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
Abstract
AIM

To explore hepatitis C virus (HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.

METHODS

A series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and cell lysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets (LDs) with NS5A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5A was analyzed further.

RESULTS

The plasmids named JFH1-mE2, JFH1-mp7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, JFH1-mE2/p7/NS5A, and mJFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units (FFUs)/mL. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) were 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. However, the six adaptive mutations were not required for the LD localization of NS5A proteins or the phosphorylation of NS5A.

CONCLUSION

In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/mL, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.

Keywords: Hepatitis C virus; JFH1; Adaptive mutation; RNA replication; Virion release; Lipid droplet localization

Core tip: In this study, we explored hepatitis C virus (HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigated the underlying mechanisms. We generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units (FFUs)/mL, and confirmed that the adaptive mutations could enhance viral replication and release. The results were established at the levels of infectious particle titers, HCV RNA, protein expression, virus release, lipid droplet, and NS5A co-localization, and further the ratio between p56 and p58 phosphoforms of NS5A.