A178 ARGONAUTE 2 IS DISPENSABLE FOR EFFICIENT HEPATITIS C VIRUS REPLICATION IN HUH 7.5 CELLS. (1st March 2018) - Amador, Y
Abstract: Background: A liver-specific microRNA, miR-122, protects the Hepatitis C virus (HCV) genome from degradation and promotes its replication. Therefore, the virus is deemed highly dependent on this miRNA. This makes it a very attractive therapeutic target that has shown promise in the clinical setting. Nevertheless, the underlying mechanism and the potential role of other host factors are still poorly understood. Argonaute (Ago) proteins are host multifunctional proteins that can be found at the heart of the RNA-Induced Silencing Complex. Humans express 4 Ago isoforms (Ago1-4) and Ago2, the only human Ago capable of endonucleolytic cleavage, has generally been viewed as the primary Ago involved in the HCV life cycle. Aims: This work aimed to investigate the specific role of Ago2 and to determine the impact of the other Ago isoforms in the HCV life cycle. Methods: To fulfill this objective we made use of the CRISPR/Cas9 technology to generate hepatoma-derived Ago2 knockout Huh7.5 cells. Results: We have generated two cell lines with confirmed biallelic indel mutations in the Ago2 gene and both showed undetectable levels of Ago2 expression by Western blot. We also confirmed that the Ago2 knockout cells were devoid of knockdown activity by assaying for small interfering RNA (siRNA) directed cleavage activity. Somewhat unexpectedly, Ago2 knockout cells are able to support HCV replication, albeit to lower levels (50–70%) than the wild-type cells. Importantly, in the absence of Ago2, two siRNAs targeting miR-122 binding site 1 rescue HCV replication that had been inhibited by blocking the activity of miR-122. Conclusions: These results indicate that the other human Ago isoforms (Ago1, 3 and/or 4) are able to sustain HCV replication in the absence of Ago2, and that at least one, but potentially all, of the other Ago proteins can mediate miR-122 promotion of the HCV life cycle. Additionally, our data suggest that Ago2's endonucleolytic cleavage activity is not required for miR-122 promotion of HCV replication. At present, we are generating cell lines in which combinations of all 4 Ago isoforms are knocked out in order to assess the roles of each Ago isoform in the HCV life cycle, and generate an Ago null cell line to be used for trans-complementation assays to investigate the mechanism by which Ago and miR-122 promote the HCV life cycle. These novel cell lines constitute valuable tools in the field of HCV research. They will allow the identification of other host factors for the design of multi-target therapeutic approaches, with potentially reduced long-term side effects and higher barriers to resistance compared to single target approaches. Funding Agencies: Saskatchewan Health Research Foundation and the Canadian Network on Hepatitis C