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

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

Figure 1

Schematic representation of adaptive mutations used in this study (A) and the electrophoresis results of each mutant virus RNA (B). A: Both nucleotide substitutions (2310, 2681, 6132, 7160, 7359, and 7658) and amino acid substitutions (D657G, H781Y, N1931S, C2274R, I2340T, and V2440L) are shown; B: HCV RNA (500 ng) was analyzed using formaldehyde agarose gel electrophoresis. Lane 1: JFH1; Lane 2: JFH1-mE2; Lane 3: JFH1-mP7; Lane 4: JFH1-mNS4B; Lane 5: JFH1-mNS5A; Lane 6: JFH1-mE2/NS5A; Lane 7: JFH1-mp7/NS5A; Lane 8: JFH1-mNS4B/NS5A; Lane 9: mJFH1; Lane 10: JFH1-mE2/p7/NS5A; M: RNA marker. HCV: hepatitis C virus.

Figure 2

Generation of high titer cell culture-adaptive JFH1 virus. Hepatitis C virus RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus in cell culture. The transfected cells were passaged every three days. The infectivity titers of the culture supernatants at day 3 (P1) and day 9 (P3) were measured. Viral titers are expressed as focus-forming units per milliliter (FFUs/mL). The data are presented as mean ± SD (n = 3). HCV: Hepatitis C virus. ^aP < 0.05; ^bP < 0.01.

Figure 3

Effects of the adaptive mutations on the hepatitis C virus RNA replication. A: Hepatitis C virus (HCV) RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. The transfected cells were passaged every 3 d. Cells were fixed 48 h after passage and infected cells were identified by fluorescence immunostaining and microscopy. Nuclear DNA was stained with DAPI (blue); B: HCV RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus in cell culture. The transfected cells were passaged every 3 d. Cells were lysed at 72 h after passage. The HCV NS3 protein levels were analysis by Western blot. ^bP < 0.01; C: HCV RNA levels in cells 3 d after transfection. Intracellular HCV RNA levels were analyzed by quantitative RT-PCR. The mean ± SD for three independent experiments are presented (qPCR assays, n = 3). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 4

Effect of the adaptive mutations on the virion release. A: Hepatitis C virus (HCV) RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. At 72 h after transfection, the infectivity titers of the culture supernatants and cell lysates were measured. Viral titers are expressed as FFUs/mL. The data are presented as mean ± SD (n = 3); B: HCV RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. At 72 h after transfection, the infectivity titers of the culture media and cell lysates were measured. The extracellular and intracellular viral titers were measured. The relative ratios of infectious virions are shown. The results were from three independent experiments; C: The naive Huh7.5 cells were infected with the culture media and cell lysates. At 72 h after infection, cells were lysed with RIPA buffer, and analyzed by Western blot.

Figure 5

Colocalization analysis of lipid droplets and hepatitis C virus NS5A. JFH1 and mJFH1 RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. At 48 h after transfection, the cells were fixed. Lipid droplets were stained with LipidTOXRed (Red). The HCV NS5A was stained with anti-NS5A antibody (Green). The nucleus was stained with DAPI (Blue). Each triplicate sample of 25 cells was analyzed using Image J software. The degree of co-localization was quantified and compared using Pearson’s correlation coefficients.

Figure 6

Phosphorylation of NS5A during JFH1 and mJFH1 replication. Huh7.5 cells were transfected with JFH1 or mJFH1 RNA. After three days of culture, cells were lysed for western blot using anti-NS5A and anti-β-actin antibodies. The quantity of p56 and p58 was determined using Image J software and the ratios of p56/p58 are shown. Data are presented as mean ± SD (n = 3). JFH1-AM120 was used as the positive control.