Figure 1

Construction and characterization of the recombinant VT7-HCV7.9 virus. A: Generation of recombinant VT7-HCV7.9. A 7.9 Kb DNA fragment containing the structural (C, E1, E2 and p7) and nonstructural (NS3, NS4A, NS4B, NS5A and the amino terminal region of NS5B) proteins of HCV from genotype 1b was cloned into a unique EcoRI restriction site of pVOTE.1 to make the plasmid transfer vector pVOTE.1-HCV7.9. BSC40 cells infected with the recombinant VT7lacOI (VT7), were transfected with the plasmid pVOTE.1-HCV7.9 as described in Materials and Methods to generate the recombinant VT7-HCV7.9. B: Expression of HCV inhibits protein synthesis in mammalian cells. Monolayers of BSC40 cells were infected at 5 PFU/cell with either the parental VT7 or the recombinant VT7-HCV7.9 viruses in the presence (+) or absence (-) of the inductor IPTG. Uninfected (U) and infected cells were metabolically labelled with ³⁵S-Met-Cys Promix (100 μCi/mL) from 4 to 24 h.p.i. as described in Materials and Methods. Approximately 100 μg of total cell protein extracted from uninfected (U) and infected cells, was fractionated by SDS-PAGE followed by autoradiography. (*) represents new additional polypeptides corresponding to the HCV proteins. C: Inducible expression of HCV proteins by recombinant VT7-HCV7.9 virus. BSC40 cells were infected as described above. Total cell protein lysates from uninfected (U) and infected cells at 24 h.p.i. were analysed by Western blot using a human anti-HCV antibody from an infected patient. The protein band migration of Core, E2, NS4B and NS5A, as determined with specific antibodies, is indicated.