Ubiquitination Is Required for Effective Replication of Coxsackievirus B3

Xiaoning Si,Guang Gao,Jerry Wong,Yahong Wang,Jingchun Zhang,Honglin Luo,Dong-Yan Jin

doi:10.1371/journal.pone.0002585

Abstract

BackgroundProtein ubiquitination and/or degradation by the ubiquitin/proteasome system (UPS) have been recognized as critical mechanisms in the regulation of numerous essential cellular functions. The importance of the UPS in viral pathogenesis has become increasingly apparent. Using murine cardiomyocytes, we have previously demonstrated that the UPS plays a key role in the replication of coxsackievirus B3 (CVB3), an important human pathogen associated with various diseases. To further elucidate the underlying mechanisms, we examined the interplay between the UPS and CVB3, focusing on the role of ubiquitination in viral lifecycle.Methodology/Principal FindingsAs assessed by in situ hybridization, Western blot, and plaque assay, we showed that proteasome inhibition decreased CVB3 RNA replication, protein synthesis, and viral titers in HeLa cells. There were no apparent changes in 20S proteasome activities following CVB3 infection. However, we found viral infection led to an accumulation of protein-ubiquitin conjugates, accompanied by a decreased protein expression of free ubiquitin, implicating an important role of ubiquitination in the UPS-mediated viral replication. Using small-interfering RNA, we demonstrated that gene-silencing of ubiquitin significantly reduced viral titers, possibly through downregulation of protein ubiquitination and subsequent alteration of protein function and/or degradation. Inhibition of deubiquitinating enzymes apparently enhances the inhibitory effects of proteasome inhibitors on CVB3 replication. Finally, by immunoprecipitation, we showed that coxsackieviral polymerase 3D was post-translationally modified by ubiquitination and such modification might be a prerequisite for its function in transcriptional regulation of viral genome.ConclusionCoxsackievirus infection promotes protein ubiquitination, contributing to effective viral replication, probably through ubiquitin modification of viral polymerase.

Highlights

Coxsackievirus B3 (CVB3), a small RNA virus in the picornaviridae family, is an important human pathogen associated with various diseases, including myocarditis, aseptic meningitis, pancreatitis and possibly insulin-dependent diabetes
We have previously shown that coxsackievirus B3 (CVB3) infection results in downregulation of several host proteins [7,9], such as cell-cycle protein cyclin D1, tumor suppressor p53, and transcription activator b-catenin in infected HeLa cells
We further showed that knockdown of ubiquitin expression by small-interfering RNA decreased CVB3 infection, likely through the downregulation of ubiquitination and subsequent alteration of protein function and/or degradation

Summary

Introduction

Coxsackievirus B3 (CVB3), a small RNA virus in the picornaviridae family, is an important human pathogen associated with various diseases, including myocarditis, aseptic meningitis, pancreatitis and possibly insulin-dependent diabetes. We and others have shown that CVB3 infection leads to activation of several intracellular signaling pathways [1,2,3,4,5,6,7], and downregulation of host proteins likely through the ubiquitin/proteasome system (UPS) [7,8,9]. It is well-established that the UPS is the major intracellular proteolytic system of all eukaryotic cells [10,11]. To further elucidate the underlying mechanisms, we examined the interplay between the UPS and CVB3, focusing on the role of ubiquitination in viral lifecycle

Methods

Results

Discussion

Conclusion