FIG. 6.

The HCV 3′-UTR enhances translation of full-length genomes. (A) Structure of the transfected RNAs. Full-length HCV genome RNAs including the 3′-UTR (upper panel) or not (lower panel) were in vitro transcribed using T7 RNA polymerase. Precise 3′ ends were generated after transcription by autocatalytic RNA cleavage mediated by the HDV genomic ribozyme fused downstream to the HCV sequence. (B) Western blots with extracts of Huh-7 cells harvested at different times after electroporation transfection with the genomic HCV RNAs shown in panel A. Proteins were detected with antibodies directed against HCV NS5B protein (upper panel) or actin (lower panel). (C) RNA stability control. Total RNA was harvested from Huh-7 cells at the indicated times after electroporation as in panel B, purified, and analyzed by RNase protection assay using 0.35 pmol of the [α-³²P]UTP-labeled antisense NS5B RNA probe specified in panel A. It contains 15 nucleotides of linker sequence, 29 nucleotides of the variable region, and 298 nucleotides of NS5B sequence (upper panel). As a control, the same RNA samples were analyzed using GAPDH antisense RNA (lower panel). In lanes 1, 10% of the undigested antisense RNA probes was loaded. (D) Western blotting as in panel B with antibodies directed against HCV NS5B protein (upper panel) or actin (lower panel). However, the HCV genomes used were the same as shown in panel A but with an NS5B polymerase GND mutation. hrs p.t., hours posttransfection.