AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication. METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon, and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model. All sequences were computer- predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA. Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls. Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and West blotting, respectively. To evaluate possible antisense effects, complementary RNAs spanning a mimic were prepared. RESULTS: In the BB7 genotype 1b replicon system, mimics of the polymerase (NS-5B), X and BA regions inhibited replication by more than 90%, 50%, and 60%, respectively. In the JFH-1 genotype 2 infection system, mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%, respectively, as effectively as a mimic with complete identity to HCV genotype 2a. The inhibitory effects were confirmed by NS3 protein levels. Antisense RNA molecules spanning the 74% identical mimic had no significant effects. CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.