Hepatitis C Virus NS2 Protein Triggers Endoplasmic Reticulum Stress and Suppresses its Own Viral Replication

Annette von dem Bussche a, Raiki Machida a, Ke Li a, Gideon Loevinsohn a b, Amrin Khander a b, Jianguo Wanga b, Takaji Wakita b, Jack R. Wands a, Jisu Li a

Received 28 December 2009; received in revised form 22 April 2010; accepted 6 May 2010. published online 02 August 2010.
Accepted Manuscript

Abstract

Background & Aims
We previously reported that the NS2 protein of hepatitis C virus (HCV) inhibits the expression of reporter genes driven by a variety of cellular and viral promoters. The aim of the study was to determine whether the broad transcriptional repression is caused by endoplasmic reticulum (ER) stress.

Methods
Phosphorylation of the translation initiation factor eIF2α and HCV replication were detected by Western and Northern blot, respectively. De novo protein synthesis was measured by metabolic labeling. Activation of ER stress responsive genes was determined by promoter reporter assay, as well as mRNA and protein measurement by real time PCR and Western blot.

Results
Transient or inducible NS2 protein expression increased eIF2α phosphorylation and reduced de novo protein synthesis. It up-regulated promoter activities and transcript levels of ER stress inducible genes including GRP78, ATF6, and GADD153, as well as GRP78 protein level. The same effect was observed when NS2 was synthesized as part of the core-E1-E2-p7-NS2 polypeptide. NS2 protein also inhibited reporter gene expression from the HCV internal ribosome entry site and consequently reduced HCV replication. The full-length HCV replicon activated GRP78, ATF6, and GADD153 promoters more efficiently than the subgenomic replicon lacking the coding sequence for both the structural proteins and NS2. Abrogation of HCV infection/replication, by an inhibitor of the NS3 protease, relieved ER stress.

Conclusions
HCV infection can induce ER stress, with NS2 protein being a major mediator. The stress can be relieved by a feedback mechanism.

Abbreviations: HCV, hepatitis C virus, NS2, no-structural protein 2, ER, endoplasmic reticulum, eIF2α, eukaryotic translation initiation factor 2, GRP78, glucose regulated protein 78, ATF6, activating transcription factor 6, GADD153, growth arrest and DNA damage induced gene-153, CMV, cytomegalovirus, SV40, simian virus 40, TNFα, tumor necrosis factor alpha, NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells, HBV, hepatitis B virus, FL, full-length, SG, subgenomic, SEAP, secreted alkaline phosphatase, ECL, enhanced chemiluminescence, PERK, PKR-like ER kinase, IRE1, inositol-requiring enzyme 1, IRES, internal ribosomal entry site, DGD, glycine decarboxylase, p.t., post transfection

Keywords: HCV infection, ER stress, viral pathogenesis, NS2 protein

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a Liver Research Center, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, Rhode Island 02903, USA

b Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan

PII: S0168-8278(10)00622-7
doi:10.1016/j.jhep.2010.05.022
© 2010 Published by Elsevier Inc.

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