Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release  

作　　者：Qi Wang Yue Li Shun-Ai Liu Wen Xie Jun Cheng 

机构地区：[1]Center of Liver Diseases,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China [2]Department of Pathology,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China [3]Institute of Infectious Diseases,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China [4]Beijing Key Laboratory of Emerging Infectious Diseases,Beijing 100015,China

出　　处：《World Journal of Gastroenterology》2018年第12期1299-1311,共13页世界胃肠病学杂志（英文版）

基　　金：Beijing Natural Science Foundation,No.7161006;Beijing Municipal Administration of Hospitals’ Youth Program,No.QML20161801 and No.QML20171801

摘　　要：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 NS5 A 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 celllysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets(LDs) with NS5 A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5 A was analyzed further.RESULTS The plasmids named JFH1-m E2, JFH1-mp7, JFH1-m NS4 B, JFH1-m NS5 A, JFH1-m E2/NS5 A, JFH1-mp7/NS5 A, JFH1-m NS4 B/NS5 A, JFH1-m E2/p7/NS5 A, and m JFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units(FFUs)/m L. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5 A(C2274 R, I2340 T, and V2440 L) and p7(H781 Y) were critical adaptive mutations. The effect of NS5 A(C2274 R, I2340 T, and V2440 L), p7(H781 Y), and NS4 B(N1931 S) 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 NS5 A proteins or the phosphorylation of NS5 A.CONCLUSION In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/m L, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.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 NS5 A 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 celllysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets(LDs) with NS5 A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5 A was analyzed further.RESULTS The plasmids named JFH1-m E2, JFH1-mp7, JFH1-m NS4 B, JFH1-m NS5 A, JFH1-m E2/NS5 A, JFH1-mp7/NS5 A, JFH1-m NS4 B/NS5 A, JFH1-m E2/p7/NS5 A, and m JFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units(FFUs)/m L. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5 A(C2274 R, I2340 T, and V2440 L) and p7(H781 Y) were critical adaptive mutations. The effect of NS5 A(C2274 R, I2340 T, and V2440 L), p7(H781 Y), and NS4 B(N1931 S) 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 NS5 A proteins or the phosphorylation of NS5 A.CONCLUSION In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/m L, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.

关 键 词：HEPATITIS C virus JFH1 Adaptive mutation RNA REPLICATION VIRION RELEASE Lipid droplet localization 

分 类 号：R57[医药卫生—消化系统]