Internal initiation sites of de novo RNA synthesis within the hepatitis C virus polypyrimidine tract.
A variety of 3'-untranslated regions (UTRs) were cloned from infectious hepatitis C virus human samples and examined in NS5B polymerase de novo initiation reactions. We isolated and characterized four distinct 3'-UTRs that harbor the conserved terminal 98 nucleotides, but have poly(U/UC) tracts of 25, 93, 98, and 101 nucleotides, respectively. Reconstitution of de novo initiation by the mature NS5B with the different 3'-UTR RNA substrates revealed distinctively sized products that are consistent with internal initiation at specific sites within the polypyrimidine tract. These sites were further mapped by demonstrating that nucleotide substitutions of the cytidylate stretches within the poly(U/UC) template eliminate specific products of de novo synthesis and by showing that these products could be radiolabeled by [gamma-32P]GTP. We also examine analogs that can substitute for GTP in this reaction.
Pellerin
Charles
C
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Research and Development, 2100 rue Cunard, Laval, Qué., Canada H7S 2G5.
Lefebvre
Sylvain
S
Little
Michael J
MJ
McKercher
Ginette
G
Lamarre
Daniel
D
Kukolj
George
G
D020413:3' Untranslated Regions; D001483:Base Sequence; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D006160:Guanosine Triphosphate / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
D020413:3' Untranslated Regions; D006160:Guanosine Triphosphate; D012313:RNA; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase