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