Abstract
The human cytomegalovirus early promoter for the UL4 gene, which codes for an early viral envelope glycoprotein designated gpUL4, requires immediate-early viral protein two (IE2) synthesis to be activated (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281, 1989). We investigated the cis-acting and trans-acting factors that regulate transcription from this UL4 promoter. In transient transfection assays, the viral IE2 protein negated the effect of an upstream cis-acting negative element and enhanced downstream gene expression. A cis-acting positive element contributed to the activity of the viral promoter when an upstream cis-acting negative element was deleted or when the viral IE2 protein was present. The cellular protein(s) that binds to the cis-acting negative element requires further investigation. The cellular protein that binds to the cis-acting positive element was characterized. Two DNA sequence-specific protein complexes were detected with DNA probes spanning the region containing the cis-acting positive element and human cytomegalovirus-infected human fibroblast cell nuclear extracts. The more slowly migrating complex was labeled complex A, and the faster was labeled complex B. Only complex B was detected with mock-infected cell nuclear extracts. Competition experiments confirmed the specificity of the A and B complexes. The protein bound to the DNA in both the complexes contacts a CCAAT box imperfect dyad symmetry (5'CCAATCACTGG3'). Either CCAAT box within the dyad symmetry could compete for binding the nuclear factor. Mutation of the CCAAT box dyad symmetry resulted in a decrease of the transcriptional activity from the UL4 promoter. A cellular transcription factor, antigenically related to nuclear factor-Y (NF-Y), was found in both complexes A and B. Events associated with viral infection caused phosphorylation of protein complex A. Dephosphorylation of the DNA-binding protein converts complex A to complex B. The effect of phosphorylation of NF-Y is not known.
Full text
PDF









Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akira S., Isshiki H., Sugita T., Tanabe O., Kinoshita S., Nishio Y., Nakajima T., Hirano T., Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990 Jun;9(6):1897–1906. doi: 10.1002/j.1460-2075.1990.tb08316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Alam T., An M. R., Papaconstantinou J. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response. J Biol Chem. 1992 Mar 15;267(8):5021–5024. [PubMed] [Google Scholar]
- Becker D. M., Fikes J. D., Guarente L. A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1968–1972. doi: 10.1073/pnas.88.5.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berkhout B., Jeang K. T. Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1992 Jan;66(1):139–149. doi: 10.1128/jvi.66.1.139-149.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birkenmeier E. H., Gwynn B., Howard S., Jerry J., Gordon J. I., Landschulz W. H., McKnight S. L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev. 1989 Aug;3(8):1146–1156. doi: 10.1101/gad.3.8.1146. [DOI] [PubMed] [Google Scholar]
- Bowers W. J., Ruddell A. a1/EBP: a leucine zipper protein that binds CCAAT/enhancer elements in the avian leukosis virus long terminal repeat enhancer. J Virol. 1992 Nov;66(11):6578–6586. doi: 10.1128/jvi.66.11.6578-6586.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burns L. J., Waring J. F., Reuter J. J., Stinski M. F., Ginder G. D. Only the HLA class I gene minimal promoter elements are required for transactivation by human cytomegalovirus immediate early genes. Blood. 1993 Mar 15;81(6):1558–1566. [PubMed] [Google Scholar]
- Chang C. P., Malone C. L., Stinski M. F. A human cytomegalovirus early gene has three inducible promoters that are regulated differentially at various times after infection. J Virol. 1989 Jan;63(1):281–290. doi: 10.1128/jvi.63.1.281-290.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chang C. P., Vesole D. H., Nelson J., Oldstone M. B., Stinski M. F. Identification and expression of a human cytomegalovirus early glycoprotein. J Virol. 1989 Aug;63(8):3330–3337. doi: 10.1128/jvi.63.8.3330-3337.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
- Chodosh L. A., Baldwin A. S., Carthew R. W., Sharp P. A. Human CCAAT-binding proteins have heterologous subunits. Cell. 1988 Apr 8;53(1):11–24. doi: 10.1016/0092-8674(88)90483-7. [DOI] [PubMed] [Google Scholar]
- DeMarchi J. M. Post-transcriptional control of human cytomegalovirus gene expression. Virology. 1983 Jan 30;124(2):390–402. doi: 10.1016/0042-6822(83)90355-0. [DOI] [PubMed] [Google Scholar]
- DeMarchi J. M., Schmidt C. A., Kaplan A. S. Patterns of transcription of human cytomegalovirus in permissively infected cells. J Virol. 1980 Aug;35(2):277–286. doi: 10.1128/jvi.35.2.277-286.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Depto A. S., Stenberg R. M. Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products. J Virol. 1989 Mar;63(3):1232–1238. doi: 10.1128/jvi.63.3.1232-1238.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DiDonato J. A., Muller M. T. DNA binding and gene regulation by the herpes simplex virus type 1 protein ICP4 and involvement of the TATA element. J Virol. 1989 Sep;63(9):3737–3747. doi: 10.1128/jvi.63.9.3737-3747.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dignam J. D., Lebovitz R. M., Roeder R. G. 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. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dorn A., Bollekens J., Staub A., Benoist C., Mathis D. A multiplicity of CCAAT box-binding proteins. Cell. 1987 Sep 11;50(6):863–872. doi: 10.1016/0092-8674(87)90513-7. [DOI] [PubMed] [Google Scholar]
- Faber M., Sealy L. Rous sarcoma virus enhancer factor I is a ubiquitous CCAAT transcription factor highly related to CBF and NF-Y. J Biol Chem. 1990 Dec 25;265(36):22243–22254. [PubMed] [Google Scholar]
- Forsburg S. L., Guarente L. Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev. 1989 Aug;3(8):1166–1178. doi: 10.1101/gad.3.8.1166. [DOI] [PubMed] [Google Scholar]
- Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hagemeier C., Walker S. M., Sissons P. J., Sinclair J. H. The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements. J Gen Virol. 1992 Sep;73(Pt 9):2385–2393. doi: 10.1099/0022-1317-73-9-2385. [DOI] [PubMed] [Google Scholar]
- 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. doi: 10.1128/jvi.66.7.4452-4456.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
- Hermiston T. W., Malone C. L., Witte P. R., Stinski M. F. 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. doi: 10.1128/jvi.61.10.3214-3221.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hooft van Huijsduijnen R., Li X. Y., Black D., Matthes H., Benoist C., Mathis D. Co-evolution from yeast to mouse: cDNA cloning of the two NF-Y (CP-1/CBF) subunits. EMBO J. 1990 Oct;9(10):3119–3127. doi: 10.1002/j.1460-2075.1990.tb07509.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horikoshi N., Maguire K., Kralli A., Maldonado E., Reinberg D., Weinmann R. Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5124–5128. doi: 10.1073/pnas.88.12.5124. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imbalzano A. N., DeLuca N. A. Substitution of a TATA box from a herpes simplex virus late gene in the viral thymidine kinase promoter alters ICP4 inducibility but not temporal expression. J Virol. 1992 Sep;66(9):5453–5463. doi: 10.1128/jvi.66.9.5453-5463.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ingles C. J., Shales M., Cress W. D., Triezenberg S. J., Greenblatt J. Reduced binding of TFIID to transcriptionally compromised mutants of VP16. Nature. 1991 Jun 13;351(6327):588–590. doi: 10.1038/351588a0. [DOI] [PubMed] [Google Scholar]
- Johnson P. F., Landschulz W. H., Graves B. J., McKnight S. L. Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses. Genes Dev. 1987 Apr;1(2):133–146. doi: 10.1101/gad.1.2.133. [DOI] [PubMed] [Google Scholar]
- Jupp R., Hoffmann S., Stenberg R. M., Nelson J. A., Ghazal P. Human cytomegalovirus IE86 protein interacts with promoter-bound TATA-binding protein via a specific region distinct from the autorepression domain. J Virol. 1993 Dec;67(12):7539–7546. doi: 10.1128/jvi.67.12.7539-7546.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kinoshita S., Akira S., Kishimoto T. A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1473–1476. doi: 10.1073/pnas.89.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klucher K. M., Rabert D. K., Spector D. H. 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. doi: 10.1128/jvi.63.12.5334-5343.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klucher K. M., Sommer M., Kadonaga J. T., Spector D. H. In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins. Mol Cell Biol. 1993 Feb;13(2):1238–1250. doi: 10.1128/mcb.13.2.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klucher K. M., Spector D. H. 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. doi: 10.1128/jvi.64.9.4189-4198.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuwabara M. D., Sigman D. S. Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. Biochemistry. 1987 Nov 17;26(23):7234–7238. doi: 10.1021/bi00397a006. [DOI] [PubMed] [Google Scholar]
- Li X. Y., Hooft van Huijsduijnen R., Mantovani R., Benoist C., Mathis D. Intron-exon organization of the NF-Y genes. Tissue-specific splicing modifies an activation domain. J Biol Chem. 1992 May 5;267(13):8984–8990. [PubMed] [Google Scholar]
- Lieberman P. M., Berk A. J. The Zta trans-activator protein stabilizes TFIID association with promoter DNA by direct protein-protein interaction. Genes Dev. 1991 Dec;5(12B):2441–2454. doi: 10.1101/gad.5.12b.2441. [DOI] [PubMed] [Google Scholar]
- Lim L. C., Swendeman S. L., Sheffery M. Molecular cloning of the alpha-globin transcription factor CP2. Mol Cell Biol. 1992 Feb;12(2):828–835. doi: 10.1128/mcb.12.2.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu B., Hermiston T. W., Stinski M. F. A cis-acting element in the major immediate-early (IE) promoter of human cytomegalovirus is required for negative regulation by IE2. J Virol. 1991 Feb;65(2):897–903. doi: 10.1128/jvi.65.2.897-903.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- López-Cabrera M., Letovsky J., Hu K. Q., Siddiqui A. Transcriptional factor C/EBP binds to and transactivates the enhancer element II of the hepatitis B virus. Virology. 1991 Aug;183(2):825–829. doi: 10.1016/0042-6822(91)91019-d. [DOI] [PubMed] [Google Scholar]
- Maity S. N., Sinha S., Ruteshouser E. C., de Crombrugghe B. Three different polypeptides are necessary for DNA binding of the mammalian heteromeric CCAAT binding factor. J Biol Chem. 1992 Aug 15;267(23):16574–16580. [PubMed] [Google Scholar]
- Maity S. N., de Crombrugghe B. Biochemical analysis of the B subunit of the heteromeric CCAAT-binding factor. A DNA-binding domain and a subunit interaction domain are specified by two separate segments. J Biol Chem. 1992 Apr 25;267(12):8286–8292. [PubMed] [Google Scholar]
- Malone C. L., Vesole D. H., Stinski M. F. 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. doi: 10.1128/jvi.64.4.1498-1506.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mantovani R., Pessara U., Tronche F., Li X. Y., Knapp A. M., Pasquali J. L., Benoist C., Mathis D. Monoclonal antibodies to NF-Y define its function in MHC class II and albumin gene transcription. EMBO J. 1992 Sep;11(9):3315–3322. doi: 10.1002/j.1460-2075.1992.tb05410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McDonough S. H., Spector D. H. Transcription in human fibroblasts permissively infected by human cytomegalovirus strain AD169. Virology. 1983 Feb;125(1):31–46. doi: 10.1016/0042-6822(83)90061-2. [DOI] [PubMed] [Google Scholar]
- Mermod N., O'Neill E. A., Kelly T. J., Tjian R. The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain. Cell. 1989 Aug 25;58(4):741–753. doi: 10.1016/0092-8674(89)90108-6. [DOI] [PubMed] [Google Scholar]
- Olesen J. T., Guarente L. The HAP2 subunit of yeast CCAAT transcriptional activator contains adjacent domains for subunit association and DNA recognition: model for the HAP2/3/4 complex. Genes Dev. 1990 Oct;4(10):1714–1729. doi: 10.1101/gad.4.10.1714. [DOI] [PubMed] [Google Scholar]
- Olsen H. S., Rosen C. A. Contribution of the TATA motif to Tat-mediated transcriptional activation of human immunodeficiency virus gene expression. J Virol. 1992 Sep;66(9):5594–5597. doi: 10.1128/jvi.66.9.5594-5597.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ozer J., Faber M., Chalkley R., Sealy L. Isolation and characterization of a cDNA clone for the CCAAT transcription factor EFIA reveals a novel structural motif. J Biol Chem. 1990 Dec 25;265(36):22143–22152. [PubMed] [Google Scholar]
- Pizzorno M. C., Mullen M. A., Chang Y. N., Hayward G. S. The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal. J Virol. 1991 Jul;65(7):3839–3852. doi: 10.1128/jvi.65.7.3839-3852.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Santomenna L. D., Colberg-Poley A. M. Induction of cellular hsp70 expression by human cytomegalovirus. J Virol. 1990 May;64(5):2033–2040. doi: 10.1128/jvi.64.5.2033-2040.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scott L. M., Civin C. I., Rorth P., Friedman A. D. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood. 1992 Oct 1;80(7):1725–1735. [PubMed] [Google Scholar]
- Spector D. H., Klucher K. M., Rabert D. K., Wright D. A. Human cytomegalovirus early gene expression. Curr Top Microbiol Immunol. 1990;154:21–45. doi: 10.1007/978-3-642-74980-3_2. [DOI] [PubMed] [Google Scholar]
- Staprans S. I., Rabert D. K., Spector D. H. 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. doi: 10.1128/jvi.62.9.3463-3473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stasiak P. C., Mocarski E. S. Transactivation of the cytomegalovirus ICP36 gene promoter requires the alpha gene product TRS1 in addition to IE1 and IE2. J Virol. 1992 Feb;66(2):1050–1058. doi: 10.1128/jvi.66.2.1050-1058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stenberg R. M., Fortney J., Barlow S. W., Magrane B. P., Nelson J. A., Ghazal P. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains. J Virol. 1990 Apr;64(4):1556–1565. doi: 10.1128/jvi.64.4.1556-1565.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stinski M. F. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides. J Virol. 1978 Jun;26(3):686–701. doi: 10.1128/jvi.26.3.686-701.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stringer K. F., Ingles C. J., Greenblatt J. Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature. 1990 Jun 28;345(6278):783–786. doi: 10.1038/345783a0. [DOI] [PubMed] [Google Scholar]
- Wade E. J., Klucher K. M., Spector D. H. An AP-1 binding site is the predominant cis-acting regulatory element in the 1.2-kilobase early RNA promoter of human cytomegalovirus. J Virol. 1992 Apr;66(4):2407–2417. doi: 10.1128/jvi.66.4.2407-2417.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wathen M. W., Stinski M. F. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. J Virol. 1982 Feb;41(2):462–477. doi: 10.1128/jvi.41.2.462-477.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang B. S., Gilbert J. D., Freytag S. O. Overexpression of Myc suppresses CCAAT transcription factor/nuclear factor 1-dependent promoters in vivo. Mol Cell Biol. 1993 May;13(5):3093–3102. doi: 10.1128/mcb.13.5.3093. [DOI] [PMC free article] [PubMed] [Google Scholar]