Hepatitis C Virus Nonstructural 4B Protein Modulates Sterol Regulatory Element-binding Protein Signaling via the AKT Pathway

Chul-Yong Park,Hyun-Jeong Jun,Takaji Wakita,Jae Hun Cheong,Soon B Hwang

doi:10.1074/jbc.m808773200

Abstract

Hepatitis C virus (HCV) infection is often associated with hepatic steatosis and yet the molecular mechanisms of HCV-associated steatosis are poorly understood. Because sterol regulatory element-binding proteins (SREBPs) are the major transcriptional factors in lipogenic gene expression including fatty acid synthase (FAS), we examined the effects of HCV nonstructural proteins on the signaling pathways of SREBP. In this study, we demonstrated that HCV nonstructural 4B (NS4B) protein increased the transcriptional activities of SREBPs. We also showed that HCV NS4B enhanced the protein expression levels of SREBPs and FAS. This was further confirmed in the context of viral RNA replication and HCV infection. The up-regulation of both SREBP and FAS by NS4B protein required phosphatidylinositol 3-kinase activity. We also demonstrated that NS4B protein induced a lipid accumulation in hepatoma cells. In addition, NS4B protein synergistically elevated the transcriptional activity of HCV core-mediated SREBP-1. These results strongly suggest that NS4B may play an important role in HCV-associated liver pathogenesis by modulating the SREBP signaling pathway.

Highlights

Steatosis is more frequent in patients infected with Hepatitis C virus (HCV) genotype 3 than in patients infected with other HCV genotypes, even though not all patients infected with genotype 3 have steatosis [15, 16]
HCV nonstructural 4B (NS4B) Protein Increases the Transcriptional Activity of Sterol regulatory element-binding proteins (SREBPs)-1—Because SREBPs are the major transcription factors for lipogenic gene expression, we investigated the effects of HCV nonstructural proteins on transcriptional activity of SREBP-1
We investigated whether promoter activity of SREBP-1 was increased by the NS4B protein

Summary

EXPERIMENTAL PROCEDURES

Plasmids—NS4B-Myc of HCV (genotype 1b) was amplified by PCR using the Korean isolate of HCV [22] as a template and subcloned into the pEF6B/His-Myc (Invitrogen) vector. Huh cells transfected with empty vector (pEF6B only) were selected as described above and used as a control. The membrane was blocked in phosphate-buffered saline (PBS) containing 5% nonfat dry milk for 1 h and incubated 2 h at room temperature with one of following antibodies: anti-FAS antibody (BD Transduction Laboratories), anti-SREBP-1 antibody (BD Transduction Laboratories, Santa Cruz Biotechnology, Inc.), anti-SREBP-2 antibody (BD Transduction Laboratories), anti-AKT and p-AKT antibody (Cell signaling Technology, Beverly, MA), anti-␤-actin and anti-FLAG antibody (Sigma), anti-Myc and anti-HA antibody (Santa Cruz Biotechnology, Inc.), anti-NS4B antibody (Virostat), and rabbit anti-NS5A polyclonal antibody in Tris-buffered saline/Tween (20 mmol/liter Tris-HCl (pH 7.5), 500 mmol/liter NaCl, and 0.05% Tween 20). RNA Isolation and Reverse Transcription-PCR—Total RNAs were isolated from vector, NS4B-myc stable, IFN-cured, and HCV subgenomic-replicon cells using TRIzol reagent (Invitrogen). The Student’s t test was used for statistical analysis. p Ͻ 0.05 was considered statistically significant

RESULTS

DISCUSSION

We have further shown that HCV