Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1

Abstract

Since the first report of frameshifting in HCV-1, its sequence has been the paradigm for examining the mechanism that directs alternative translation of the hepatitis C virus (HCV) genome. The region encoding the core protein from this strain contains a cluster of 10 adenines at codons 8-11, which is thought to direct programmed ribosomal frameshifting (PRF), but formal evidence for this process has not been established unequivocally. To identify the mechanisms of frameshifting, this study used a bicistronic dual luciferase reporter system in a coupled transcription/translation in vitro assay. This approach revealed +1 as well as -1 frameshifting, whereas point mutations, selectively introduced between codons 8 and 11, demonstrated that PRF did not readily account for frameshifting in strain HCV-1. Sequence analysis of cDNAs derived from RNA transcribed by T7 RNA polymerase in the dual luciferase reporter system, as well as in both a subgenomic replicon and an infectious clone derived from strain JFH1, identified additions and deletions of adenines between codons 8 and 11 due to transcriptional slippage (TS). Moreover, RNA isolated from cells infected with virus generated by JFH1 containing the A-rich tract also contained heterogeneity in the adenine sequence, strongly suggesting TS by the NS5B viral polymerase. These findings have important implications for insight into frameshifting events in HCV-1 and demonstrate for the first time the involvement of transcriptional slippage in this recoding event.

The Fidelity of Transcription

“…Additional observations of transcriptional slippage on homopolymeric tracts have been reported in bacteriophages (10), prokaryotes (11)(12)(13)(14), viruses (15), and eukaryotes (16,17). A clear demonstration of in vivo transcriptional slippage during elongation was provided by Wagner et al (18), who showed that Escherichia coli RNA polymerase made slippage RNA products from templates with a homopolymeric tract greater than 9 nucleotides.…”

RNA Polymerase Slippage as a Mechanism for the Production of Frameshift Gene Products in Plant Viruses of the Potyviridae Family

“…In the case of viruses, polymerase slippage results in the alteration of newly synthesized RNA. The mechanism has been well characterized in animal RNA viruses such as Ebolavirus (1) (EBOV) or Hepatitis C virus (HCV) (2). For plant viruses of the Potyviridae family, polymerase slippage has been proposed as a general process of evolution (3), although a lack of experimental systems has precluded confirmatory data, and most pieces of evidence are indirect (4).…”

Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

“…*, P Ͻ 0.02 (Student's t test). 5=-GGCGTCTTCCCCAGATCTCCATCCTGCCCAGCC-3= coreϩ1 630 JFH1 5=-GGCGTCTTCCCCAGATCTGCCATCCTGCCCAGCC-3= core-1 630 JFH1 5=-GGCGTCTTCCCCAGATCTTCACCTGCCCAGCC-3= coreϩ1 630 H77 5=-GGCGTCTTCCCCAGATCTGCCATCCCGCCCACCC-3= N328 frameshift at codons 8 to 11 (7)(8)(9)33), internal initiation at codon 26 (12,15), a frameshift at codon 42 (16), and a shorter form initiating at codons 85/87 (13,15). As shown in Fig.…”