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Updated: Jun 9, 2026
A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
Hepatitis C virus NS2 protein triggers endoplasmic reticulum stress and suppresses its own viral replication
Annette von dem Bussche1, Raiki Machida, Ke Li
1Liver Research Center, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.
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 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.
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.
Insights
Hepatitis C virus (HCV) NS2 protein induces endoplasmic reticulum (ER) stress, impacting protein synthesis and viral replication. This ER stress response can be alleviated through a feedback mechanism, offering insights into HCV pathogenesis.
Area of Science:
- Virology
- Cellular Biology
- Molecular Biology
Background:
- Hepatitis C virus (HCV) NS2 protein previously shown to inhibit reporter gene expression from various promoters.
- The study investigates if this broad transcriptional repression is mediated by endoplasmic reticulum (ER) stress.
Purpose of the Study:
- To determine if HCV NS2 protein-mediated transcriptional repression is caused by ER stress.
- To elucidate the role of NS2 protein in inducing ER stress during HCV infection.
Main Methods:
- Western and Northern blot analyses for eIF2α phosphorylation and HCV replication.
- Metabolic labeling to measure de novo protein synthesis.
- Promoter reporter assays, real-time PCR, and Western blot to assess ER stress responsive genes.
Main Results:
- NS2 protein expression elevated eIF2α phosphorylation, reduced protein synthesis, and upregulated ER stress genes (GRP78, ATF6, GADD153).
- HCV replication, particularly full-length replicons, enhanced ER stress gene activation.
- Inhibiting HCV replication with an NS3 protease inhibitor relieved ER stress.
Conclusions:
- HCV infection induces ER stress, with NS2 protein identified as a key mediator.
- A feedback mechanism exists to relieve ER stress during HCV infection.
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