly, Seneca Valley Virus (SVV) 3C protease cleaves sPro-IL-1ß to produce mature IL-1ß, while it cleaves hPro-IL-1ß but does not produce mature IL-1ß in a specific manner. When the inflammasome is blocked, SVV 3C continues to activate IL-1ß through direct cleavage in porcine alveolar macrophages (PAMs). Through molecular modeling and mutagenesis studies, we discovered that the pro-domain of sPro-IL-1ß serves as an 'exosite' with its hydrophobic residues docking into a positively charged 3C protease pocket, thereby directing the substrate to the active site. The cleavage of sPro-IL-1ß generates a monomeric and active form of IL-1ß, initiating the downstream signaling. Thus, these studies provide IL-1ß is an inflammatory sensor that directly detects viral protease through an independent pathway operating in parallel with host inflammasomes.

Enterovirus D68 3C protease antagonizes type I interferon signaling by cleaving signal transducer and activator of transcription 1.

Following the successful control of poliovirus, the re-emergence of respiratory enterovirus D68 (EV-D68), a prominent non-polio enterovirus, has become a serious public health concern worldwide. Host innate immune responses are the primary defense against EV-D68 invasion; however, the mechanism underlying viral evasion of the antiviral activity of interferons (IFN) remains unclear. In this study, we found that EV-D68 inhibited type I IFN signaling by cleaving signal transducer and activator of transcription 1 (STAT1), a crucial factor in cellular responses to interferons and other cytokines. We observed that the prototype and circulating EV-D68 strains conserved their ability to induce STAT1 cleavage and attenuate IFN signal transduction. Further investigation revealed that EV-D68 3C protease cleaves STAT1 at the 131Q residue. Interestingly, not all enterovirus-encoded 3C proteases exhibited this ability. EV-D68 and poliovirus 3C proteases efficiently induced STAT1 cleavage; whereas, 3C proteases from EV-A71, coxsackievirus A16, and echoviruses did not. STAT1 cleavage also abolished the nuclear translocation capacity of STAT1 in response to IFN stimulation to activate downstream signaling elements. Overall, these results suggest that STAT1, targeted by viral protease 3C, is utilized by EV-D68 to subvert the host's innate immune response.IMPORTANCEEnterovirus D68 (EV-D68) has significantly transformed over the past decade, evolving from a rare pathogen to a potential pandemic pathogen. The interferon (IFN) signaling pathway is an important defense mechanism and therapeutic target for the host to resist viral invasion. Previous studies have reported that the EV-D68 virus blocks or weakens immune recognition and IFN production in host cells through diverse strategies; however, the mechanisms of EV-D68 resistance to IFN signaling have not been fully elucidated. Our study revealed that EV-D68 relies on its own encoded protease, 3C, to directly cleave signal transducer and activator of transcription 1 (STAT1), a pivotal transduction component in the IFN signaling pathway, disrupting the IFN-mediated antiviral response. Previous studies on human enteroviruses have not documented direct cleavage of the STAT1 protein to evade cellular immune defenses. However, not all enteroviral 3C proteins can cleave STAT1. These findings highlight the diverse evolutionary strategies different human enteroviruses employ to evade host immunity.

Foot-and-mouth disease virus (FMDV) negatively regulates ZFP36 protein expression to alleviate its antiviral activity.

Zinc finger protein 36 (ZFP36) is a key regulator of inflammatory and cytokine production. However, the interplay between swine zinc-finger protein 36 (sZFP36) and foot-and-mouth disease virus (FMDV) has not yet been reported. Here, we demonstrate that overexpression of sZFP36 restricted FMDV replication, while the knockdown of sZFP36 facilitated FMDV replication. To subvert the antagonism of sZFP36, FMDV decreased sZFP36 protein expression through its non-structural protein 3C protease (3Cpro). Our results also suggested that 3Cpro-mediated sZFP36 degradation was dependent on its protease activity. Further investigation revealed that both N-terminal and C-terminal-sZFP36 could be degraded by FMDV and FMDV 3Cpro. In addition, both N-terminal and C-terminal-sZFP36 decreased FMDV replication. Moreover, sZFP36 promotes the degradation of FMDV structural proteins VP3 and VP4 via the CCCH-type zinc finger and NES domains of sZFP36. Together, our results confirm that sZFP36 is a host restriction factor that negatively regulates FMDV replication.IMPORTANCEFoot-and-mouth disease (FMD) is an infectious disease of animals caused by the pathogen foot-and-mouth disease virus (FMDV). FMD is difficult to prevent and control because there is no cross-protection between its serotypes. Thus, we designed this study to investigate virus-host interactions. We first demonstrate that swine zinc-finger protein 36 (sZFP36) impaired FMDV structural proteins VP3 and VP4 to suppress viral replication. To subvert the antagonism of sZFP36, FMDV and FMDV 3Cpro downregulate sZFP36 expression to facilitate FMDV replication. Taken together, the present study reveals a previously unrecognized antiviral mechanism for ZFP36 and elucidates the role of FMDV in counteracting host antiviral activity.

An efficient trans complementation system for in vivo replication of defective poliovirus mutants.

The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in cis). Efficient complementation in vivo of replication complex formation by viral proteins provided in trans, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient trans complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in cis. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3Cpro protease between 3Cpro and 3Dpol (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in trans. Only a defect of 3Dpol activity of the replicon could be rescued in trans; inactivating mutations in 2CATPase/hel, 3B, and 3Cpro of the replicon completely abrogated the trans-rescued replication. An intact N-terminus of the 3Cpro domain of the 3CDpro provided in trans was essential for the trans-active function. By using this trans complementation system, a high-titer defective PV pseudovirus (PVpv) (>107 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on trans complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection. IMPORTANCE: Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in trans) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in cis) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.

Identification of the proteolytic signature in CVB3-infected cells.

Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase in vitro. Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3Cpro-targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection. IMPORTANCE: RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis.

Impaired interferon response in senecavirus A infection and identification of 3Cpro as an antagonist.

Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3Cpro) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3Cpro selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3Cpro cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3Cpro partially differ from previously reported classical motifs recognized by other picornaviral 3Cpro, highlighting the distinct characteristics of SVA 3Cpro. Together, these results reveal a mechanism by which SVA 3Cpro antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3Cpro.IMPORTANCESenecavirus A (SVA), the only member in the Senecavirus genus within the Picornaviridae family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3Cpro, a protease of SVA, functions as an antagonist for the IFN response. 3Cpro utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3Cpro as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.

Transcription factor EB (TFEB) interaction with RagC is disrupted during enterovirus D68 infection.

Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is crucial for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus at glutamine 60 (Q60) immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC-binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells but not in autophagy-defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. IMPORTANCE: Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.

Allosteric regulation of Senecavirus A 3Cpro proteolytic activity by an endogenous phospholipid.

Seneca virus A (SVA) is an emerging novel picornavirus that has recently been identified as the causative agent of many cases of porcine vesicular diseases in multiple countries. In addition to cleavage of viral polyprotein, the viral 3C protease (3Cpro) plays an important role in the regulation of several physiological processes involved in cellular antiviral responses by cleaving critical cellular proteins. Through a combination of crystallography, untargeted lipidomics, and immunoblotting, we identified the association of SVA 3Cpro with an endogenous phospholipid molecule, which binds to a unique region neighboring the proteolytic site of SVA 3Cpro. Our lipid-binding assays showed that SVA 3Cpro displayed preferred binding to cardiolipin (CL), followed by phosphoinositol-4-phosphate (PI4P) and sulfatide. Importantly, we found that the proteolytic activity of SVA 3Cpro was activated in the presence of the phospholipid, and the enzymatic activity is inhibited when the phospholipid-binding capacity decreased. Interestingly, in the wild-type SVA 3Cpro-substrate peptide structure, the cleavage residue cannot form a covalent binding to the catalytic cysteine residue to form the acyl-enzyme intermediate observed in several picornaviral 3Cpro structures. We observed a decrease in infectivity titers of SVA mutants harboring mutations that impaired the lipid-binding ability of 3Cpro, indicating a positive regulation of SVA infection capacity mediated by phospholipids. Our findings reveal a mutual regulation between the proteolytic activity and phospholipid-binding capacity in SVA 3Cpro, suggesting that endogenous phospholipid may function as an allosteric activator that regulate the enzyme's proteolytic activity during infection.

Theilovirus 3C Protease Cleaves the C-Terminal Domain of the Innate Immune RNA Sensor, Melanoma Differentiation-Associated Gene 5, and Impairs Double-Stranded RNA-Mediated IFN Response.

Melanoma differentiation-associated gene 5 (MDA5), a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), has pivotal roles in innate immune responses against many positive-stranded RNA viruses, including picornavirus and coronavirus. Upon engagement with dsRNA derived from viral infection, MDA5 initiates coordinated signal transduction leading to type I IFN induction to restrict viral replication. In this study, we describe a targeted cleavage events of MDA5 by the 3C protease from Theilovirus. Upon ectopic expression of theilovirus 3C protease from Saffold virus or Theiler's murine encephalomyelitis virus but not encephalomyocarditis virus, fragments of cleaved MDA5 were observed in a dose-dependent manner. When enzymatically inactive Theilovirus 3C protease was expressed, MDA5 cleavage was completely abrogated. Mass spectrometric analysis identified two cleavage sites at the C terminus of MDA5, cleaving off one of the RNA-binding domains. The same cleavage pattern was observed during Theilovirus infection. The cleavage of MDA5 by Theilovirus protease impaired ATP hydrolysis, RNA binding, and filament assembly on RNA, resulting in dysfunction of MDA5 as an innate immune RNA sensor for IFN induction. Furthermore, the cleavage-resistant MDA5 mutant against the 3C protease showed an enhanced IFN response during Saffold virus infection, indicating that Theilovirus has a strategy to circumvent the antiviral immune response by cleaving MDA5 using 3C protease. In summary, these data suggest MDA5 cleavage by 3C protease as a novel immune evasive strategy of Theilovirus.

Seneca Valley virus 3Cpro antagonizes type I interferon response by targeting STAT1-STAT2-IRF9 and KPNA1 signals.

IMPORTANCE: Type I interferon (IFN) signaling plays a principal role in host innate immune responses against invading viruses. Viruses have evolved diverse mechanisms that target the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway to modulate IFN response negatively. Seneca Valley virus (SVV), an emerging porcine picornavirus, has received great interest recently because it poses a great threat to the global pork industry. However, the molecular mechanism by which SVV evades host innate immunity remains incompletely clear. Our results revealed that SVV proteinase (3Cpro) antagonizes IFN signaling by degrading STAT1, STAT2, and IRF9, and cleaving STAT2 to escape host immunity. SVV 3Cpro also degrades karyopherin 1 to block IFN-stimulated gene factor 3 nuclear translocation. Our results reveal a novel molecular mechanism by which SVV 3Cpro antagonizes the type I IFN response pathway by targeting STAT1-STAT2-IRF9 and karyopherin α1 signals, which has important implications for our understanding of SVV-evaded host innate immune responses.

Cleavage of 14-3-3ε by the enteroviral 3C protease dampens RIG-I-mediated antiviral signaling.

Viruses have evolved diverse strategies to evade the host innate immune response and promote infection. The retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and MDA5 are antiviral factors that sense viral RNA and trigger downstream signal via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon expression. 14-3-3ε is a key component of the RIG-I translocon complex that interacts with MAVS at the mitochondrial membrane; however, the exact role of 14-3-3ε in this pathway is not well understood. In this study, we demonstrate that 14-3-3ε is a direct substrate of both the poliovirus and coxsackievirus B3 (CVB3) 3C proteases (3Cpro) and that it is cleaved at Q236↓G237, resulting in the generation of N- and C-terminal fragments of 27.0 and 2.1 kDa, respectively. While the exogenous expression of wild-type 14-3-3ε enhances IFNB mRNA production during poly(I:C) stimulation, expression of the truncated N-terminal fragment does not. The N-terminal 14-3-3ε fragment does not interact with RIG-I in co-immunoprecipitation assays, nor can it facilitate RIG-I translocation to the mitochondria. Probing the intrinsically disordered C-terminal region identifies key residues responsible for the interaction between 14-3-3ε and RIG-I. Finally, overexpression of the N-terminal fragment promotes CVB3 infection in mammalian cells. The strategic enterovirus 3Cpro-mediated cleavage of 14-3-3ε antagonizes RIG-I signaling by disrupting critical interactions within the RIG-I translocon complex, thus contributing to evasion of the host antiviral response. IMPORTANCE Host antiviral factors work to sense virus infection through various mechanisms, including a complex signaling pathway known as the retinoic acid-inducible gene I (RIG-I)-like receptor pathway. This pathway drives the production of antiviral molecules known as interferons, which are necessary to establish an antiviral state in the cellular environment. Key to this antiviral signaling pathway is the small chaperone protein 14-3-3ε, which facilitates the delivery of a viral sensor protein, RIG-I, to the mitochondria. In this study, we show that the enteroviral 3C protease cleaves 14-3-3ε during infection, rendering it incapable of facilitating this antiviral response. We also find that the resulting N-terminal cleavage fragment dampens RIG-I signaling and promotes virus infection. Our findings reveal a novel viral strategy that restricts the antiviral host response and provides insights into the mechanisms underlying 14-3-3ε function in RIG-I antiviral signaling.

Enterovirus D68 Infection Induces TDP-43 Cleavage, Aggregation, and Neurotoxicity.

Enterovirus D68 (EV-D68), which causes severe respiratory diseases and irreversible central nervous system damage, has become a serious public health problem worldwide. However, the mechanisms by which EV-D68 exerts neurotoxicity remain unclear. Thus, we aimed to analyze the effects of EV-D68 infection on the cleavage, subcellular translocation, and pathogenic aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in respiratory or neural cells. The results showed that EV-D68-encoded proteases 2A and 3C induced TDP-43 translocation and cleavage, respectively. Specifically, 3C cleaved residue 327Q of TDP-43. The 3C-mediated cleaved TDP-43 fragments had substantially decreased protein solubility compared with the wild-type TDP-43. Hence, 3C activity promoted TDP-43 aggregation, which exerted cytotoxicity to diverse human cells, including glioblastoma T98G cells. The effects of commercially available antiviral drugs on 3C-mediated TDP-43 cleavage were screened, and the results revealed lopinavir as a potent inhibitor of EV-D68 3C protease. Overall, these results suggested TDP-43 as a conserved host target of EV-D68 3C. This study is the first to provide evidence on the involvement of TDP-43 dysregulation in EV-D68 pathogenesis. IMPORTANCE Over the past decade, the incidence of enterovirus D68 (EV-D68) infection has increased worldwide. EV-D68 infection can cause different respiratory symptoms and severe neurological complications, including acute flaccid myelitis. Thus, elucidating the mechanisms underlying EV-D68 toxicity is important to develop novel methods to prevent EV-D68 infection-associated diseases. This study shows that EV-D68 infection triggers the translocalization, cleavage, and aggregation of TDP-43, an intracellular protein closely related to degenerative neurological disorders. The viral protease 3C decreased TDP-43 solubility, thereby exerting cytotoxicity to host cells, including human glioblastoma cells. Thus, counteracting 3C activity is an effective strategy to relieve EV-D68-triggered cell death. Cytoplasmic aggregation of TDP-43 is a hallmark of degenerative diseases, contributing to neural cell damage and central nervous system (CNS) disorders. The findings of this study on EV-D68-induced TDP-43 formation extend our understanding of virus-mediated cytotoxicity and the potential risks of TDP-43 dysfunction-related cognitive impairment and neurological symptoms in infected patients.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Cleavage of Stau2 by 3C protease promotes EV-A71 replication.

BACKGROUND: Enterovirus A71 (EV-A71), as a neurotropic virus, mainly affects infants and young children under the age of 5. EV-A71 infection causes hand-foot-mouth disease and herpetic angina, and even life-threatening neurological complications. However, the molecular mechanism by which EV-A71 induces nervous system damage remains elusive. The viral protease 3C plays an important role during EV-A71 infection and is also a key intersection of virus-host interactions. Previously, we used yeast two-hybrid to screen out the host protein Double-stranded RNA-binding protein Staufen homolog 2 (Stau2), an important member involved in neuronal mRNA transport, potentially interacts with 3C. METHODS: We used coimmunoprecipitation (Co-IP) and immunofluorescence assay (IFA) to confirm that EV-A71 3C interacts with Stau2. By constructing the mutant of Stau2, we found the specific site where the 3C protease cleaves Stau2. Detection of VP1 protein using Western blotting characterized EV-A71 viral replication, and overexpression or knockdown of Stau2 exhibited effects on EV-A71 replication. The effect of different cleavage products on EV-A71 replication was demonstrated by constructing Stau2 truncates. RESULTS: In this study, we found that EV-A71 3C interacts with Stau2. Stau2 is cleaved by 3C at the Q507-G508 site. Overexpression of Stau2 promotes EV-A71 VP1 protein expression, whereas depletion of Stau2 by small interfering RNA inhibits EV-A71 replication. Stau2 is essential for EV-A71 replication, and the product of Stau2 cleavage by 3C, 508-570 aa, has activity that promotes EV-A71 replication. In addition, we found that mouse Stau2 is also cleaved by EV-A71 3C at the same site. CONCLUSIONS: Our research provides an example for EV-A71-host interaction, enriching key targets of host factors that contribute to viral replication.

Expression of virus-like particles (VLPs) of foot-and-mouth disease virus (FMDV) using Saccharomyces cerevisiae.

We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational "ribosomal skipping" sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a "ribosomal skipping" signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles. KEY POINTS: • Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae. • Proteolytic processing of a structural polyprotein from a monocistronic transcript. • Assembly of the processed viral capsid proteins into a virus-like particle.

Structure of Senecavirus A 3C Protease Revealed the Cleavage Pattern of 3C Protease in Picornaviruses.

Senecavirus A (SVA) is an emerging picornavirus infecting porcine of all age groups and causing foot and mouth disease (FMD)-like symptoms. One of its key enzymes is the 3C protease (3Cpro), which is similar to other picornaviruses and essential for virus maturation by controlling polyprotein cleavage and RNA replication. In this study, we reported the crystal structure of SVA 3Cpro at a resolution of 1.9 Å and a thorough structural comparison against all published picornavirus 3Cpro structures. Using statistical and graphical visualization techniques, we also investigated the sequence specificity of the 3Cpro. The structure revealed that SVA 3Cpro adopted a typical chymotrypsin-like fold with the S1 subsite as the most conservative site among picornavirus 3Cpro. The surface loop, A1-B1 hairpin, adopted a novel conformation in SVA 3Cpro and formed a positively charged protrusion around S' subsites. Correspondingly, SVA scissile bonds preferred Asp rather than neutral amino acids at P3' and P4'. Moreover, SVA 3Cpro showed a wide range tolerance to P4 residue volume (acceptable range: 67 Å3 to 141 Å3), such as aromatic side chain, in contrast to other picornaviruses. In summary, our results provided valuable information for understanding the cleavage pattern of 3Cpro. IMPORTANCE Picornaviridae is a group of RNA viruses that harm both humans and livestock. 3Cpro is an essential enzyme for picornavirus maturation, which makes it a promising target for antiviral drug development and a critical component for virus-like particle (VLP) production. However, the current challenge in the development of antiviral drugs and VLP vaccines includes the limited knowledge of how subsite structure determines the 3Cpro cleavage pattern. Thus, an extensive comparative study of various picornaviral 3Cpro was required. Here, we showed the 1.9 Å crystal structure of SVA 3Cpro. The structure revealed similarities and differences in the substrate-binding groove among picornaviruses, providing new insights into the development of inhibitors and VLP.

Enterovirus 3C Protease Cleaves TRIM7 To Dampen Its Antiviral Activity.

Mammalian TRIM7 is an antiviral protein that inhibits multiple human enteroviruses by degrading the viral 2BC protein. Whether TRIM7 is reciprocally targeted by enteroviruses is not known. Here, we report that the 3C protease (3Cpro) from two enteroviruses, coxsackievirus B3 (CVB3) and poliovirus, targets TRIM7 for cleavage. CVB3 3Cpro cleaves TRIM7 at glutamine 24 (Q24), resulting in a truncated TRIM7 that fails to inhibit CVB3 due to dampened E3 ubiquitin ligase activity. TRIM7 Q24 is highly conserved across mammals, except in marsupials, which instead have a naturally occurring histidine (H24) that is not subject to 3Cpro cleavage. Marsupials also express two isoforms of TRIM7, and the two proteins from koalas have distinct antiviral activities. The longer isoform contains an additional exon due to alternate splice site usage. This additional exon contains a unique 3Cpro cleavage site, suggesting that certain enteroviruses may have evolved to target marsupial TRIM7 even if the canonical Q24 is missing. Combined with computational analyses indicating that TRIM7 is rapidly evolving, our data raise the possibility that TRIM7 may be targeted by enterovirus evasion strategies and that evolution of TRIM7 across mammals may have conferred unique antiviral properties. IMPORTANCE Enteroviruses are significant human pathogens that cause viral myocarditis, pancreatitis, and meningitis. Knowing how the host controls these viruses and how the viruses may evade host restriction is important for understanding fundamental concepts in antiviral immunity and for informing potential therapeutic interventions. In this study, we demonstrate that coxsackievirus B3 uses its virally encoded protease to target the host antiviral protein TRIM7 for cleavage, suggesting a potential mechanism of viral immune evasion. We additionally show that TRIM7 has evolved in certain mammalian lineages to express protein variants with distinct antiviral activities and susceptibilities to viral protease-mediated cleavage.

Multiple-Site SUMOylation of FMDV 3C Protease and Its Negative Role in Viral Replication.

Protein SUMOylation represents an important cellular process that regulates the activities of numerous host proteins as well as of many invasive viral proteins. Foot-and-mouth disease virus (FMDV) is the first animal virus discovered. However, whether SUMOylation takes place during FMDV infection and what role it plays in FMDV pathogenesis have not been investigated. In the present study, we demonstrated that SUMOylation suppressed FMDV replication by small interfering RNA (siRNA) transfection coupled with pharmaceutical inhibition of SUMOylation, which was further confirmed by increased virus replication for SUMOylation-deficient FMDV with mutations in 3C protease, a target of SUMOylation. Moreover, we provided evidence that four lysine residues, Lys-51, -54, -110, and -159, worked together to confer the SUMOylation to the FMDV 3C protease, which may make SUMOylation of FMDV 3C more stable and improve the host's chance of suppressing the replication of FMDV. This is the first report that four lysine residues can be alternatively modified by SUMOylation. Finally, we showed that SUMOylation attenuated the cleavage ability, the inhibitory effect of the interferon signaling pathway, and the protein stability of FMDV 3C, which appeared to correlate with a decrease in FMDV replication. Taken together, the results of our experiments describe a novel cellular regulatory event that significantly restricts FMDV replication through the SUMOylation of 3C protease. IMPORTANCE FMD is a highly contagious and economically important disease in cloven-hoofed animals. SUMOylation, the covalent linkage of a small ubiquitin-like protein to a variety of substrate proteins, has emerged as an important posttranslational modification that plays multiple roles in diverse biological processes. In this study, four lysine residues of FMDV 3C were found to be alternatively modified by SUMOylation. In addition, we demonstrated that SUMOylation attenuated FMDV 3C function through multiple mechanisms, including cleavage ability, the inhibitory effect of the interferon signaling pathway, and protein stability, which, in turn, resulted in a decrease of FMDV replication. Our findings indicate that SUMOylation of FMDV 3C serves as a host cell defense against FMDV replication. Further understanding of the cellular and molecular mechanisms driving this process should offer novel insights to design an effective strategy to control the dissemination of FMDV in animals.