All ETDs from UAB

Advisory Committee Chair

Ming Luo

Advisory Committee Members

Kirill Popov

Terje Dokland

Louise Chow

Elliot Lefkowitz

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Negative strand RNA viruses (NSV) are unique because their nucleocapsids are used directly as the template for both transcription and replication. The viral genomic RNA is coated by the nucleoprotein (N) for the entirety of the NSV replication cycle. The viral polymerase, which is composed of the L and P proteins, can only recognize encapsidated RNA as the template for RNA synthesis. Our previous studies have solved the EM structure of the MuV N-RNA complex at 25Ņ. This structure revealed how the nucleocapsid is assembled and provides an initial model for examining how the viral polymerase may recognize the nucleocapsid as the template for transcription and replication. However, the low resolution of the MuV N-RNA model can only provide a limited amount of information about the MuV nucleocapsid structure. Further experiments are required to address the unique properties of paramyxovirus replication, such as the "rule of six" and post- transcriptional mRNA editing. The structure of the MuV N-RNA ring, along with structural data from several other NSVs, was used to design the experiments in this proposal. The long term goal is to delineate the interactions of the MuV N-P-L-RNA complex. A system was developed to express and purify the N and P proteins of mumps virus. Purified N-RNA complex was further examined using electron microscopy. Systematic studies were conducted to map the different domains of MuV P. Interactions between MuV P and the nucleocapsid were then determined. Additionally, the oligomerization domain of MuV P was defined and its crystal structure was solved. Next, the three dimensional structure of the authentic MuV NC was solved using cryo-electron microcopy, and the effects of phosphoprotein binding on the MuV NC structure were observed. These studies provided insights into how MuV P associates with NC and what changes are induced by P binding. From these studies, a model of the MuV replicase was created, reflecting the novel features of the mumps N and P proteins discovered in this thesis.

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