Effects of short RNA structural analogues against hepatitis C virus genotypes 2, 3 and 4 in replicon cells.

Abstract

To determine whether computer-predicted short RNA structural analogues could inhibit hepatitis C virus (HCV) genotype 2a, 3a and 4a replication in cultured cells. Short RNA sequences, X12, X12a and X12b, designed to be identical in secondary structure to the X region in the 3'-untranslated region (3'-UTR) of the HCV 1b genome, as well as shorter stem-loop components of X region, were inserted into a plasmid and transfected into separate Huh7.5 human hepatoma cells stably transfected with subgenomic replicons for genotypes 2a, 3a and 4a. All replicons included a firefly luciferase reporter gene. After 48 h of plasmid transfection, the inhibition of HCV replication was determined by HCV RNA isolation and quantification by real-time polymerase chain reaction and luciferase assays. All the secondary structural analogues to genotype 1b X region cross-inhibited genotype 2a, 3a and 4a replicons. The maximum inhibition by genotype 1b X region structural analogues was obtained against genotype 2a cells in which X12, X12a and X12b inhibited replication by 30%, 63% and 72%, respectively (P < 0.05 for all), compared to an unrelated hepatitis B viral analogue. Despite substantial sequence dissimilarity, HCV RNA genotype 1b X region analogues cross-inhibited the replication of HCV genotypes 2a, 3a and 4a. Particular conformations and not the sequence of the stem-loops of the X region are involved in HCV replication.