INTERNAL iDE NOVO/i INITIATION SITES OF THE HCV NS5B POLYMERASE AND USE THEREOF

摘要

To further define the nature of de novo initiation from the 3'-UTR, several distinct 3'-UTR's that harbor the conserved terminal 98 nucleotides, but have poly U/U-C tracts of different length were isolated and characterized. 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 mapped by demonstrating that nucleotide substitutions of the cytidylate residues in the poly U/U-C template affect the generation of specific products of the de novo initiation reaction. Moreover, initiation within the poly U/U-C template is also primed by GTP and an assay that evaluates inhibitors of this reaction as potential HCV therapeutics is claimed.