EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication.
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ABSTRACT:
SUBMITTER: Zhou X
PROVIDER: S-EPMC9243667 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date. METHODS:The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cpro or 3Dpol were tested. The interaction between EV71 3Cpro and quercetin was predicted and calculated by molecular docking. RESULTS:Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cpro, blocking viral replication, but not the activity of the protease, 2Apro, or the RNA polymerase, 3Dpol. Modeling of the molecular binding of the 3Cpro-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity. CONCLUSIONS:Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3Cpro activity, thereby blocking EV71 replication.
| S-EPMC6069798 | biostudies-literature
Project description:Enterovirus 71 (EV71) is one of the major pathogens of hand, foot, and mouth disease, which poses a major risk to public health and infant safety. 3C protease (3Cpro), a non-structural protein of EV71, promotes viral protein maturation by cleaving polyprotein precursors and facilitates viral immune escape by cleaving host proteins. In this study, we screened for human proteins that could interact with EV71 3Cpro using a yeast two-hybrid assay. Immune-associated protein TRAF3 Interacting Protein 3 (TRAF3IP3) was selected for further study. The results of co-immunoprecipitation and immunofluorescence demonstrated the interaction between TRAF3IP3 and EV71 3Cpro. A cleavage band was detected, indicating that both transfected 3Cpro and EV71 infection could cleave TRAF3IP3. 87Q-88G was identified as the only 3Cpro cleavage site in TRAF3IP3. In Jurkat and rhabdomyosarcoma (RD) cells, TRAF3IP3 inhibited EV71 replication, and 3Cpro cleavage partially resisted TRAF3IP3-induced inhibition. Additionally, the nuclear localization signal (NLS) and nuclear export signal (NES) of TRAF3IP3 were identified. The NES contributed to TRAF3IP3 alteration of 3Cpro localization and inhibition of EV71 replication. Together, these results indicate that TRAF3IP3 inhibits EV71 replication and 3Cpro resists such inhibition via proteolytic cleavage, providing a new example of virus-host interaction.
| S-EPMC9260427 | biostudies-literature
Project description:Human Rhinovirus (HRV) is a pathogen of significant medical importance, being a major cause of upper respiratory tract infections (common colds) as well as causing the majority of virus-induced asthma exacerbations. We investigated whether HRV could modulate apoptosis, an innate antiviral response. Apoptotic signals are generated either extrinsically or intrinsically and are propagated via caspase cascades that lead to cell death, reducing viral replication, which relies on cellular machinery. Using HRV16 infected cells, in combination with chemical inducers and inhibitors of extrinsic apoptosis we show that HRV16 3C protease cleaves a key intermediate in extrinsic apoptosis. Receptor-interacting protein kinase-1 (RIPK1), an extrinsic apoptosis adaptor protein, was cleaved by caspase 8, as expected, during chemical induction of apoptosis. RIPK1 was cleaved in HRV infection albeit at a different site. Caspase 8 activation, which is associated with extrinsic apoptosis, was concurrent with HRV 3C protease mediated cleavage of RIPK1, and potentially increased the accessibility of the HRV 3C cleavage site within RIPK1 in-vitro. The caspase 8 mediated RIPK1 cleavage product has a pro-apoptotic function, and further cleavage of this pro-apoptotic cleavage product by HRV 3C may provide a mechanism by which HRV limits apoptosis.
| S-EPMC5785518 | biostudies-literature
Project description:The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3C(pro) cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3C(pro), occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3C(pro)-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3C(pro) cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.
| S-EPMC3059221 | biostudies-literature
Project description: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.
| S-EPMC9472655 | biostudies-literature
Project description:For the last decade, porcine epidemic diarrhea virus (PEDV) variant strains have caused severe damage to the global pig industry. Until now, no effective antivirals have been developed for the therapeutic treatment of PEDV infection. In the present study, we found that quercetin significantly suppressed PEDV infection at noncytotoxic concentrations. A molecular docking study indicated that quercetin might bind the active site and binding pocket of PEDV 3C-like protease (3CLpro). Surface plasmon resonance (SPR) analysis revealed that quercetin exhibited a binding affinity to PEDV 3CLpro. Based on the results of the fluorescence resonance energy transfer (FRET) assay, quercetin was proven to exert an inhibitory effect on PEDV 3CLpro. Since coronavirus 3CLpro is an important drug target and participates in the viral replication process, quercetin should be developed as a novel drug in the control of PEDV infection.
| S-EPMC7662296 | biostudies-literature
Project description:Human enterovirus type 71 (EV71), the major causative agent of hand-foot-and-mouth disease, has been known to cause fatal neurological complications. Unfortunately, the reason for neurological complications that have been seen in fatal cases of the disease and the relationship between EV71 virulence and viral genetic sequences remains largely undefined. The 3C protease (3Cpro) of EV71 plays an irreplaceable role in segmenting the precursor polyprotein during viral replication, and intervening with host life activity during viral infection. In this study, for the first time, the 69th residue of 3C protease has been identified as a novel virulence determinant of EV71. The recombinant virus with single point variation, in the 69th of 3Cpro, exhibited obvious decline in replication, and virulence. We further determined the crystal structure of 3C N69D at 1.39 Ǻ resolution and found that conformation of 3C N69D demonstrated significant changes compared with a normal 3C protein, in the substrate-binding site and catalytic active site. Strikingly, one of the switch loops, essential in fixing substrates, adopts an open conformation in the 3C N69D-rupintrivir complex. Consistent with this apparent structural disruption, the catalytic activity of 3C N69D decreased sharply for host derived and viral derived substrates, detected for both in vitro and in vivo. Interestingly, in addition to EV71, Asp69 was also found in 3C proteases of other virus strains, such as CAV16, and was conserved in nearly all C type human rhinovirus. Overall, we identified a natural virulence determinant of 3C protease and revealed the mechanism of attenuated virulence is mediated by N69D substitution. Our data provides new insight into the enzymatic mechanism of a subdued 3C protease and suggests a theoretical basis for virulence determinantion of picornaviridae.
| S-EPMC5290453 | biostudies-literature
Project description:Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell-virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C(pro)) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3' pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C(pro). CstF-64 was cleaved in vitro by 3C(pro) but neither by mutant 3C(pro) (in which the catalytic site was inactivated) nor by another EV71 protease 2A(pro). Serial mutagenesis was performed in CstF-64, revealing that the 3C(pro) cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3'-end pre-mRNA processing and polyadenylation in 3C(pro)-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C(pro) cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.
| S-EPMC2742901 | biostudies-literature
Project description:BackgroundEnterovirus 71 (EV71) usually infects infants causing hand-foot-mouth disease (HFMD), even fatal neurological disease like aseptic meningitis. Effective drug for preventing and treating EV71 infection is unavailable currently. EV71 3C mediated the cleavage of many proteins and played an important role in viral inhibiting host innate immunity. Promyelocytic leukemia (PML) protein, the primary organizer of PML nuclear bodies (PML-NBs), can be induced by interferon and is involved in antiviral activity. PML inhibits EV71 replication, and EV71 infection reduces PML expression, but the molecular mechanism is unclear.MethodsThe cleavage of PMLIII and IV was confirmed by co-transfection of EV71 3C protease and PML. The detailed cleavage sites were evaluated further by constructing the Q to A mutant of PML. PML knockout cells were infected with EV71 to identify the effect of cleavage on EV71 replication. Immunofluorescence analysis to examine the interference of EV71 3C on the formation of PML-NBs.ResultsEV71 3C directly cleaved PMLIII and IV. Furthermore, 3C cleaved PMLIV at the sites of Q430-A431 and Q444-S445 through its protease activity. Overexpression of PMLIV Q430A/Q444A variant exhibited stronger antiviral potential than the wild type. PMLIV Q430A/Q444A formed normal nuclear bodies that were not affected by 3C, suggesting that 3C may impair PML-NBs production via PMLIV cleavage and counter its antiviral activities. PML, especially PMLIV, which sequesters viral proteins in PML-NBs and inhibits viral production, is a novel target of EV71 3C cleavage.ConclusionsEV71 3C cleaves PMLIV at Q430-A431 and Q444-S445. Cleavage reduces the antiviral function of PML and decomposes the formation of PML-NBs, which is conducive to virus replication.
| S-EPMC8686290 | biostudies-literature
Project description:Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (L(pro)) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C(pro), also inhibits IFN-α/β response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C(pro) of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-κB and interferon-regulatory factor signaling pathways. 3C(pro) specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3C(pro) abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C(pro) and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.
| S-EPMC3416110 | biostudies-literature