E2F Mediates Dihydrofolate Reductase Promoter Activation and Multiprotein Complex Formation in Human Cytomegalovirus Infection

Abstract

The adenovirus immediate-early protein E1A activates the adenovirus E2 promoter and several cellular gene promoters through transcription factor E2F. The immediate-early proteins of human cytomegalovirus (HCMV) can complement an E1A-deficient adenovirus mutant and activate the adenovirus E2 promoter. HCMV also has been shown to activate the adenovirus E2 promoter. On the basis of these findings, we have investigated whether HCMV can activate the promoter of the cellular dihydrofolate reductase (DHFR) gene, which requires E2F binding for maximal promoter activity. We show that HCMV activates the DHFR promoter and that products of the HCMV major immediate-early gene region mediate the activation of the promoter specifically through the E2F site. We used gel mobility shift assays to search for potential molecular mechanisms for this activation and found an "infection-specific" multimeric complex that bound to the E2F sites in the DHFR and E2 promoters in extracts from HCMV-infected cells but not in extracts from uninfected cells. Several antibodies against HCMV immediate-early gene products had no effect on this infection-specific complex. Subsequently, the complex was found to contain E2F, cyclin A, p33cdk2, and p107 and to be similar to S-phase-specific complexes that recently have been identified in several cell types. A functional role for the binding of the cyclin A-p33cdk2 complex to cellular gene promoters has yet to be demonstrated; however, HCMV infection causes the induction of both cellular DNA replication and transcription of growth-related genes containing E2F sites in their promoters. The findings described above therefore may relate to both of these effects of HCMV infection. We also provide evidence that some of the molecular events associated with adenovirus infection are different from those associated with HCMV infection.

The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclin-dependent kinase activation

“…Naturally, such a model needs to include other viral regulators supporting the cell cycle arrest (Lu and Shenk, 1999; Hayashi et al ., 2000) and the gene expression program of S phase (Margolis et al ., 1995; Poma et al ., 1996). The model is consistent with E2F‐specific gene expression in IE2‐arrested cells (Figures 4 and 5), the E2F‐dependent activation of dihydrofolate reductase (Wade et al ., 1992; Lembo et al ., 1999) and thymidylate synthase (Gribaudo et al ., 2000) in infected cells, and more generally with the dependency of HCMV replication on cdk activity (Bresnahan et al ., 1997).…”

Control of cytomegalovirus lytic gene expression by histone acetylation

“…The major immediate early (IE) genes are the first to be transcribed, resulting in two abundant proteins, IE1 p72 and IE2 p86 (Stenberg et al ., 1989), which are able to autoregulate the major immediate early promoter (MIEP; Pizzorno et al ., 1988; Cherrington and Mocarski, 1989). In addition to activating the promoters of viral early genes, the IE proteins can potently activate some cellular promoters (Hermiston et al ., 1987; Hunninghake et al ., 1989; Hagemeier et al ., 1992; M.Wade et al ., 1992; Walker et al ., 1992; Schwartz et al ., 1994, 1996; Hayhurst et al ., 1995; Caswell et al ., 1996). The resulting viral early gene expression is required for DNA replication and ultimately leads to the expression of the late virus‐assembly genes (for reviews see Anders and McCue, 1996; Gibson, 1996; Spector, 1996).…”