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.