Role of the Human Cytomegalovirus IE2 Protein on Transcriptional Regulation of Viral Promoters
type: CreativeWork
description: 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…
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…
author: Harris, Siabhon Michelle
keywords: Cytomegalovirus, Immediate early genes, Viral promoters, Molecular Biology, Virology
date published: 2009-04-01T07:00:00Z
date modified: 2026-07-24T03:34:46Z
provider: Old Dominion University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Old Dominion University