Richard M. Stenberg

The Human Cytomegalovirus Major Immediate-Early Gene

Immediate-early genes function to regulate viral and cellular gene expression during the course of virus replication. The major immediate-early gene region of human cytomegalovirus plays a key role in affecting activation and repression of viral and cellular genes. These proteins intimately associate and/or interact with viral and cellular proteins during this process. Herein, we will discuss the current understanding of this complex gene region.

Immediate-Early Genes of Human Cytomegalovirus: Organization and Function

Immediate-early (IE) genes of human cytomegalovirus are the first genes expressed after the virus infects the cell. The most extensively characterized of the IE genes is the major IE gene region. Originating from this gene is a series of overlapping mRNAs which encode proteins that have common and unique domains. Functionally, at least three IE proteins, IE 72, IE 55, and IE 86, are responsible for regulation of viral promoters. These proteins interact to enhance and repress expression of the major IE promoter resulting in a coordinate regulation that affects the balance of IE gene expression. In addition, IE proteins interact with cell factors during regulation of viral gene expression to activate early promoters. Therefore, the coordinate regulation of IE genes and their corresponding proteins, by cellular proteins in the infected cell, is likely to be the single most important determinant influencing the outcome of human cytomegalovirus infection.

Functional properties of the human cytomegalovirus IE86 protein required for transcriptional regulation and virus replication.

ABSTRACT The human cytomegalovirus (HCMV) IE86 protein is essential for HCMV replication due to its ability to transactivate critical viral early promoters. In the current study, we performed a comprehensive mutational analysis between amino acids (aa) 535 and 545 of IE86 and assessed the impact of these mutations on IE86-mediated transcriptional activation. Using transient assays and complementing analysis with recombinant HCMV clones, we show that single amino acid mutations differentially impair the ability of IE86 to mediate transactivation of essential early gene promoters. The conserved tyrosine at amino acid 544 is critical for activation of the UL54 promoter in vitro and in the context of the viral genome. In contrast, mutation of the proline at position 535 disrupted activation of the UL54 promoter in transient assays but displayed activity similar to that of wild-type (WT) IE86 when assessed in the genomic context. To examine the underlying mechanism of this differential effect, glutathione S-transferase (GST) pulldown assays were performed, revealing that Y544 is critical for binding to the TATA binding protein (TBP), suggesting that this interaction is likely necessary for the ability of IE86 to activate the UL54 promoter. In contrast, mutation of either P535 or Y544 disrupted activation of the UL112-113 promoter both in vitro and in vivo, suggesting that interaction with TBP is not sufficient for IE86-mediated activation of this early promoter. Together, these studies demonstrate that IE86 activates early promoters by distinct mechanisms.