Original Article
Copyright ©2011 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Aug 7, 2011; 17(29): 3398-3406
Published online Aug 7, 2011. doi: 10.3748/wjg.v17.i29.3398
Function of nonstructural 5A protein of genotype 2a in replication and infection of HCV with gene substitution
Yong-Zhi Wang, Wen-Bo Wang, Ming-Mei Cao, Wen Wang, Lan-Juan Zhao, Gang Xu, Hao Ren, Zhong-Tian Qi
Yong-Zhi Wang, Department of Cardiology, NO. 254 Hospital PLA, Tianjin 300142, China
Wen-Bo Wang, Ming-Mei Cao, Wen Wang, Lan-Juan Zhao, Gang Xu, Hao Ren, Zhong-Tian Qi, Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai 200433, China
Author contributions: Wang YZ and Wang WB contributed equally to this work; Ren H and Qi ZT designed the research; Wang YZ, Wang WB, Cao MM, Wang W, Zhao LJ and Xu G performed the research; Wang YZ and Ren H wrote the manuscript.
Supported by The Natural Science Foundation of China, No. 30872247 and 30600529; the PLA medical research funds of China, No. 06H021 and 06Z027 and Shanghai LAD Project (B901)
Correspondence to: Hao Ren, MD, PhD, Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. renhao980@sohu.com
Telephone: +86-21-81870996 Fax: +86-21-81870988
Received: November 3, 2010
Revised: February 26, 2011
Accepted: March 5, 2011
Published online: August 7, 2011
Abstract

AIM: To explore the function of Nonstructural 5A (NS5A) protein of genotype 2a (JFH1) in the replication and infection of hepatitis C virus (HCV).

METHODS: Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR) method and the restriction enzyme reaction. In vitro RNA transcripts of chimera, prototype J6JFH and negative control J6JFH1 (GND) were prepared and transfected into Huh-7.5 cells with liposomes. Immunofluorescence assay (IFA), fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.

RESULTS: The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point (2.58 ± 5.97 × 106vs 4.27 ± 1.72 × 104, P = 0.032). The maximal level of HCV RNA in chimera was 5.6 ± 1.8 × 104 GE/μg RNA at day 34 after transfection, while the wild type reached a peak level at day 13 which was 126 folds higher (70.65 ± 14.11 × 105vs 0.56 ± 0.90 × 105, P = 0.028). HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level. Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles, ranging from 10 ± 5 ffu/mL to 78.3 ± 23.6 ffu/mL, while that of FL-J6JFH1 ranged from 5.8 ± 1.5 × 102 ffu/mL to 2.5 ± 1.4 × 104 ffu/mL.

CONCLUSION: JFH1 NS5A might play an important role in the robust replication of J6JFH1. The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.

Keywords: Hepatitis C virus; Nonstructural 5A; Chimera; Cell culture-produced virus; Replication; Infection