Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 28, 2017; 23(28): 5158-5166
Published online Jul 28, 2017. doi: 10.3748/wjg.v23.i28.5158
Single amino acid mutation of SR-BI decreases infectivity of hepatitis C virus derived from cell culture in a cell culture model
Rong Gao, Wei Gao, Gang Xu, Jie Xu, Hao Ren
Rong Gao, Wei Gao, Gang Xu, Jie Xu, Hao Ren, Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai 200433, China
Author contributions: Gao R, Gao W and Xu J performed the experiments; Xu G and Ren H designed the study and contributed to data acquisition, analysis and interpretation; Gao R and Ren H drafted and critically revised the manuscript; all authors approved the final version of the article to be published; Gao R and Gao W contributed equally to this work.
Supported by National Natural Science Foundation of China, No. 31370196; and the National 973 Program, No. 2013CB531601.
Institutional review board statement: The study was approved by the Institutional Review Board of the Second Military Medical University.
Institutional animal care and use committee statement: No animals were used in this experiment.
Conflict-of-interest statement: The authors have no conflicts of interest regarding this article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Hao Ren, Professor, Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. renh@smmu.edu.cn
Telephone: +86-21-81870996 Fax: +86-21-81870988
Received: April 13, 2017
Peer-review started: April 18, 2017
First decision: May 16, 2017
Revised: May 24, 2017
Accepted: July 4, 2017
Article in press: July 4, 2017
Published online: July 28, 2017
Abstract
AIM

To investigate the effect of a single amino acid mutation in human class B scavenger receptor I (SR-BI) on the infectivity of cell culture-derived hepatitis C virus (HCVcc) in SR-BI knock-down Huh7-siSR-BI cells.

METHODS

Site-directed mutagenesis was used to construct the SR-BI S112F mutation, and the mutation was confirmed by nucleotide sequencing. SR-BI knock-down Huh7-siSR-BI cells were transfected with SR-BI S112F, SR-BI wild type (WT) and control plasmids, and then infected with HCVpp (HCV pseudoparticles) and hepatitis C virus derived from cell culture (HCVcc). A fluorescence assay was performed to analyze the effect of the S112F mutation on HCV entry; quantitative real-time PCR, immunofluorescence, and Western blot assays were used to analyze the effect of the S112F mutation on HCV infectivity. CHO cells expressing WT and SR-BI S112F were incubated with the HCV E2 protein expressed in HEK 293T cells, and flow cytometry was performed to examine the ability of SR-BI S112F to bind to the HCV E2 protein. Huh7-siSR-BI cells were transfected with SR-BI WT and the S112F mutant, and then DiI-HDL was added and images captured under the microscope to assess the ability of SR-BI S112F to take up HDL.

RESULTS

The SR-BI S112F mutation was successfully constructed. The S112F mutation decreased the expression of the SR-BI mRNA and protein. SR-BI S112F decreased HCV entry and HCVcc infectivity in Huh7-siSR-BI cells. The S112F mutation impaired the binding of SR-BI to HCV E2 protein and decreased the HDL uptake of SR-BI.

CONCLUSION

The S112F single amino acid mutation in SR-BI decreased the levels of the SR-BI mRNA and protein, as well as the ability of SR-BI to bind to the HCV E2 protein. Amino acid 112 in SR-BI plays important roles in HCV entry and the infectivity of HCVcc in vitro.

Keywords: Hepatitis C virus, Class B scavenger receptor I, Mutant, Infectivity

Core tip: Human class B scavenger receptor I (SR-BI) plays important roles in both host lipid metabolism and the entry of hepatitis C virus (HCV). Single nucleotide polymorphisms (SNPs) in the host genome that affect the virus-host interaction have received increasing attention in recent years. Several SR-BI SNPs have been reported to affect the high-density lipoprotein cholesterol levels in populations carrying SR-BI mutations; however, the impact of SR-BI SNPs on HCV infection has not been studied intensively. Based on our results, the S112F single amino acid mutation in SR-BI inhibited the infectivity of hepatitis C virus derived from cell culture in a cell culture model by downregulating the expression of the SR-BI protein.