Hepatitis C virus (HCV), a (+)-strand RNA virus, persistently infects ~200 million persons worldwide and, despite advances in antiviral therapy, remains the leading cause of hepatic cirrhosis and liver cancer in the U.S. In this application, we propose a continuation of a productive line of research aimed at understanding the molecular mechanism(s) underlying the unique dependence of HCV replication on the evolutionarily conserved, liver-specific host microRNA, miR-122-5p. Our published research shows that the binding of miR-122 to two conserved sites (S1 and S2) near the 5' end of the 9.7 kb (+)-sense RNA genome promotes HCV replication in two distinct ways: (1) by binding S1 and S2, miR-122 protects HCV RNA from 5' exonucleolytic decay mediated by XRN1, a cytoplasmic host 5' exoribonuclease, and (2) miR-122 directly stimulates HCV RNA synthesis by rebalancing the proportion of (+)-RNA molecules engaged in templating RNA synthesis vs. directing translation on ribosomes. Both functions require AGO2 which our data show is recruited to the genome by miR-122. We now show in new preliminary data that miR-122 also regulates circularization of the HCV genome through competition for binding with poly-(rC) binding protein 2 (PCBP2) and possibly other host RNA-binding proteins. We propose three specific aims to investigate the overarching hypothesis that a unique ribonucleoprotein complex formed by miR-122 and AGO2 at the 5' end of the HCV genome critically regulates its engagement in different stages of the viral lifecycle.
In Aim 1, we will assess the role of miR-122 in regulating strand-specific HCV RNA synthesis and packaging of newly synthesized (+)-RNA.
Aim 2 will study how viral replication is regulated by competition between miR-122 and host RNA-binding proteins, including PCBP2 and newly recognized candidate host factors identified through quantitative proteomics.
Aim 3 will characterize the role of miR-122 in modulating circularization of the HCV (+)-strand genome mediated by protein-protein interactions, and how this relates to genome stability and viral RNA synthesis. While our efforts focus on HCV specifically, these studies have broad relevance to mechanisms of replication of other pathogenic (+)-strand RNA viruses.
Hepatitis C virus (HCV) is a major cause of liver-specific morbidity and mortality worldwide, resulting in >350,000 deaths annually due to cancer and cirrhosis with especially high rates of disease among those co- infected with human immunodeficiency virus (HIV) or having a history of injection drug use. This project investigates the novel role played by a small cellular ribonucleic acid, miR-122, in replication of the virus. Or work is revealing novel aspects of the molecular mechanisms underlying the promotion of viral replication by this liver-specific micro-RNA, and is thereby enhancing the theoretical basis for use of miR-122 antagomirs as salvage therapy for patients with resistance to direct-acting antiviral agents. The proposed studies investigate fundamental aspects of replication that are relevant to a broad array of pathogenic positive-strand RNA viruses.
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|Mitchell, Jonathan K; Midkiff, Bentley R; Israelow, Benjamin et al. (2017) Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R. MBio 8:|
|Benzine, Tiffany; Brandt, Ryan; Lovell, William C et al. (2017) NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains. PLoS Pathog 13:e1006343|
|Yamane, Daisuke; Selitsky, Sara R; Shimakami, Tetsuro et al. (2017) Differential hepatitis C virus RNA target site selection and host factor activities of naturally occurring miR-122 3? variants. Nucleic Acids Res 45:4743-4755|
|Bility, Moses T; Nio, Kouki; Li, Feng et al. (2016) Chronic hepatitis C infection-induced liver fibrogenesis is associated with M2 macrophage activation. Sci Rep 6:39520|
|Selitsky, Sara R; Dinh, Timothy A; Toth, Cynthia L et al. (2015) Transcriptomic Analysis of Chronic Hepatitis B and C and Liver Cancer Reveals MicroRNA-Mediated Control of Cholesterol Synthesis Programs. MBio 6:e01500-15|
|Li, You; Yamane, Daisuke; Lemon, Stanley M (2015) Dissecting the roles of the 5' exoribonucleases Xrn1 and Xrn2 in restricting hepatitis C virus replication. J Virol 89:4857-65|
|Selitsky, Sara R; Baran-Gale, Jeanette; Honda, Masao et al. (2015) Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C. Sci Rep 5:7675|
|Zhou, Kali; Hu, Fengyu; Wang, Charles et al. (2015) Genotypic distribution and hepatic fibrosis among HIV/HCV co-infected individuals in Southern China: a retrospective cross-sectional study. BMC Infect Dis 15:401|
|Mauger, David M; Golden, Michael; Yamane, Daisuke et al. (2015) Functionally conserved architecture of hepatitis C virus RNA genomes. Proc Natl Acad Sci U S A 112:3692-7|
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