The objective of this study is to elucidate the interactions between cellular and viral factors which results in the progression of human cytomegalovirus (HCMV) infection and activation of the cell cycle. Specific attention will be devoted to understanding how HCMV initiates its gene expression and how HCMV major immediate-early (IE) proteins affect cellular factors to regulate downstream viral gene expression and activate macromolecular synthesis. Our working hypotheses are (a) upon infection, interactions of HCMV glycoprotein gB and/or gH with their cellular receptors trigger signal transduction pathways resulting in IkB phosphorylation, the nuclear translocation of NF-kB and transactivation of the HCMV majore IE promoter (among others), and (b) the resultant IE2 product(s) acting alone, or in cooperation with IE1, transactivate the expression of a second tier of viral and cellular transcription factors, including NF-kB. Target promoters include viral as well as those regulating the expression of enzymes involved in DNA synthesis and cell proliferation. To test these hypotheses, we propose the following approaches: (1) To overexpress and purify HCMV gB and gH from insect or mammalian cell system, and examine their effects on the activation of nuclear transcription factors, including NF-kB, in uninfected cells. Antibodies against these proteins and inhibitors of kinases associated with signal transduction will be used in attempts to block these effects and elucidate their mechanisms of activation. (2) To identify the virion-associated protein kinase(s) capable of phosphorylating IkB, and investigate the regulation of (i) cytosolic pools of IkB/NF-kB and (ii) nuclear NF-kB activity upon HCMV infection. We postulate that the second tier of NF-kB activation (Hypothesis (b)) is due to increased expression of genes encoding NF-kB proteins by the newly synthesized viral IE proteins. Therefore, we shall study the kinetics of NF-kB induction, determine the means by which NF-kB is released at IE times and investigate the mechanism by which the second phase of NF-Kb induction occurs. Promoters of p65 and p50 subunits of NF-kB, and promoter with SP-1 consensus sequence will be used to examine their responsiveness to IE proteins transactivation; (3) To detect and identify important viral and cellular proteins directly or indirectly interacting with IE2 proteins which are responsible for the activation of electromobility shift assay (EMSA), immuno-coprecipitation, DNA footprinting, Western analyses and in vitro transcription will be used to study the interactions. (4) To identify the interacting domains between IE2 and cellular factors, such as TFIID, SP-1, etc. Attention will be devoted to study interactions among IE2, RB, cyclins and E2F, between IE2 and TFIID, and between IE2 and SP-1. DNA sequence-specific binding of IE2 via SP-1, E2F, or NF-kB will be studied by EMSA, DNA footprinting and electron microscopic analysis.
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