All ETDs from UAB

Advisory Committee Chair

Sunnie R Thompson

Advisory Committee Members

Louise T Chow

Elliot J Lefkowitz

Casey D Morrow

Peter E Prevelige

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

A significant number of pathogens of economical and medical importance are positive sense RNA viruses. Their genomes enter host cells and subvert them to support virus growth. These large-scale changes involve the co-opting of cellular proteins through interactions with viral RNA and proteins. Characterizing these interactions leads to a better understanding of viral life cycles and the identification of new antiviral therapies. The order Picornavirales contains viruses that infect a wide array of eukaryotes. Their genomes serve as a messenger RNA (mRNA) that is translated by host ribosomes cap-independently through an internal ribosome entry site (IRES). Most members of the order have a monocistronic genome with a single IRES much like the prototypical family, Picornaviridae. In contrast, the genomes of the family Dicistroviridae have two open reading frames, each with its own IRES. The intergenic region (IGR) IRESs can bind ribosomes in vitro and initiate translation unaided by any eukaryotic initiation factors (eIFs). We have tested this model in vivo using yeast genetics combined with dual luciferase reporters. Our studies show that dicistroviral IGR IRESs do not require eIFs in vivo, rather, diminished eIF activity enhances IGR IRES-dependent translation. Our current understanding of the host factors required throughout a virus life cycle relies upon in vitro assays with limited scope and in vivo siRNA screens that miss essential proteins. We have developed a novel method to identify RNA-binding host factors in vivo during live infections in mammalian cells. We validated the method during a poliovirus infection by identifying viral proteins and known host factors for the family Picornaviridae. The assay also robustly identified cellular proteins with unknown functions during a picornaviral life cycle. We have shown that some of these proteins enhance poliovirus amplification. This method has the potential to aid in the study of many RNA viruses and identify new targets for antiviral drugs.

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