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Mol Cell Biol. 1992 October; 12(10): 4364–4374.
PMCID: PMC360360

E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection.


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.

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  • AbuBakar S, Au WW, Legator MS, Albrecht T. Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells. Environ Mol Mutagen. 1988;12(4):409–420. [PubMed]
  • Albrecht T, Boldogh I, Fons M, Lee CH, AbuBakar S, Russell JM, Au WW. Cell-activation responses to cytomegalovirus infection relationship to the phasing of CMV replication and to the induction of cellular damage. Subcell Biochem. 1989;15:157–202. [PubMed]
  • Albrecht T, Fons MP, Boldogh I, AbuBakar S, Deng CZ, Millinoff D. Metabolic and cellular effects of human cytomegalovirus infection. Transplant Proc. 1991 Jun;23(3 Suppl 3):48–55. [PubMed]
  • Albrecht T, Nachtigal M, St Jeor SC, Rapp F. Induction of cellular DNA synthesis and increased mitotic activity in syrian hamster embryo cells abortively infected with human cytomegalovirus. J Gen Virol. 1976 Feb;30(2):167–177. [PubMed]
  • Albrecht T, Rapp F. Malignant transformation of hamster embryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology. 1973 Sep;55(1):53–61. [PubMed]
  • Babiss LE. The cellular transcription factor E2f requires viral E1A and E4 gene products for increased DNA-binding activity and functions to stimulate adenovirus E2A gene expression. J Virol. 1989 Jun;63(6):2709–2717. [PMC free article] [PubMed]
  • Bagchi S, Raychaudhuri P, Nevins JR. Phosphorylation-dependent activation of the adenovirus-inducible E2F transcription factor in a cell-free system. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4352–4356. [PubMed]
  • Bagchi S, Raychaudhuri P, Nevins JR. Adenovirus E1A proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for E1A trans-activation. Cell. 1990 Aug 24;62(4):659–669. [PubMed]
  • Bagchi S, Weinmann R, Raychaudhuri P. The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell. 1991 Jun 14;65(6):1063–1072. [PubMed]
  • Bandara LR, Adamczewski JP, Hunt T, La Thangue NB. Cyclin A and the retinoblastoma gene product complex with a common transcription factor. Nature. 1991 Jul 18;352(6332):249–251. [PubMed]
  • Bandara LR, La Thangue NB. Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature. 1991 Jun 6;351(6326):494–497. [PubMed]
  • Baeuerle PA, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. [PubMed]
  • Benson JD, Huang ES. Human cytomegalovirus induces expression of cellular topoisomerase II. J Virol. 1990 Jan;64(1):9–15. [PMC free article] [PubMed]
  • Blake MC, Azizkhan JC. Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo. Mol Cell Biol. 1989 Nov;9(11):4994–5002. [PMC free article] [PubMed]
  • Blake MC, Jambou RC, Swick AG, Kahn JW, Azizkhan JC. Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter. Mol Cell Biol. 1990 Dec;10(12):6632–6641. [PMC free article] [PubMed]
  • Boldogh I, AbuBakar S, Albrecht T. Activation of proto-oncogenes: an immediate early event in human cytomegalovirus infection. Science. 1990 Feb 2;247(4942):561–564. [PubMed]
  • Boldogh I, AbuBakar S, Deng CZ, Albrecht T. Transcriptional activation of cellular oncogenes fos, jun, and myc by human cytomegalovirus. J Virol. 1991 Mar;65(3):1568–1571. [PMC free article] [PubMed]
  • Boldogh I, AbuBakar S, Millinoff D, Deng CZ, Albrecht T. Cellular oncogene activation by human cytomegalovirus. Lack of correlation with virus infectivity and immediate early gene expression. Arch Virol. 1991;118(3-4):163–177. [PubMed]
  • Cao L, Faha B, Dembski M, Tsai LH, Harlow E, Dyson N. Independent binding of the retinoblastoma protein and p107 to the transcription factor E2F. Nature. 1992 Jan 9;355(6356):176–179. [PubMed]
  • Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevins JR. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. [PubMed]
  • Chittenden T, Livingston DM, Kaelin WG., Jr The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell. 1991 Jun 14;65(6):1073–1082. [PubMed]
  • Davis MG, Kenney SC, Kamine J, Pagano JS, Huang ES. Immediate-early gene region of human cytomegalovirus trans-activates the promoter of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8642–8646. [PubMed]
  • DeMarchi JM. Correlation between stimulation of host cell DNA synthesis by human cytomegalovirus and lack of expression of a subset of early virus genes. Virology. 1983 Sep;129(2):274–286. [PubMed]
  • Devoto SH, Mudryj M, Pines J, Hunter T, Nevins JR. A cyclin A-protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F-cyclin A complex. Cell. 1992 Jan 10;68(1):167–176. [PubMed]
  • Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. [PMC free article] [PubMed]
  • Elledge SJ, Spottswood MR. A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. EMBO J. 1991 Sep;10(9):2653–2659. [PubMed]
  • Estes JE, Huang ES. Stimulation of cellular thymidine kinases by human cytomegalovirus. J Virol. 1977 Oct;24(1):13–21. [PMC free article] [PubMed]
  • Ewen ME, Faha B, Harlow E, Livingston DM. Interaction of p107 with cyclin A independent of complex formation with viral oncoproteins. Science. 1992 Jan 3;255(5040):85–87. [PubMed]
  • Ewen ME, Xing YG, Lawrence JB, Livingston DM. Molecular cloning, chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma gene product-related protein. Cell. 1991 Sep 20;66(6):1155–1164. [PubMed]
  • Faha B, Ewen ME, Tsai LH, Livingston DM, Harlow E. Interaction between human cyclin A and adenovirus E1A-associated p107 protein. Science. 1992 Jan 3;255(5040):87–90. [PubMed]
  • Geder KM, Lausch R, O'Neill F, Rapp F. Oncogenic transformation of human embryo lung cells by human cytomegalovirus. Science. 1976 Jun 11;192(4244):1134–1137. [PubMed]
  • Gerster T, Roeder RG. A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6347–6351. [PubMed]
  • Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. [PMC free article] [PubMed]
  • Graham FL, van der Eb AJ. Transformation of rat cells by DNA of human adenovirus 5. Virology. 1973 Aug;54(2):536–539. [PubMed]
  • Hagemeier C, Walker S, Caswell R, Kouzarides T, Sinclair J. The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID. J Virol. 1992 Jul;66(7):4452–4456. [PMC free article] [PubMed]
  • Hardy S, Engel DA, Shenk T. An adenovirus early region 4 gene product is required for induction of the infection-specific form of cellular E2F activity. Genes Dev. 1989 Jul;3(7):1062–1074. [PubMed]
  • Hardy S, Shenk T. E2F from adenovirus-infected cells binds cooperatively to DNA containing two properly oriented and spaced recognition sites. Mol Cell Biol. 1989 Oct;9(10):4495–4506. [PMC free article] [PubMed]
  • Helin K, Lees JA, Vidal M, Dyson N, Harlow E, Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. [PubMed]
  • Hermiston TW, Malone CL, Witte PR, Stinski MF. Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter. J Virol. 1987 Oct;61(10):3214–3221. [PMC free article] [PubMed]
  • Hiebert SW, Blake M, Azizkhan J, Nevins JR. Role of E2F transcription factor in E1A-mediated trans activation of cellular genes. J Virol. 1991 Jul;65(7):3547–3552. [PMC free article] [PubMed]
  • Hiebert SW, Chellappan SP, Horowitz JM, Nevins JR. The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev. 1992 Feb;6(2):177–185. [PubMed]
  • Hiebert SW, Lipp M, Nevins JR. E1A-dependent trans-activation of the human MYC promoter is mediated by the E2F factor. Proc Natl Acad Sci U S A. 1989 May;86(10):3594–3598. [PubMed]
  • Hirai K, Watanabe Y. Induction of alpha type DNA polymerases in human cytomegalovirus-infected WI-38 cells. Biochim Biophys Acta. 1976 Oct 18;447(3):328–339. [PubMed]
  • Hu QJ, Bautista C, Edwards GM, Defeo-Jones D, Jones RE, Harlow E. Antibodies specific for the human retinoblastoma protein identify a family of related polypeptides. Mol Cell Biol. 1991 Nov;11(11):5792–5799. [PMC free article] [PubMed]
  • Huang ES. Human cytomegalovirus. III. Virus-induced DNA polymerase. J Virol. 1975 Aug;16(2):298–310. [PMC free article] [PubMed]
  • Huang ES, Chen ST, Pagano JS. Human cytomegalovirus. I. Purification and characterization of viral DNA. J Virol. 1973 Dec;12(6):1473–1481. [PMC free article] [PubMed]
  • Huang MM, Hearing P. The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex. Genes Dev. 1989 Nov;3(11):1699–1710. [PubMed]
  • Isom HC. Stimulation of ornithine decarboxylase by human cytomegalovirus. J Gen Virol. 1979 Feb;42(2):265–278. [PubMed]
  • Kaelin WG, Jr, Krek W, Sellers WR, DeCaprio JA, Ajchenbaum F, Fuchs CS, Chittenden T, Li Y, Farnham PJ, Blanar MA, et al. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. [PubMed]
  • Kamiya S, Tanaka J, Ogura T, Ogura H, Sato H, Hatano M. Rabbit kidney cells abortively infected with human cytomegalovirus are arrested in mitotic phase. Arch Virol. 1986;89(1-4):131–144. [PubMed]
  • Klucher KM, Rabert DK, Spector DH. Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene. J Virol. 1989 Dec;63(12):5334–5343. [PMC free article] [PubMed]
  • Klucher KM, Spector DH. The human cytomegalovirus 2.7-kilobase RNA promoter contains a functional binding site for the adenovirus major late transcription factor. J Virol. 1990 Sep;64(9):4189–4198. [PMC free article] [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Lafemina RL, Pizzorno MC, Mosca JD, Hayward GS. Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes. Virology. 1989 Oct;172(2):584–600. [PubMed]
  • Lipp M, Schilling R, Bernhardt G. Trans-activation of human MYC: the second promoter is target for the stimulation by adenovirus E1a proteins. Oncogene. 1989 May;4(5):535–541. [PubMed]
  • Loeken MR, Brady J. The adenovirus EIIA enhancer. Analysis of regulatory sequences and changes in binding activity of ATF and EIIF following adenovirus infection. J Biol Chem. 1989 Apr 15;264(11):6572–6579. [PubMed]
  • Lüleci G, Sakízlí M, Günalp A. Selective chromosomal damage caused by human cytomegalovirus. Acta Virol. 1980 Sep;24(5):341–345. [PubMed]
  • Maguire HF, Hoeffler JP, Siddiqui A. HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein-protein interactions. Science. 1991 May 10;252(5007):842–844. [PubMed]
  • Malone CL, Vesole DH, Stinski MF. Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins. J Virol. 1990 Apr;64(4):1498–1506. [PMC free article] [PubMed]
  • Marton MJ, Baim SB, Ornelles DA, Shenk T. The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA. J Virol. 1990 May;64(5):2345–2359. [PMC free article] [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Mudryj M, Devoto SH, Hiebert SW, Hunter T, Pines J, Nevins JR. Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A. Cell. 1991 Jun 28;65(7):1243–1253. [PubMed]
  • Mudryj M, Hiebert SW, Nevins JR. A role for the adenovirus inducible E2F transcription factor in a proliferation dependent signal transduction pathway. EMBO J. 1990 Jul;9(7):2179–2184. [PubMed]
  • Neill SD, Hemstrom C, Virtanen A, Nevins JR. An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F. Proc Natl Acad Sci U S A. 1990 Mar;87(5):2008–2012. [PubMed]
  • Pagano M, Draetta G, Jansen-Dürr P. Association of cdk2 kinase with the transcription factor E2F during S phase. Science. 1992 Feb 28;255(5048):1144–1147. [PubMed]
  • Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G. Cyclin A is required at two points in the human cell cycle. EMBO J. 1992 Mar;11(3):961–971. [PubMed]
  • Pearson BE, Nasheuer HP, Wang TS. Human DNA polymerase alpha gene: sequences controlling expression in cycling and serum-stimulated cells. Mol Cell Biol. 1991 Apr;11(4):2081–2095. [PMC free article] [PubMed]
  • Pines J, Hunter T. Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature. 1990 Aug 23;346(6286):760–763. [PubMed]
  • Raychaudhuri P, Bagchi S, Devoto SH, Kraus VB, Moran E, Nevins JR. Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes. Genes Dev. 1991 Jul;5(7):1200–1211. [PubMed]
  • Raychaudhuri P, Bagchi S, Neill SD, Nevins JR. Activation of the E2F transcription factor in adenovirus-infected cells involves E1A-dependent stimulation of DNA-binding activity and induction of cooperative binding mediated by an E4 gene product. J Virol. 1990 Jun;64(6):2702–2710. [PMC free article] [PubMed]
  • Reichel R, Kovesdi I, Nevins JR. Activation of a preexisting cellular factor as a basis for adenovirus E1A-mediated transcription control. Proc Natl Acad Sci U S A. 1988 Jan;85(2):387–390. [PubMed]
  • Reichel R, Neill SD, Kovesdi I, Simon MC, Raychaudhuri P, Nevins JR. The adenovirus E4 gene, in addition to the E1A gene, is important for trans-activation of E2 transcription and for E2F activation. J Virol. 1989 Sep;63(9):3643–3650. [PMC free article] [PubMed]
  • Shirodkar S, Ewen M, DeCaprio JA, Morgan J, Livingston DM, Chittenden T. The transcription factor E2F interacts with the retinoblastoma product and a p107-cyclin A complex in a cell cycle-regulated manner. Cell. 1992 Jan 10;68(1):157–166. [PubMed]
  • Spector DJ, Tevethia MJ. Identification of a human cytomegalovirus virus DNA segment that complements an adenovirus 5 immediate early mutant. Virology. 1986 Jun;151(2):329–338. [PubMed]
  • Staprans SI, Rabert DK, Spector DH. Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene. J Virol. 1988 Sep;62(9):3463–3473. [PMC free article] [PubMed]
  • Jeor SC, Albrecht TB, Funk FD, Rapp F. Stimulation of cellular DNA synthesis by human cytomegalovirus. J Virol. 1974 Feb;13(2):353–362. [PMC free article] [PubMed]
  • St Jeor SC, Hutt R. Cell DNA replication as a function in the synthesis of human cytomegalovirus. J Gen Virol. 1977 Oct;37(1):65–73. [PubMed]
  • Swick AG, Blake MC, Kahn JW, Azizkhan JC. Functional analysis of GC element binding and transcription in the hamster dihydrofolate reductase gene promoter. Nucleic Acids Res. 1989 Nov 25;17(22):9291–9304. [PMC free article] [PubMed]
  • Tevethia MJ, Spector DJ. Complementation of an adenovirus 5 immediate early mutant by human cytomegalovirus. Virology. 1984 Sep;137(2):428–431. [PubMed]
  • Tevethia MJ, Spector DJ, Leisure KM, Stinski MF. Participation of two human cytomegalovirus immediate early gene regions in transcriptional activation of adenovirus promoters. Virology. 1987 Dec;161(2):276–285. [PubMed]
  • Thalmeier K, Synovzik H, Mertz R, Winnacker EL, Lipp M. Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter. Genes Dev. 1989 Apr;3(4):527–536. [PubMed]
  • Tsai LH, Harlow E, Meyerson M. Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase. Nature. 1991 Sep 12;353(6340):174–177. [PubMed]
  • Wright DA, Spector DH. Posttranscriptional regulation of a class of human cytomegalovirus phosphoproteins encoded by an early transcription unit. J Virol. 1989 Jul;63(7):3117–3127. [PMC free article] [PubMed]
  • Wright DA, Staprans SI, Spector DH. Four phosphoproteins with common amino termini are encoded by human cytomegalovirus AD169. J Virol. 1988 Jan;62(1):331–340. [PMC free article] [PubMed]
  • Závada V, Erban V, Rezácová D, Vonka V. Thymidine-kinase in cytomegalovirus infected cells. Arch Virol. 1976;52(4):333–339. [PubMed]

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