Hepatitis C virus NS2 protein triggers endoplasmic reticulum stress and suppresses its own viral replication.
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.



Phosphorylation of the translation initiation factor eIF2α and HCV replication was 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.



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.



HCV infection can induce ER stress, with NS2 protein being a major mediator. The stress can be relieved by a feedback mechanism.
von dem Bussche
Annette
A
Liver Research Center, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.
Machida
Raiki
R
Li
Ke
K
Loevinsohn
Gideon
G
Khander
Amrin
A
Wang
Jianguo
J
Wakita
Takaji
T
Wands
Jack R
JR
Li
Jisu
J
D051702:Activating Transcription Factor 6 / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D051742:Transcription Factor CHOP / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication
C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; C497249:DDIT3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C097010:NS2 protein, Hepatitis C virus; D017361:Viral Nonstructural Proteins; D051742:Transcription Factor CHOP
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