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Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis.

ABSTRACT: Enterovirus 71 (EV71), a member of the Picornaviridae family, may cause serious clinical manifestations associated with the central nervous system. Enterovirus 3C protease is required for virus replication and can trigger host cell apoptosis via cleaving viral polyprotein precursor and cellular proteins, respectively. Although the role of the 3C protease in processing viral and cellular proteins has been established, very little is known about the modulation of EV71 3C function by host cellular factors. Here, we show that sumoylation promotes EV71 3C protein ubiquitination for degradation, correlating with a decrease of EV71 in virus replication and cell apoptosis. SUMO E2-conjugating enzyme Ubc9 was identified as an EV71 3C-interacting protein. Further studies revealed that EV71 3C can be SUMO (small ubiquitin-like modifier)-modified at residue Lys-52. Sumoylation down-regulated 3C protease activity in vitro and also 3C protein stability in cells, in agreement with data suggesting 3C K52R protein induced greater substrate cleavage and apoptosis in cells. More importantly, the recombinant EV71 3C K52R virus infection conferred more apoptotic phenotype and increased virus levels in culture cells, which also correlated with a mouse model showing increased levels of viral VP1 protein in intestine and neuron loss in the spinal cord with EV71 3C K52R recombinant viral infection. Finally, we show that EV71 3C amino acid residues 45-52 involved in Ubc9 interaction determined the extent of 3C sumoylation and protein stability. Our results uncover a previously undescribed cellular regulatory event against EV71 virus replication and host cell apoptosis by sumoylation at 3C protease.

SUBMITTER: Chen SC

PROVIDER: S-EPMC3173077 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis.

Chen Shu-Chuan SC Chang Luan-Yin LY Wang Yi-Wei YW Chen Yi-Chun YC Weng Kuo-Feng KF Shih Shin-Ru SR Shih Hsiu-Ming HM

The Journal of biological chemistry 20110722 36

Enterovirus 71 (EV71), a member of the Picornaviridae family, may cause serious clinical manifestations associated with the central nervous system. Enterovirus 3C protease is required for virus replication and can trigger host cell apoptosis via cleaving viral polyprotein precursor and cellular proteins, respectively. Although the role of the 3C protease in processing viral and cellular proteins has been established, very little is known about the modulation of EV71 3C function by host cellular ...[more]

PMID: 21784861

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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.

Project description:Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCE:Infection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication.

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Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis.

Publications

Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis.

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