Modulation of hepatitis C virus RNA abundance by the liver-specific microRNA miR-122
Abstract/Contents
- Abstract
- Hepatitis C virus (HCV) is a global health problem, infecting approximately 2% of the world's population. The virus is hepatotropic, replicating in liver cells, and its only known hosts are humans and chimpanzees. HCV is an unusual virus in that it requires the liver-specific host microRNA (miRNA) miR-122 for HCV RNA accumulation. Though the precise mechanism by which miR-122 upregulates HCV RNA is still under investigation, it is known that miR-122 must bind to two adjacent sites in the 5' end of the HCV genome. In this dissertation, a stepwise mutational analysis of the entire sequence of miR-122 was performed to identify residues important for HCV RNA accumulation. All mutant miRNAs were tested in canonical miRNA reporter assays and in HCV RNA accumulation assays. The identities of two nucleotides within miR-122, at positions 15 and 16, were shown to be dispensable for canonical miRNA and siRNA activity but required for HCV RNA accumulation. Compensatory mutations in the HCV genome upstream of the original binding sites uncovered supplementary binding sites for nucleotides 15 and 16 of miR-122. This analysis led to a new model for miR-122-HCV RNA interactions. To further define the requirements of HCV for miR-122, we investigated whether the predecessor of mature miR-122, a long hairpin precursor designated pre-miR-122, was also able to mediate HCV RNA accumulation. The function of pre-miR-122 was tested in miRNA, siRNA, and HCV RNA accumulation assays. Inhibition of pre-miR-122 processing was achieved by substituting deoxyribonucleotides into the loop of pre-miR-122 to prevent Dicer-mediated cleavage. Full-length pre-miR-122 was demonstrated to be functional in miRNA and siRNA assays and to be sufficient for HCV RNA accumulation. Pre-miR-122 also required traditional components of the RNA-induced silencing complex (RISC) for activity. Taken together, this research has uncovered novel requirements of miR-122 for HCV RNA accumulation. Components shown to be dispensable for canonical miRNA interactions were necessary for this unusual microRNA-target RNA interaction. Uncovering hepatitis C virus's stringent requirements for the mature and precursor forms of miR-122 will pave the way for new antiviral therapies targeting a host factor.
Description
Type of resource | text |
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Form | electronic; electronic resource; remote |
Extent | 1 online resource. |
Publication date | 2012 |
Issuance | monographic |
Language | English |
Creators/Contributors
Associated with | Cox, Erica Machlin | |
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Associated with | Stanford University, Department of Microbiology and Immunology. | |
Primary advisor | Sarnow, P. (Peter) | |
Thesis advisor | Sarnow, P. (Peter) | |
Thesis advisor | Arvin, Ann M | |
Thesis advisor | Chen, Chang-Zheng | |
Thesis advisor | Fire, Andrew Zachary | |
Advisor | Arvin, Ann M | |
Advisor | Chen, Chang-Zheng | |
Advisor | Fire, Andrew Zachary |
Subjects
Genre | Theses |
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Bibliographic information
Statement of responsibility | Erica Machlin Cox. |
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Note | Submitted to the Department of Microbiology and Immunology. |
Thesis | Ph.D. Stanford University 2012 |
Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Erica Machlin Cox
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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