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

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.

Characterization of the human cytomegalovirus UL75 (glycoprotein H) late gene promoter.

Glycoprotein H (gH, UL75) of human cytomegalovirus (HCMV) is an essential envelope glycoprotein that functions in viral entry and the activation of gene expression. To understand the regulation of this important viral gene, the promoter of the UL75 late gene was characterized in HCMV-infected cells at the late stages of viral infection. Primer extension analysis revealed a single major start site located 26 bp downstream of a putative TATA element. Deletion analysis showed the presence of a dominant activation domain from +14 to +35 that masked regulatory sequences upstream of the TATA element. Mutational analysis demonstrated that a PEA3-like element in this downstream domain was important for promoter activation. In addition, gel shift analysis revealed direct protein binding to the PEA3-like element. Together, these studies reveal that the gH promoter is regulated in a complex manner with sequences both upstream and downstream of the cap site influencing promoter activation.

Identification and characterization of novel murine cytomegalovirus M112-113 (e1) gene products.

The human cytomegalovirus (HCMV) UL112-113 gene products play important roles in viral DNA replication and transcriptional regulation. In this report, we characterize two novel transcripts originating from the homologous M112-113 (e1) region of the murine cytomegalovirus (MCMV) genome. These transcripts of 2.0 and 2.4 kb represent alternatively spliced products of the e1 gene region. Analysis of the e1 proteins demonstrates the presence of a previously unidentified 87-kDa protein that is likely encoded by the 2.4-kb transcript. All four protein products derived from the e1 gene region are expressed with early kinetics, are coordinately regulated, and localize predominantly to the nucleus of MCMV-infected cells. The expression pattern and localization of the e1 proteins show significant similarity to those of the HCMV UL112-113 proteins, signifying that MCMV e1 will serve as a useful model for assessing the role of this early gene region during viral infection.