Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1.
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.
Ratinier
Maxime
M
IBCP (Institut de Biologie et Chimie des Protéines), CNRS, UMR 5086, Université de Lyon, IFR 128, 7 passage du Vercors, F-69367 Lyon, France.
Boulant
Steeve
S
MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
Combet
Christophe
C
IBCP (Institut de Biologie et Chimie des Protéines), CNRS, UMR 5086, Université de Lyon, IFR 128, 7 passage du Vercors, F-69367 Lyon, France.
Targett-Adams
Paul
P
MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
McLauchlan
John
J
MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
Lavergne
Jean-Pierre
JP
IBCP (Institut de Biologie et Chimie des Protéines), CNRS, UMR 5086, Université de Lyon, IFR 128, 7 passage du Vercors, F-69367 Lyon, France.
D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018965:Frameshifting, Ribosomal; D017930:Genes, Reporter; D016174:Hepacivirus / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics*
D018076:DNA, Complementary; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D008156:Luciferases