Date of Award

5-2018

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biological Science

Advisor/Committee Chair

Cara Pager

Committee Member

Gabriele Fuchs

Abstract

Hepatitis C virus (HCV) infected 30,500 people in the United States alone in 2014. As a positive-sense single-stranded RNA virus, the HCV genome facilitates translation and replication of the virus within the host cell. During infection, HCV subverts numerous host proteins to aid in viral production. One such protein is DDX6, a DEAD-box helicase that is associated with the decapping complex and microRNA Induced Silencing Complex (miRISC). Previous studies in our lab have shown that DDX6 is required for HCV expression. Specifically, DDX6 modulates the interaction of miR-122 with the HCV 5’ UTR and promotes stability and replication of the viral genome. To elucidate which enzymatic activities of DDX6 are required to promote HCV gene expression, we used site-directed mutagenesis to disrupt the enzymatic activities of DDX6. Specifically, we mutated the ATPase, RNA binding and helicase domains within 3x-Flag-DDX6Δsi plasmid DNA. We next used lentivirus transduction to stably express the 3xFlag-tagged DDX6 mutants in Huh7 cells, in which expression of endogenous DDX6 was deleted by gene editing using CRISPR-Cas9. Analysis by immunofluorescence staining and confocal microscopy of uninfected cells, shows wild-type 3xFlag-DDX6 localizing in processing bodies, while the enzymatic mutants show diffuse cytoplasmic staining. DDX6 stable cell lines were infected with the HCV infectious clone JFH-1, and at three-days post-infection cells were harvested and viral and cellular protein and RNA abundance analyzed by western and northern blotting, respectively. Surprisingly, only DDX6 knock-out cells expressing the wild-type 3xFlag-DDX6 were infected with HCV. These data show that the enzymatic activities of DDX6 are critical for HCV infection. Since RNA helicases unwind RNA, it is possible that the helicase and RNA binding activities of RCK/p54 are necessary to remodel the complex secondary structures found on the 5’ and 3’ untranslated regions of the HCV genome, and may play an important role in the transition between translation and replication.

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