Hepatitis C virus (HCV) is a major cause of liver-specific morbidity and mortality worldwide, resulting in >350,000 deaths annually due to cirrhosis and cancer with high rates of disease among those co-infected with human immunodeficiency virus (HIV) or having a history of injection drug use. microRNA-122 (miR-122), an abundant, liver-specific miRNA is essential for replication of infectious HCV and thus plays a novel role in the pathogenesis of chronic hepatitis C. Recent chimpanzee studies have validated miR-122 silencing as an effective antiviral strategy, yet how miR-122 promotes HCV replication is not well understood. Recent work shows that miR-122 promotes viral translation through direct interactions with a miRNA response element at the 5'end of (+)-strand RNA, while new preliminary data indicate that it also acts to stabilize the RNA. This project will investigate the hypothesis that miR-122, in association with Ago and possibly other cellular proteins, forms a ribonucleoprotein complex (the "miRNA-associated stabilization complex", or MASC) at the 5'end of (+)-strand HCV RNA. The MASC complex has several proposed functions: (i) it protects (+)-strand RNA within the cytoplasmic compartment from degradation, thereby increasing RNA available for translation and assembly into membrane-bound replicase complexes;(ii) it may directly enhance the translational activity of HCV RNA;and (iii), it may promote new viral RNA synthesis by facilitating macroassembly of replicase complexes and/or de novo initiation of RNA synthesis. A series of interrelated aims will rigorously test these hypotheses, determine RNA and protein components of the MASC, and further characterize the novel functions of this unique ribonucleoprotein complex.
Aim 1 will determine: (a) how miR-122 base-pairs with (+)- strand HCV RNA within the MASC complex;(b) whether RNA stabilization and MASC-increased translation can be functionally uncoupled, and;(c) whether MASC function requires HCV sequences outside the response element.
Aim 2 will: (a) combine RNA affinity selection and quantitative proteomics analysis to identify protein components of the MASC complex;(b) validate the presence of these proteins in the MASC complex by IP and assess their role in miR-122 function by RNA interference;(c) develop a cell-free system in which MASC function is recapitulated and the role of host proteins can be further characterized, and;(d) ascertain whether HCV RNA is stabilized by tethering of Ago or other proteins to the 5'RNA.
Aim 3 will determine: (a) whether miR-122 regulates macroassembly or stability of replication vesicles by live cell imaging, (b) whether miR-122 is required for negative-strand RNA synthesis, and (c) whether miR-122 facilitates separation of duplexed strands within replicative forms of HCV RNA. Collectively, these studies will provide novel insights into functions of a miRNA that: (a) are important in the pathogenesis of human disease, (b) represent a well-validated but poorly understood therapeutic target, and (c) are unique not only among mammalian viruses but among the Metazoa.

Public Health Relevance

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 will investigate the novel role played by a small cellular ribonucleic acid, miR-122, in this disease. This liver-specific micro-RNA is essential for replication of HCV, and it is a well-validated but very poorly understood target for antiviral therapy. The studies proposed will provide novel insight into how it functions in HCV replication and in the pathogenesis of chronic hepatitis C, thereby enhancing the scientific background on which it can be exploited as a therapeutic target.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI095690-03
Application #
8444519
Study Section
Virology - A Study Section (VIRA)
Program Officer
Koshy, Rajen
Project Start
2011-04-15
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
3
Fiscal Year
2013
Total Cost
$346,286
Indirect Cost
$111,286
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Masaki, Takahiro; Matsunaga, Satoko; Takahashi, Hirotaka et al. (2014) Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-? in infectious virus production. J Virol 88:7541-55
Li, You; Masaki, Takahiro; Shimakami, Tetsuro et al. (2014) hnRNP L and NF90 interact with hepatitis C virus 5'-terminal untranslated RNA and promote efficient replication. J Virol 88:7199-209
Rusyn, Ivan; Lemon, Stanley M (2014) Mechanisms of HCV-induced liver cancer: what did we learn from in vitro and animal studies? Cancer Lett 345:210-5
McGivern, David R; Masaki, Takahiro; Williford, Sara et al. (2014) Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors. Gastroenterology 147:453-62.e7
Yi, MinKyung; Hu, Fengyu; Joyce, Michael et al. (2014) Evolution of a cell culture-derived genotype 1a hepatitis C virus (H77S.2) during persistent infection with chronic hepatitis in a chimpanzee. J Virol 88:3678-94
Yamane, Daisuke; McGivern, David R; Wauthier, Eliane et al. (2014) Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation. Nat Med 20:927-35
Spaniel, Carolyn; Honda, Masao; Selitsky, Sara R et al. (2013) microRNA-122 abundance in hepatocellular carcinoma and non-tumor liver tissue from Japanese patients with persistent HCV versus HBV infection. PLoS One 8:e76867
Honegger, Jonathan R; Kim, Seungtaek; Price, Aryn A et al. (2013) Loss of immune escape mutations during persistent HCV infection in pregnancy enhances replication of vertically transmitted viruses. Nat Med 19:1529-33
Li, You; Masaki, Takahiro; Lemon, Stanley M (2013) miR-122 and the Hepatitis C RNA genome: more than just stability. RNA Biol 10:919-23
Li, Kui; Lemon, Stanley M (2013) Innate immune responses in hepatitis C virus infection. Semin Immunopathol 35:53-72

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