Two cis-acting elements in negative RNA strand of Hepatitis C virus involved in synthesis of positive RNA strand in vitro.
Sequences at the 3'-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3'-terminal region of negative RNA strand ((-)3'T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3'-cytidylate (3'C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3'-proximal stem-loop resulted in an approximately 90% decrease in RNA synthesis. Disruption of the 3'-proximal stem-loop structure by nucleotide substitutions led to a 70-80% decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3'C by guanylate (G) led to a 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80% decrease in the RNA synthesis only. These findings demonstrate that the 3'-proximal stem-loop and the first 3'C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.
Ye
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State Key Laboratory of Virology, Ministry of Education, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P.R. China.
Timani
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KA
Ye
L
L
Kong
L
L
Yang
X
X
Liao
Q
Q
Wu
J
J
D020413:3' Untranslated Regions; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014779:Virus Replication
D020413:3' Untranslated Regions; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase