Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Mar 28, 2018; 24(12): 1299-1311
Published online Mar 28, 2018. doi: 10.3748/wjg.v24.i12.1299
Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release
Qi Wang, Yue Li, Shun-Ai Liu, Wen Xie, Jun Cheng
Qi Wang, Wen Xie, Jun Cheng, Center of Liver Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
Qi Wang, Shun-Ai Liu, Jun Cheng, Beijing Key Laboratory of Emerging Infectious Diseases, Beijing 100015, China
Yue Li, Department of Pathology, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
Shun-Ai Liu, Jun Cheng, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
Author contributions: Wang Q and Li Y contributed equally to this work; Wang Q and Li Y performed the experiments and analyzed the data; Wang Q and Cheng J designed the research; Wang Q and Li Y wrote the manuscript; Xie W revised the manuscript; Liu SA provided vital reagents and analytical tools.
Supported by Beijing Natural Science Foundation, No. 7161006; and Beijing Municipal Administration of Hospitals’ Youth Program, No. QML20161801 and No. QML20171801.
Institutional review board statement: This study did not involve any animal experiments or human specimens, and thus was exempted from ethical review according to the Human Research Management Stipulation of the Beijing Ditan Hospital Affiliated to the Capital University of Medical Sciences.
Conflict-of-interest statement: The authors declare that there are no conflicts of interest in this study.
Data sharing statement: Technical appendix, statistical code, and data set available from the corresponding author at chengj0817@ccmu.edu.cn. 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: Jun Cheng, MD, PhD, Professor, Center of Liver Diseases, Beijing Ditan Hospital, Capital Medical University, No. 8, Jingshun East Street, Chaoyang District, Beijing 100015, China. chengj0817@ccmu.edu.cn
Telephone: +86-10-84322006 Fax: +86-10-84397196
Received: December 1, 2017
Peer-review started: December 1, 2017
First decision: December 20, 2017
Revised: January 4, 2018
Accepted: January 17, 2018
Article in press: January 17, 2018
Published online: March 28, 2018
Processing time: 115 Days and 5.2 Hours
ARTICLE HIGHLIGHTS
Research background

Hepatitis C virus (HCV) causes acute and chronic hepatitis, and leads to permanent liver damage and hepatocellular carcinoma. The infectious HCV JFH1 cell culture system represents a major advance in anti-HCV drug discovery research and facilitates the study of HCV life cycle. However, HCV JFH1 (genotype 2a) merely generates relatively low viral titers. JFH1-∆V3-EGFP, which includes six mutations located in the E2, p7, NS4B, and NS5A regions, could produce higher titers of HCV-EGFP reporter virus. However, there were no data about which mutations or combinations thereof are responsible for enhanced viral production and the underlying mechanisms.

Research motivation

This JFH1 model generated infectious viral particles in cell culture and facilitated the study of the HCV life cycle, but the low infectious virion titer limits its application range. Some previous studies have confirmed that adaptive mutations could enhance the virion titer, but the mechanism has not yet been fully elucidated. In this study, we focused on the positive effect of six adaptive mutations located in the E2, p7, NS4B, and NS5A regions, and found that the mechanism was different among them during the procession. These results gave us some new insights into the infectious HCV cell culture system and adaptive mutations.

Research objectives

The main objective of this study was to establish an infectious HCV cell culture system with a robust titer, and to discuss the underlying mechanisms of the adaptive mutations found in previous studies. The results of this study have supplied the researchers with a useful tool. We hope it will be used for the study of viral structure, virus-host interaction, anti-HCV drug discovery, and vaccine development.

Research methods

We investigated JFH1-mE2, JFH1-mp7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, JFH1-mE2/p7/ NS5A, and mJFH1, carrying all the six mutations. We analyzed the infectious HCV titer, HCV RNA and NS3 protein levels, viral release capacity, assembly and hyper-phosphorylation of NS5A to determine the role of these mutations in the HCV life cycle. These methods were the routine ways adopted widely in virological and molecular biological research.

Research results

The main findings in this study were as follows: (1) we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/mL; (2) The six adaptive mutations increased the HCV particle production at varying levels. The NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) are critical adaptive mutations. The effect of NS5A (C2274R, I2340T, and V2440L), p7 (H781Y), and NS4B (N1931S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release; and (3) the six adaptive mutations were all not required for the lipid droplet localization of NS5A proteins or the phosphorylation of NS5A. To our knowledge, this is a new robust titer related to adaptive mutations from JFH1. The problems that remain to be solved in the future include: (1) how could the adaptive mutations be translated to clinical conditions? (2) are these mutation patterns observed in vivo? and (3) would these results be relevant to the resistance to direct-acting antivirals (DAAs)?

Research conclusions

First, this study generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/mL. Second, all of the six adaptive mutations increased the HCV particle production at varying levels. Third, the NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) were critical adaptive mutations, but they were not required for the LD localization of NS5A proteins or the phosphorylation of NS5A. Based on the new findings of this study, we proposed that more important adaptive mutations would be addressed in the future, and unknown mechanism of the HCV life cycle would be explained.

Research perspectives

This study re-confirmed that the JFH1 was still a promising system to study the HCV life cycle. To use adaptive mutations was an effective way to establish a new system with higher infectious HCV virion titer. In addition, we also re-confirmed that the molecular mechanism of interaction between viral proteins and/or host-cell proteins is more complex and important.