Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription

Abstract

Three polypeptides are produced from the major immediate-early (IE) region of human cytomegalovirus by alternative splicing. The IE gene products regulate subsequent viral and cellular gene expression. We previously reported that cotransfection of a genomic clone of the major IE region stimulated transient expression of chloramphenicol acetyltransferase driven by the dihydrofolate reductase (DHFR) promoter and that an intact E2F site was required for the trans activation (M. Wade, T. F. Kowalik, M. Mudryj, E.-S. Huang, and J. C. Azizkhan, Mol. Cell. Biol. 12:4364-4374, 1992). With the availability of cDNA clones for the individual major IE proteins, we sought to determine which of these proteins exerted this effect and whether the IE protein(s) interacted with E2F. In this study, we use cotransfection to demonstrate that the 55-and 86-kDa major IE proteins from the IE2 region can each moderately trans activate the DHFR promoter and that the 72-kDa IE1 protein stimulates DHFR transcription to a much higher level. Furthermore, trans activation through the 72-kDa IE1 protein is in part E2F dependent, while activation by the 55-and 86-kDa IE proteins is E2F independent. We also demonstrate by in vitro pull-down assays that the 72-kDa IE1 protein can specifically interact with the DNA binding domain of E2F1 (amino acids 88 to 191) in the presence of nuclear extract. Moreover, antibodies to either E2F1 or IE72 will immunoprecipitate both E2F and IE72 from cells that stably express IE72, and antibody to E2F1 will immunoprecipitate IE72 from normal human fibroblast cells infected with human cytomegalovirus.

Downregulation of GFAP, TSP‐1, and p53 in human glioblastoma cell line, U373MG, by IE1 protein from human cytomegalovirus

“…Our findings are somewhat surprising in that the expression of genes affected by IE1 was expected to be upregulated rather than downregulated, because IE1 has been known to be a transactivator of various cellular and viral promoters (Stenberg and Stinski, 1985;Tevethia et al, 1987;Davis et al, 1987;Cherrington and Mocarski, 1989;Sambucetti et al, 1989;Stenberg et al, 1989;Iwamoto et al, 1990;Hagemeier et al, 1992;Monick et al, 1992;Michelson et al, 1994;Margolis et al, 1995;Yur-ochko et al, 1995;Kim et al, 1999;Murayama et al, 2000). However, most affected genes harbored in the cDNA chip were downregulated by the IE1 protein.…”

“…This 72-kDa phosphoprotein is abundantly expressed immediately after infection (Plachter et al, 1996). In transient transfection assays, IE1 has been shown to be involved in the autoregulation of its own promoter and also in the transactivation of viral and cellular promoters, alone or in concert with other viral or cellular proteins (Stenberg and Stinski, 1985;Davis et al, 1987;Tevethia et al, 1987;Cherrington and Mocarski, 1989;Sambucetti et al, 1989;Stenberg et al, 1989;Iwamoto et al, 1990;Hagemeier et al, 1992;Monick et al, 1992;Michelson et al, 1994;Margolis et al, 1995;Yurochko et al, 1995;Kim et al, 1999;Murayama et al, 2000). It was also reported that the IE1 gene product could cooperate with another IE gene product, IE2, and adenoviral E1A to transform primary baby rat kidney (BRK) cells (Shen et al, 1997).…”

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts

“…IEp72 is known to be a DNA-binding protein, with regulatory functions on viral gene expression [Isomura and Stinski, 2003]. Furthermore, it is able to interfere with several cellular processes, including gene regulation, cell cycle progression, signal transduction, apoptosis, interaction with specific nuclear domains, such as promielocytic leukemia (PML) bodies [Margolis et al, 1995;Zhu et al, 1995;Wilkinson et al, 1998;McElroy et al, 2000;Castillo and Kowalik, 2002]. Specifically, IEp72 was previously reported to accumulate in PML bodies and to trigger their disruption about 4 h after infection [Kelly et al, 1995;Ahn and Hayward, 1997;Muller and Dejean, 1999].…”

The S phase of the cell cycle and its perturbation by human cytomegalovirus

“…Thus, HCMV is similar to adenovirus (E1A oncoprotein), SV40 (large T antigen), papillomavirus (E7 oncoprotein) and EBV nuclear antigen 3C in targeting this pathway [65][66][67][68]. Furthermore, IE72 directly interacts with E2F1 [69] and its inherent kinase activity is also believed to phosphorylate p107 and p130 (but not pRb) releasing the E2F4 family member, and, through a direct interaction, phosphorylates E2Fs À1, À2 and À3. This leads to stimulation of cellular DHFR expression to maintain abundant resources within the cell for the virus to exploit for high-level viral DNA synthesis [59].…”