Regulation of the HCMV 72 kD immediate early protein by phosphorylation
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Human cytomegalovirus (HCMV) infection disrupts the E2F-pRb pathway and thereby affects host cell cycle regulation. Disruption of this pathway contributes to the altered cell cycle profile observed in infected cells and increased expression of host and viral genes regulated by the transcription factor E2F. One specific viral gene product has been implicated in targeting this pathway, the major immediate early protein IE1/IE72. IE72 is phosphorylated both during HCMV infection and when expressed by transfection. Since the activities of many transcriptional activators are modulated by phosphorylation, the effect of phosphorylation on IE72 activity was investigated. IE72 phosphorylation was examined during HCMV infection and during the cell cycle. IE72 is phosphorylated at early times during infection, and the phosphorylation drops as infection proceeds. In growth arrested cells, IE72 is highly phosphorylated, and this phosphorylation drops upon serum stimulation. The sites of phosphorylation were mapped to nine serine residues in the C-terminal region, and these sites were mutated in various combinations and together to study the effects of loss of phosphorylation on IE72 activity. It was found that IE72 phosphorylation is required for the full activation of some E2F-dependent promoters, while it had no effect on other promoters. IE72 can also cooperate with other HCMV immediate early proteins to activate transcription in a synergistic manner; phosphorylation is required for synergism with IE19, whereas it has no role in synergism with IE86. Finally, the loss of IE72 phosphorylation was found to have no effect on its ability to complement an IE72-deficient HCMV strain. These data indicate that phosphorylation of IE72 modulates many of its activities, and that these functions may not all be required during HCMV infection.