Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Immunology and Microbiology

First Advisor

Philip E. Pellett

Abstract

THE ROLES OF HUMAN CYTOMEGALOVIRUS TEGUMENT PROTEINS pUL48 AND pUL103 DURING LYTIC INFECTION

by

DANIEL A. ORTIZ

December 2015

Advisor: Dr. Philip E. Pellett

Major: Immunology and Microbiology

Degree: Doctor of Philosophy

Human cytomegalovirus (HCMV) is a large double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, HCMV is able to reconfigure the host cell machinery to establish a virus producing factory, termed the cytoplasmic virion assembly complex (cVAC). Generating drugs that affect cVAC development or function provides an alternative mode of action for HCMV antivirals that can essentially eliminate virion production. The objective of this work is aimed at identifying regulators of cVAC biogenesis that may serve as potential drug targets.

We identified three HCMV genes (UL48, UL94, and UL103) whose silencing or protein degradation had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. In addition, we found that mutant viruses expressing an unstable form of the UL103 protein causes a reduction in plaque size and the aberrant formation of intracellular virions.

To help define the mechanisms of action and predict additional functions of pUL103, we investigated its viral and cellular protein-protein interactions. We applied a dual method approach, co-immunoprecipitation and proximity biotinylation affinity purification followed by mass spectrometry to enrich for pUL103 interaction partners in uninfected and HCMV infected cells. This led to identification of pUL103 in nuclei, delineation of a novel ALIX binding domain, and a role for pUL103 in ALIX localization in infected cells.

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