The relationship between hepatitis C virus (HCV) and the liver specific microRNA miR- 122 is unusual. While most miRNAs inhibit translation when bound to their targets, miR- 122 increases HCV protein expression by binding to its RNA genome to enhance RNA stability and replication. Developing a cell system that supports the HCV lifecycle took almost 10 years of research. Huh-7 cells, which are one of the only cell lines found to support the entire HCV lifecycle, are also one of the only cell lines found to express miR-122. We hypothesized that insufficient miR-122 expression in most other hepatocyte derived cell lines was the reason they could not support HCV replication. Indeed, when we engineered HepG2 cells to express miR-122, HCV replication was greatly enhanced. We went on to show that HepG2 cells overexpressing both the missing entry factor CD81 in addition to miR-122 supported the entire HCV lifecycle. We found a striking difference in these cells' ability to sustain HCV infection and spread when compared to Huh-7 and Huh-7.5 cells, which was accompanied by robust antiviral and proinflammatory response reminiscent of HCV infections in vivo. Furthermore, blocking RIG-I like receptor and IFN-λ signaling pathways promoted HCV infection and spread in these cells. These studies further solidify the importance of IFN-λ in the hepatic response to HCV infection and revealed non-redundant roles of RIG-I and MDA5 in HCV recognition and antiviral response. Finally, we set out to better understand how miR-122 inhibition influences HCV replication over time and if HCV can develop resistance to miR-122 inhibition. We found a single nucleotide change at position 28 (G28A) of the HCV genome, which falls between the two miR-122 seed- binding sites, and enhanced HCV replication in cells with low miR-122 activity. Naturally occurring HCV isolates encoding an A at nucleotide 28 were similarly resistant to miR- 122 inhibition, indicating that subtle differences in viral sequence, even outside the seed-binding site, greatly influence HCV's requirement for miR-122. These studies provide insight into the interaction between miR-122 and HCV, and have important implications for the development of anti-miR-122-based HCV drugs. Taken together, these studies demonstrate how manipulation of miR-122 can reveal new and important information about HCV host-cell interactions.
|Advisor:||Evans, Matthew J.|
|Commitee:||Lemon, Stanley M., Lim, Jean K., Simon, Viviana A., Ting, Adrian T., tenOever, Benjamin|
|School:||Icahn School of Medicine at Mount Sinai|
|School Location:||United States -- New York|
|Source:||DAI-B 76/02(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Virology, Immunology|
|Keywords:||HepG2, MDA5, RIG-I, Type iii interferon, hepatitis c virus, miR-122|
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