Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1

Abstract

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.

Fig 1

Replication and virus production by MA/JFH-1 chimeras in Huh7.5.1 cells. (A) Schematic structures of JFH-1, MA, and two MA/JFH-1 chimeras (MA/JFH-1.1 and MA/JFH-1.2). The junction of JFH-1 and MA in the 5′ UTR is an AgeI site, and the junction of MA and JFH-1 in the NS2 region is a BsaBI site. A, AgeI; B, BsaBI; X, XbaI. (B to E) Chimeric HCV RNA replication in Huh7.5.1 cells. HCV core protein level in cells (B) and culture medium (C) and HCV RNA levels in medium (D) and infectivity of culture medium (E) from HCV RNA-transfected Huh7.5.1 cells are shown. Ten micrograms of HCV RNA was transfected into Huh7.5.1 cells, and cells and culture medium were harvested on days 1, 2, and 3. n.d., not determined. Assays were performed three times independently, and data are presented as means ± standard deviation. Dashed line indicates detection limit. wt, wild type.

Fig 2

Long-term culture of MA/JFH-1.1 and MA/JFH-1.2 RNA-transfected cells. Ten micrograms of HCV RNA was transfected into Huh7.5.1 cells, and cells were passaged every 2 to 5 days, depending on cell status. Culture medium was collected after every passage, and HCV core protein levels were measured. Transfection was performed twice for each chimeric RNA (1 and 2 for each construct). (A) HCV core protein levels in culture medium from MA/JFH-1.1 and MA/JFH-1.2 RNA-transfected cells. (B) Immunostained cells at 3 days after transfection (a to d), at 21 days after transfection (e to h), and at the time of peak core levels (days 42 to 49). Infected cells were visualized with anti-core protein antibody (green), and nuclei were visualized with DAPI (blue). (C) Infection of naïve cells by culture medium at an MOI of 0.001. (D) Immunostained cells at 15 days after infection with medium at peak core protein levels (Fig. 2A) at an MOI of 0.001. Infected cells were visualized with anti-core antibody (green), and nuclei were visualized with DAPI (blue).

Fig 3

Effects of R167G on replication and virus production of MA/JFH-1.2 in Huh7.5.1 cells. Ten micrograms of HCV RNA was transfected into Huh7.5.1 cells, and cells and medium were harvested on days 1, 2, and 3. HCV core protein levels in the cells (A) and culture medium (B) and HCV RNA levels in the medium (C) and the infectivity of culture medium (D) from HCV RNA-transfected Huh7.5.1 cells are shown. n.d., not determined. Dashed line indicates the detection limit. Assays were performed three times independently, and data are presented as means ± standard deviation. (E) HCV core protein levels in culture medium from cells infected with medium at 3 days posttransfection at an MOI of 0.005. (F) Immunostained cells at 19 days postinfection. Infected cells were visualized with anti-core antibody (green), and nuclei were visualized with DAPI (blue).

Fig 4

Effects of R167G on replication and virus production of MA/JFH-1.2 and JFH-1 in Huh7-25 cells. Ten micrograms of HCV RNA was transfected into Huh7-25 cells, and cells and medium were harvested on days 1, 2, and 3. HCV core protein levels in cells (A and D) and in medium (B and E) were measured, and infectivity of medium (C and F) was determined. n.d., not determined. Dashed line indicates the detection limit. (G) Intracellular specific infectivity and virus secretion efficiency of chimeric HCV RNA-transfected cells. Intracellular and extracellular infectivity of day 3 samples was determined, and specific infectivity and virus secretion rate were calculated. Assays were performed three times independently, and data are presented as means ± standard deviation. NA, not available.

Fig 5

Replication and virus production of MA/N3H+N5BX-JFH1/R167G in Huh7.5.1 cells. (A) Schematic structures of JFH-1, MA, and MA/N3H+N5BX-JFH1. The junction of JFH-1 and MA in the 5′ UTR is an AgeI site; the junctions of MA and JFH-1 in the NS3 regions are ClaI and EcoT22I sites, and the junction in the NS5B region is a BsrGI site. A, AgeI; X, XbaI. (B to G) Chimeric HCV RNA replication in Huh7.5.1 cells. Ten micrograms of HCV RNA was transfected into Huh7.5.1 cells, and cells and medium were harvested on days 1, 3, and 5. HCV core protein levels in cells (B) and in medium (C) and HCV RNA levels in medium (D) were measured, and infectivity of medium (E) was determined. Assays were performed three times independently, and data are presented as means ± standard deviation. n.d., not determined. Dashed line indicates the detection limit. (F) Immunostained cells. Huh7.5.1 cells were infected with concentrated medium from RNA-transfected cells on day 5. Infected cells were visualized with anti-core antibody (green), and nuclei were visualized with DAPI (blue). (G) Infectivity of concentrated culture medium from HCV RNA-transfected cells. Culture medium was concentrated by 20 times. Infectivities of original and concentrated culture media were determined. Dashed line indicates detection thelimit.

Fig 6

Cell culture-adaptive mutations enhanced infectious virus production of MA/N3H+N5BX-JFH1/R167G. (A) Long-term culture of MA/N3H+N5BX-JFH1 and MA/N3H+N5BX-JFH1/R167G RNA-transfected cells. Ten micrograms of HCV RNA was transfected into Huh7.5.1 cells, and cells were passaged every 2 to 5 days, depending on cell status. Culture medium was collected after every passage, and HCV core protein levels were measured. HCV core protein levels in culture medium from MA/N3H+N5BX-JFH1 and MA/N3H+N5BX-JFH1/R167G RNA-transfected cells are presented. (B) Immunostained cells on days 5 and 54 after transfection. Infected cells were visualized with anti-core antibody (green), and nuclei were visualized with DAPI (blue). (C to F) Effect of four additional cell culture-adaptive mutations on virus production. Ten micrograms of HCV RNA was transfected into Huh7.5.1 cells, and cells and medium were harvested on days 1, 3, and 5. HCV core levels in cells (C) and in medium (D) and HCV RNA levels in medium (E) were measured, and infectivity of medium (F) was determined. Assays were performed three times independently, and data are presented as means ± standard deviation. n.d., not determined. Dashed line indicates the detection limit.

Fig 7

Comparisons of interferon sensitivity between JFH-1, MA/JFH-1.2/R167G and MA/N3H+N5BX-JFH1/5am. Two micrograms of HCV RNA was transfected into Huh7.5.1 cells, and interferon was added at the indicated concentrations at 4 h after transfection. HCV core protein levels in cells (A) and in medium (B) on day 3 were measured, and data are expressed as percent versus untreated cells (0 IU/ml). Assays were performed three times independently, and data are presented as means ± standard deviation.