|dc.description.abstract||Generally Hepatitis C Virus tropism is limited to hepatocytes. This limited tropism is a result of the receptors HCV requires for cellular entry and other host cellular factors including, uniquely, a liver specific miRNA, miR-122. The relationship between HCV and miR-122 is interesting, as commonly, miRNA are associated with suppression of function, but in the case of HCV, miR-122 actively promotes HCV proliferation. In-depth studies have demonstrated that miR-122 along with the RNA induced silencing complex (RISC) protein Argonaute 2 (Ago2) binds directly to two seed sequences separated by 8-9 nucleotides on HCV 5’UTR. Binding to the 5’UTR results in an increase in viral replication and translation. The method by which miR-122 promotes HCV translation and replication is not fully understood but evidence suggests that part of the function of miR-122 is to stabilize the HCV genome and protect it from exonuclease degradation by Xrn1, but other mechanisms remain to be identified. The reliance of HCV on miR-122 is best exemplified by the fact that removal of miR-122 by a miR-122 antagonist drastically impedes HCV viral titers in Chimpanzees and humans with no indication of escape mutants.
The observation that HCV augmentation of the HCV life cycle by miR-122 requires Ago2 suggests that other components downstream in the miRNA suppression pathway may also be part of the mechanism of action. Our studies focused specifically on the processing body (p-body) associated DEAD-box helicase DDX6. DDX6 is essential for p-body assembly, required for robust miRNA suppression activity and elevated in HCV associated hepatocellular carcinomas. As such we hypothesized that DDX6 and p-bodies were directly or in-directly associated with the mechanism of action of miR-122.
Knocking down DDX6 with siRNA indicated that DDX6 augments both HCV replication and translation. To examine whether DDX6 augmentation of HCV replication was related to the effects of miR-122 on the HCV life cycle, HCV replication and translation were assessed in the presence or absence of miR-122 when DDX6 was knocked down. Our data indicated that HCV replication and translation were augmented equally by miR-122 whether DDX6 was present or not. Our data also demonstrated that HCV replication and translation that was occurring independent of miR-122 was also still affected by DDX6 knockdown. Taken together our observations strongly suggest that the role DDX6 has on HCV is independent of HCV and miR-122’s relationship.
In order to better understand miR-122’s relationship with HCV, we hypothesized that targeting the miR-122 binding region with siRNA would inhibit HCV replication initially, but that over the course of several rounds of treatment with the same siRNA, HCV would mutate to escape the siRNA, producing escape mutants that replicate without a dependency on miR-122. These escape mutants could be evaluated on how they replicate without using miR-122, shedding light on miR-122 and HCV’s relationship. Conversely if no escape mutants arose the siRNA could be further studied as a potential therapeutic for HCV.
siRNA designed to target the miR-122 binding region inhibited HCV replication, confirming that the designed siRNAs could access the miR-122 binding region and function as an siRNA. Interestingly, when the siRNAs were used against a replication competent HCV RNA having a single nucleotide mutation in the first miR-122 binding site, instead of abolishing siRNA knockdown, two of the siRNA showed enhanced inhibition activity. The target sequences of these siRNAs spanned both miR-122 binding sites and we speculate that their inhibitory activity was due to competition for miR-122 binding to site 2. This observation indicates that siRNA targeting the miR-122 binding region have dual activity, by siRNA induced cleavage, and as a competitive inhibitor of miR-122 binding.
Selection for viral escape mutants of the miR-122-binding site targeting siRNAs revealed viral RNAs having mutations within the miR-122 binding sites, in the surrounding region, and to other areas within the HCV IRES. The mutant viruses will be used to assess the influence of miR-122 binding site mutations on HCV replicative fitness, and to determine if the virus can evolve to replicate independent from augmentation by miR-122.||en_US