We have previously shown that many cell cycle regulatory gene products are markedly affected by
infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). One of these
proteins,
cyclin E, is a key determinant of cell cycle progression during G(1), and its
mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729-3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous
cyclin E promoter during the course of
infection. In vivo
dimethyl sulfate footprinting of the
cyclin E promoter revealed several regions of protection and
hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced
cyclin E transcripts initiate downstream of the start site identified in mock-infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral
immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous
cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of
cyclin E expression. Moreover, IE1-72 does not appear to be required, as
infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of
cyclin E expression. Using electrophoretic mobility shift assays with infected
cell extracts and a region of the
cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an
infection-specific banding pattern. One of the
infection-specific bands contained the
proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4-DP-1-p130 complex along with viral early gene expression may play a role in the transcriptional regulation of
cyclin E mRNA during HCMV
infection.