HCV RNA-dependent RNA polymerase replicates in vitro the 3 ' terminal region of the minus-strand viral RNA more efficiently than the 3 ' terminal region of the plus RNA

The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus ty pe C, catalyzes the replication of the viral genomic RNA. Little is known a bout the recognition domains of the viral genome by the NS5B. To better und erstand the initiation of RNA synthesis on HCV genomic RNA, we used in vitr o transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3' end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were sy nthesized by using the 3' UTR of the (+) strand RNA. In contrast the 341 nu cleotides at the 3' end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to b e involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3' end of the minus-strand RNA: (a) th e presence of a C residue as the 3' terminal nucleotide; (b) one or two G r esidues at positions +2 and +3; (c) other sequences and/or structures insid e the following 42-nucleotide stretch. These results indicate that the 3' e nd of the minus-strand RNA of HCV possesses some sequences and structure el ements well recognized by the purified NS5B.