The SV40 oncoprotein T antigen (T Ag) affects many cellular functions and is a promiscuous transcriptional activator. Similarly, the HCMV major immediate early proteins (MIEPs) also affect numerous cellular functions and we have found that they share many aspects of transcriptional activation with T Ag. In the past grant period, we established that T Ag aids in the formation and stabilization of the basal transcription complex and remains associated with TFIID, where it performs a co-activator or TAF (TBP-associated factor)- like function. In parallel studies, we found that the MIEPs also associate with TFIID and also perform a TAF-like function. T Ag and the MIEPs also affect the cell cycle and apoptosis; our recent studies indicate that these effects result in part from alterations of intracellular pathways, which mediate metabolism. In other studies, we have begun a detailed mapping of phosphorylation sites on the MIEPs, and the identification of functions mediated by phosphorylation.
In Aim 1, the PI will continue to examine transcriptional control by T Ag and the MIEPs. He will determine functional domains of T Ag needed to enhance the formation and stabilization of the basal transcription complex, and he will determine whether the MIEPs also mediate this effect. He will determine the involvement of phosphorylation (see Aim 3) and DnaJ domains in the functions of T Ag and the MIEPs, and the effects of T Ag and the MIEPs on histone acetyltransferase activities and chromatin remodeling will also be examined.
In Aim 2, he will examine the effects of T Ag and the MIEPs on cellular metabolism, the cell cycle and apoptosis using the Ts13 cell model system, as well as permissive cells. He will determine the mechanisms of p21 gene expression control mediated by the MIEPs and examine the effects of T Ag and the MIEPs on the cellular kinase Akt and the pathways controlled by it. Akt is a regulator of many metabolic and apoptotic processes.
In Aim 3 the PI will continue mapping phosphorylation sites on the MIEPS in order to obtain a battery of mutants in which sites have been mutated singly or in combination. These will be functionally analyzed in the studies of Aims 1 and 2 using transfection and viral infection. In parallel studies the effects of phosphorylation of T Ag will be studied. These studies will provide new insights into the mechanisms by which T Ag and the MIEPs manipulate cellular transcription, metabolism, the cell cycle and apoptosis.
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