All ETDs from UAB

Advisory Committee Chair

Gail W Wertz

Advisory Committee Members

Andrew Ball

Jay Brown

Ming Luo

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The template for transcription and RNA replication for vesicular stomatitis virus (VSV) and other negative-strand RNA viruses is a ribonucleoprotein (RNP) complex consisting of the viral RNA genome and associated nucleocapsid (N) protein. The structure of the RNP-like complex of VSV showed that the RNA is sequestered between two lobes of the N protein, and adjacent N monomers are linked by an extensive network of interactions. Long-range interactions mediated by the N-terminal arm and C-terminal loop of the N protein stabilize the side-to-side contacts between adjacent N monomers, and are required for RNA encapsidation. It is unclear how the polymerase gains access to the RNA within the RNP complex during polyribonucleotide synthesis, but it was postulated that polymerase binding to the RNP template destabilizes contacts between the C-terminal loop and N-terminal arm, and local dissociation of the N-terminal arm causes a conformational change in the N-terminal lobe to expose the RNA to the polymerase. Using the structure of the VSV N protein complexed with RNA, we identified residues in the C-terminal loop of the N protein that were postulated to form inter- and intra-molecular contacts that may affect the function of the N protein in RNP templates. We investigated the role of individual amino acid residues in the C-terminal loop in the formation of RNP complexes and tested their ability to function as templates for viral RNA synthesis. Mutations in the C-terminal loop, designed to destabilize loop integrity, produced templates that supported up to10-fold higher levels of RNA replication than wild-type templates without affecting transcription. In contrast, mutations predicted to disrupt contacts between the C-terminal loop and N-terminal arm yielded decreased levels of transcription without affecting RNA replication. We recovered recombinant VSV engineered to contain the above mutations in the C-terminal loop to investigate the effect of these N gene mutations on overall virus replication and to take advantage of the disparate effects of these mutations on transcription and RNA replication to probe the N protein interactions with the other components of the viral replication machinery.

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