An interaction between glutathione and the capsid is required for the morphogenesis of C-cluster enteroviruses.

Hsin-Chieh Ma,Chunling Wang,Ying-Han Chen,Nina Rehage,Eckard Wimmer,Nihal Altan-Bonnet,Michael Strauss,Aniko V Paul,Ping Jiang,Steffen Mueller,James Hogle,Ying Liu,Richard J Kuhn

doi:10.1371/journal.ppat.1004052

Abstract

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.

Highlights

Glutathione (GSH), c-L-glutamyl-L-cysteinylglycine, is an important cellular reducing agent, which prevents damage to cellular components caused by free radicals or peroxides
We describe the role of glutathione, an important cellular reducing agent, in enterovirus morphogenesis by studying the inhibition of GSH biosynthesis with buthionine sulfoximine (BSO) on viral proliferation
We propose a model to explain the role of GSH in enterovirus morphogenesis, which is to stabilize the capsid precursors and the mature virus during and after the encapsidation of the progeny viral RNA by direct interaction with capsid proteins

Summary

Introduction

Glutathione (GSH), c-L-glutamyl-L-cysteinylglycine, is an important cellular reducing agent, which prevents damage to cellular components caused by free radicals or peroxides. GSH has roles in signal transduction, gene expression and apoptosis [1]. The thiol group (SH) of GSH’s cysteine serves as a proton donor and is responsible for the biological activity of GSH. Glutathione is present in several forms in cells, tissues, and plasma, at a high concentration of about 5 mM. It primarily exists in free form either in a reduced (GSH) or in an oxidized state (GSSG). GSH is capable of forming disulfide bonds with cysteine residues in proteins and in its bound form it regulates protein function [1]

Objectives

Methods

Results

Conclusion