Student Theses and Dissertations

Date of Award

2015

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darnell Robert Laboratory

Abstract

Hepatitis C virus (HCV) uniquely requires the liver specific microRNA-122 (miR- 122) for replication, yet global effects on endogenous microRNA (miRNA) targets during infection are unexplored. In this body of work, we employed highthroughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection. We demonstrate robust AGO binding on the 5' untranslated region of HCV RNA at known and predicted miR-122 sites, thereby establishing conclusive biochemical evidence of endogenous miR-122 action on HCV RNA that firmly agrees with previous genetic evidence. We further characterize novel AGO binding on HCV RNA to determine its dependence on miR-122, miRNAs generally, replication competence and time. These results establish an unbiased interaction landscape between HCV RNA and cellular miRNAs, mostly miR-122. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 "sponge" effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. Based on these results, we describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets. This in turn may provide an environment fertile for the long-term oncogenic potential of HCV. This last point presented a fitting entree into miR-122 biology, given its known tumor suppressive activity in the liver. To conclude this work, we performed AGO-CLIP in miR-122 knockout mouse livers as well as in human liver samples, to determine the in vivo targetome for this miRNA across two species. Surprisingly, we discovered widespread and non-canonical miR-122 binding throughout the transcriptome. Furthermore, a substantial fraction of this binding was not conserved between mouse and human transcriptomes, despite the fact that miR-122 is highly conserved. These results, in concert with AGOCLIP in HCV infected cells, point to a model where HCV may have evolved the use of miR-122 for its high abundance and its well buffered capacity to be inhibited with minimal detrimental effects to the host, and perhaps benefits for the virus. In sum, this thesis reveals how miR-122 is redistributed in the cell following HCV infection. As a molecular mechanism, chronic inhibition of miR-122 by HCV RNA is proposed to impact, and may very well help induce, the complex constellation of liver diseases that characterize this infection in humans.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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