Date of Award

Spring 2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Julie Kerry

Committee Member

Ann Campbell

Committee Member

Richard Drake

Committee Member

laura Hanson

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous pathogen able to cause severe mortality and morbidity in immuno-compromised individuals. Successful infection by HCMV is dependent on expression of viral genes essential for replication. Immediate early (IE) gene products are the first subset of viral genes to be expressed during infection and function as key transcriptional regulators. IE2 is one the most predominantly expressed IE proteins and is essential for HCMV infection. IE2 transactivates several viral promoters, including those of the essential viral DNA polymerase (UL54) and UL112-113 gene regions. IE2 is also able to autoregulate is own expression and repress expression of the major IE gene products. This study aims to investigate the role of IE2 and cellular proteins in regulation of viral promoters in order to gain a better understanding of early events required for HCMV replication. Through the use of both transient assays and complementing assays in recombinant HCMV clones, we show that single amino acid mutations in the C-terminus of IE2 impair both IE2-mediated transactivation of early gene promoters and autorepression of the major IE promoter. These mutations in IE2 also result in nonviable recombinant viruses, emphasizing the importance of IE2 in HCMV replication. GST pulldown assays demonstrate that mutagenesis of Tyrosine 544 in IE2 reduces IE2 interactions with TATA binding protein (TBP) when compared to the wildtype IE2 protein. Using ChIP assays, we demonstrate that wildtype IE2 is recruited to the UL54 promoter in transiently transfected cells. However, mutagenesis in the IE2 protein at Proline 535 and Tyrosine 544 significantly decrease recruitment of IE2 to the UL54 promoter. Interestingly, these functional defects in mutated IE2 protein had no effect on RNA polymerase II recruitment, suggesting that IE2 may function in transcriptional regulation alter formation of the transcriptional pre-initiation complex. In this dissertation, we have further characterized regions and functional properties of IE2 essential for UL54 activation and HCMV replication. Our studies are significant in understanding the regulation of viral genes essential for replication of HCMV and in the development of novel HCMV therapies.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/kzx1-8q27

ISBN

9781109217483

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