Date of Award

4-15-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Teryl K. Frey - Committee Chair

Second Advisor

Dr. Margo A. Brinton - Committee Member

Third Advisor

Dr. Phang C. Tai - Committee Member

Fourth Advisor

Dr. Susanna F. Greer - Committee Member

Abstract

Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis.

DOI

https://doi.org/10.57709/1392609

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