Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis.
The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (-) RNA synthesis have been identified in the 3' non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (-) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3'-end of the (-) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.
Mahias
Kathleen
K
CNRS UMR 5234, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
Ahmed-El-Sayed
Neveen
N
Masante
Cyril
C
Bitard
Juliette
J
Staedel
Cathy
C
Darfeuille
Fabien
F
Ventura
Michel
M
Astier-Gin
Thérèse
T
D001483:Base Sequence; D002460:Cell Line; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry*; D038621:Regulatory Sequences, Ribonucleic Acid
D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid