Figure 1.
Basic characteristics of the HCV-RMT clone.
Change in HCV copy number in chimeric mice. (A) Two mice were intravenously infected with 10 µl patient serum HCG9. (B) Three mice per group were directly injected with 30 µg HCV RNAs of the HCV-RMT strain or the JFH-1 strain into the liver. Data are indicated as the mean ± S.D. (C) Schematic representation of construction of the replicon and the sites of adaptive mutations. (D) Colony formation assay of replicon clones with adaptive mutations. Each RNA (1 µg) was electroporated into HuH7-K4 cells. (E) Western blot analysis of replicon cells. Each culture of replicon RNA-electroporated cells was maintained and passaged with G418 selection for 2 weeks. Cell lysates (10 µg) were loaded onto an SDS-PAGE gel.
Figure 2.
In vitro replication ability of HCV-RMT derivative genomes.
(A) Schematic representation of construction of the HCV genome and the sites of adaptive mutations (red bars). (B) Electroporation of the generated HCV-RNA genomes of wild-type HCV-RMT (closed triangles), HCV-RMT with triple mutations (HCV-RMTtri; closed circles), and the JFH-1 strain (open circles) into Huh-7.5.1 or HuH7-K4 cells. The experiments were carried out in duplicate. (C) Comparison of the in vitro replication ability of each HCV-RMT derivative in HuH7-K4 cells. The experiments were carried out in duplicate. Wild type: open squares, E1202G: open circles, E1056V: open triangles, A2199T: closed squares, E1056V and A2199T: closed triangles, triple mutations: closed circles. (D) Immunostaining for the HCV core protein in HCV-RNA-electroporated cells. Scale bar = 100 µm. The percent of HCV core protein-positive cells (%) was calculated as an average of ten observed areas.
Figure 3.
Establishment of HCV-RMTtri highly replicating #11 cell and infectivity of its supernatant on naïve HuH7-K4 cells.
(A) Immunostaining for the HCV core protein in HCV-RMTtri-electroporated parental cells and the cell clone (#11) obtained by limiting dilution cloning. Scale bar = 100 µm. (B) Immunostaining for the HCV core protein in naïve HuH7-K4 cells infected with supernatants of HCV-RMTtri- or JFH-1-replicating cells. Scale bar = 50 µm. (C) Infection with the HCV-RMTtri supernatant was inhibited with anti-CD81 antibody in a similar manner as JFH-1. Control IgG (normal mouse IgG1): open circles, anti-CD81 mAb (JS-81): closed circles. Data are indicated as the mean ± S.D. (D) Replication of HCV-RMTtri in HuH7-K4 cells was inhibited by HCV replication inhibitors such as cyclosporin A and interferon-α. Drugs were added to #11 cells in 96-well plates 1 day after passaging, and cells were harvested after 72 h of treatment. NT: no treatment.
Figure 4.
In vivo replication ability of HCV-RMT derivatives.
(A) Change in HCV copy numbers in the serum of chimeric mice in which the HCV genome was directly injected into the livers. HCV-RMT (wild type): closed circles, HCV-RMT (E1056V and A2199T): open circles, HCV-RMT (E1202G): closed triangles, HCV-RMTtri: closed squares, JFH-1: open triangles. Data are indicated as the mean ± S.D. (B) HCV copy number in the livers. N.D.: not detected. (C) Serum/liver ratio of HCV copy number.
Table 1.
Relationship between the in vitro and in vivo replication ability of HCV-RMT derivatives.