Characterization of Cis-acting partners within the cytomegalovirus major immediate-early enhancer that strengthen MIE gene expression and viral fitness Courtney Searcey Galle , University of Iowa Follow

[摘要] Human cytomegalovirus infects approximately 50% of adults in the United States and in most cases is asymptomatic. However, in the case of immune compromised persons such as AIDS patients, transplant patients, and newborn babies, life threatening CMV disease can occur. The HCMV major immediate-early enhancer functions as a master regulatory switch, whose activation is essential for the expression of the major IE transactivating proteins, IE1 p72 and IE2 p86. While critical to the viral lifecycle, regulation of MIE enhancer activation is very complex and not yet fully understood. I characterized the role of cis-acting partners within the MIE enhancer that function to strengthen MIE gene expression and discovered a novel mode of post-IE enhancer regulation. These results add significantly to our understanding the inner workings of the HCMV MIE enhancer/promoter in lytically infected cells. The distal portion of the MIE enhancer is composed of two functionally redundant segments, which are necessary for MIE gene expression at low multiplicity of infection (MOI). Using an unbiased genetic approach I identified a previously unrecognized cis-acting TGGGCA/G repeat that is inextricably linked to GC-box repeats, which together form an enhancer-spanning network. This network of elements (TG network) is conserved in nonhuman primate CMV MIE enhancers. HCMV constructs lacking the entire enhancer TG network inadequately sustain MIE gene expression at low MOI at post-immediate early (IE) times of infection (≥8 h pi). An MIE enhancer-specified mode of post-IE regulation has not been described before and suggests a cis-regulatory code specialization that has evolved to sustain rather than to initiate MIE gene expression. I hypothesized that another cis-acting element(s) function together with the TG network to form a multi-network system that senses and integrates a variety of cellular environmental signals to modulate efficiency in the initiation and/or maintenance of MIE enhancer-dependent gene expression. Using recombinant viruses with mutations in either the cyclic AMP response element (CRE) network, NFkB network, TG network, or a combination of these networks, I show that the TG-C and TG-K partnerships are the most important for conferring the greatest level of MIE enhancer-dependent MIE gene expression, frequency and size of viral plaques in HFF cells, while the TG-K partnership is most important to DNT-2 cells. Additionally, I conclude that the C-K partnership functions through an alternate mechanism than that of the TG network. Together these results suggest that the strength of enhancement by cis-acting network pair interactions forms a multi-network system that modulates efficiency of MIE enhancer-dependent gene expression and which differs in relation to cell type during lytic infection.