Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication.

Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase.