HCV genome and ribosomal frameshifts. (A) HCV genomic organization. Codons 8 to 14, which contain the frameshift signal, are italicized. Bolded letters indicate termination codons in the −1/+2 reading frame. The two consensus −1 ribosomal frameshift sequences are underlined. The amino acids encoded by all three reading frames are also shown. Asterisks denote the locations of stop codons. The HCV-1 sequence is used for the figure (). (B) Protein expression from pCMV-G-Core (lane 1) and pCMV-6xHis-G-Core (lane 2). The latter contains the insertion of the hexahistidine tag at the 5′ end of the G-core coding sequence. (C) Protein expression from pCMV-G-Core (lane 1) and pCMV-G-Coremt (lane 2). The latter contains the mutation that removed the termination codon at codon 14 of the −1/+2 reading frame. RNA was synthesized from DNA constructs by using T7 RNA polymerase and translated in vitro, using rabbit reticulocyte lysates in the presence of [³⁵S]methionine. The proteins synthesized were analyzed on a polyacrylamide gel. The arrow denotes the G-1.5-kDa protein made from the pCMV-G-Coremt construct.

Analysis of the −1/+2 frameshift from the 9A-Core sequences. (A) p21 core and 9A core RNAs, synthesized from pCMV-Core (lane 1) and pGEM-9ACore (lane 2) constructs, were translated by using wheat germ extracts in the presence of [³⁵S]methionine and [³H]lysine and analyzed by gel electrophoresis, as described in Materials and Methods. [³H]lysine was included as the label to enhance the signal of the core protein synthesized from the 9ACore sequence, which otherwise would be difficult to detect (see Fig. ). RNA amounts used in lanes 1 and 2 were not equivalent. (B) pEF-9ACore (lane 1), pEF-9ACoreE1 (lane 2), and the pCDEF control vector (lane 3) were transfected into Huh7 cells and analyzed by Western blot analysis about 42 h posttransfection, using antibodies against p21 core protein (bottom panel) or E1 envelope protein (upper panel). The electrophoretic mobility of the p21 core protein produced from the 9ACore constructs was indistinguishable from that expressed from the wild-type core protein construct (data not shown).

Analysis of −1/+2 and −2/+1 frameshift efficiencies in Huh7 cells. (A) The first 14 codons of the core protein sequence, with (pEF-FSLuc) or without (pEF-FSmtLuc) the termination codon at codon 14, were fused to the three reading frames of the luciferase reporter (indicated by an asterisk). (B) Huh7 cells transfected by the reporter DNA plasmids were harvested 48 h after transfection for the luciferase assays. Transfection efficiency was monitored by cotransfection with a human growth hormone expression plasmid. Frameshift efficiencies, which are shown in the log scale, were calculated as described in Materials and Methods. Experiments were done either in duplicates or triplicates and repeated at least four times. Numbers represent the average of all experiments combined.

Synthesis of F protein by the HCV-1 genomic RNA. HCV-1 RNA was synthesized from pHCV-1 and then translated using rabbit reticulocyte lysates and labeled with [³⁵S]methionine in the absence (lane 2) or presence (lane 3) of canine microsomal membranes (CMM). Protein products were analyzed on a 15% gel. The F protein synthesized from pGEM-9ACore was used as a marker (lane 4). p21 core protein was not readily detected in lane 4, as its detection usually required radiolabeling with [³H]lysine (see Fig. ). Lane 1 was the translation conducted in the absence of RNA.

Regulation of frameshifting by puromycin. (A) pEF-FSmtLuc and pEF-FSPSmtLuc constructs were transfected into Huh7 cells along with the growth hormone plasmid, which was used to monitor the transfection efficiency. After 24 h, cells were treated with puromycin for another 24 h, harvested, and analyzed for luciferase activity. The experiment was done in duplicates, which were repeated at least three times. Data represent the mean ± SEM of a typical set of experiments. The fold increase in frameshift efficiency was calculated by dividing the frameshift efficiency of puromycin-treated samples by that of the untreated samples. (B) pCMV-G-Core and pCMV-G-CoreΔ constructs were used for the synthesis of RNA, which was then translated in vitro, using wheat germ extracts in the presence of [³⁵S]methionine and 0 to 5 μM puromycin. Products were analyzed by gel electrophoresis, followed by autoradiography. (C) Protein products shown in panel B were quantitated by densitometry. Frameshift efficiency was calculated by determining the intensity of G-F protein or G-1.5-kDa protein bands divided by that of the G-core protein at each puromycin concentration, taking into consideration the number of methionine residues in each protein. The fold changes in these frameshift efficiencies from that of the untreated control (0 μM puromycin) were then plotted against the puromycin concentration. Similar results were obtained using rabbit reticulocyte lysates (data not shown).

Analysis of the consensus −1 frameshift signals in ribosomal frameshift. (A) The A-rich HCV-1 frameshift signal. The two consensus −1 frameshift signals are underlined. The A-to-G substitution at nt 376 (AΔG) and the A-to-C substitution at nt 382 (AΔC) are also shown. (B) Effect of the AΔG substitution on ribosomal frameshift in vitro. G-Core and G-Core(AΔG) RNAs were synthesized from pCMV-G-Core and pCMV-G-Core(AΔG), respectively, translated in wheat germ extracts in the presence of [³⁵S]methionine, and analyzed on a polyacrylamide gel. (C) Effect of the AΔG and AΔC double nucleotide substitutions on the frameshift efficiencies in Huh7 cells. pEF-FSPSmtLuc and pEF-FSPSmt(AΔG, AΔC)Luc constructs were transiently transfected into Huh7 cells along with the growth hormone plasmid, which monitored the transfection efficiency, and analyzed for luciferase activity at 42 to 48 h posttransfection. Experiments were done in either duplicates or triplicates. Experiments were repeated at least twice. Data represent the average of all experiments. pEF-FSPSmtLuc constructs gave frameshift efficiencies of 0.54% ± 0.07% for −2/+1 frameshift and 2.47% ± 0.18% for −1/+2 frameshift, which were taken as 100%. (D) pEF-FSPSmt(AΔG, AΔC)Luc constructs were transfected into Huh7 cells with human growth hormone plasmid for 24 h and then treated with 1 and 5 μM puromycin for another 24 h. Cells were harvested and analyzed for frameshift efficiencies by the luciferase assays. Experiments were done in duplicates and repeated three times. Data represent the mean ± SEM of a typical set of experiments. The fold increase of frameshift efficiency was calculated by dividing the frameshift efficiency of the puromycin-treated samples by that of the untreated samples.

Mechanisms of HCV ribosomal frameshifts in the absence of the consensus −1 frameshift signals. The RNA sequence and structure are based on the pH 77c sequence (), which contains G and C residues at nt 376 and 382, respectively. The A-rich sequence is shaded. The two loops of the double stem-loop structure can potentially form base pairs to produce a pseudoknot structure. In this model, ribosomes, reaching the double stem-loop structure, will stall in the presence of puromycin or other factors, thus facilitating the slippage of peptidyl-tRNA at the P site either forward (+1) or backward (−1). The −1 slippage may occur one more time (dashed arrow), leading to the decoding of the F reading frame.