Secondary Structural Elements of the HCV X-region Involved in Viral Replication

BACKGROUND AND AIMS: The noncoding regions in the 3′-untranslated region (UTR) of the hepatitis C virus (HCV) genome contain secondary structures that are important for replication. The aim of this study was to identify detailed conformational elements of the X-region involved in HCV replication. METHODS: Ribonucleic acid (RNA) structural analogs X94, X12, and X12c were constructed to have identical conformation but 94%, 12%, and 0% sequence identity, respectively, to the X region of HCV genotype 2a. Effects of structural analogs on replication of HCV genotypes 1b and 2a HCV RNA were studied by quantitative reverse transcriptase polymerase chain reaction. RESULTS: In replicon BB7 cells, a constitutive replication model, HCV RNA levels decreased to 55%, 52%, 53%, and 54% after transfection with expression plasmids generating RNA structural analogs 5B-46, X-94, X-12, and X-12c, respectively (p<0.001 for all). In an HCV genotype 2a infection model, RNA analogs 5B-46, X-94, and X-12 in hepatic cells inhibited replication to 11%, 9%, and 12%, respectively. Because the X-12 analog was only 12% identical to the corresponding sequence of HCV genotype 2a, the sequence per se, or antisense effects were unlikely to be involved. CONCLUSIONS: The data suggest that conformation of secondary structures in 3′-UTR of HCV RNA genome is required for HCV replication. Stable expression of RNA analogs predicted to have identical stem-loop structures might inhibit HCV infection of hepatocytes in liver and may represent a novel approach to design anti-HCV agents.