Abstract
The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the binding of both DEF-A and DEF-B complexes to the downstream elements. Finally, as revealed by immunomicroscopy, pIVa2 is targeted to the nucleus, where it distributes to restricted locations in the nucleoplasm, as well as to the nucleoli. Altogether, these results demonstrate that pIVa2 plays a critical role in the transition from the early to the late phase of the lytic cycle. Furthermore, pIVa2 may serve additional functions yet to be uncovered, as suggested by its presence within the cell nucleolus.
Full Text
The Full Text of this article is available as a PDF (1.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akusjärvi G. Proteins with transcription regulatory properties encoded by human adenoviruses. Trends Microbiol. 1993 Aug;1(5):163–170. doi: 10.1016/0966-842x(93)90085-6. [DOI] [PubMed] [Google Scholar]
- Alonso-Caplen F. V., Katze M. G., Krug R. M. Efficient transcription, not translation, is dependent on adenovirus tripartite leader sequences at late times of infection. J Virol. 1988 May;62(5):1606–1616. doi: 10.1128/jvi.62.5.1606-1616.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Banerji J., Rusconi S., Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981 Dec;27(2 Pt 1):299–308. doi: 10.1016/0092-8674(81)90413-x. [DOI] [PubMed] [Google Scholar]
- Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell. 1979 Aug;17(4):935–944. doi: 10.1016/0092-8674(79)90333-7. [DOI] [PubMed] [Google Scholar]
- Bocco J. L., Reimund B., Chatton B., Kedinger C. Rb may act as a transcriptional co-activator in undifferentiated F9 cells. Oncogene. 1993 Nov;8(11):2977–2986. [PubMed] [Google Scholar]
- Crossland L. D., Raskas H. J. Identification of adenovirus genes that require template replication for expression. J Virol. 1983 Jun;46(3):737–748. doi: 10.1128/jvi.46.3.737-748.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Flint S. J. Expression of adenoviral genetic information in productively infected cells. Biochim Biophys Acta. 1982 Apr 29;651(2-3):175–208. doi: 10.1016/0304-419x(82)90011-7. [DOI] [PubMed] [Google Scholar]
- Green S., Issemann I., Sheer E. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res. 1988 Jan 11;16(1):369–369. doi: 10.1093/nar/16.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hen R., Sassone-Corsi P., Corden J., Gaub M. P., Chambon P. Sequences upstream from the T-A-T-A box are required in vivo and in vitro for efficient transcription from the adenovirus serotype 2 major late promoter. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7132–7136. doi: 10.1073/pnas.79.23.7132. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jansen-Durr P., Boeuf H., Kédinger C. Replication-induced stimulation of the major late promoter of adenovirus is correlated to the binding of a factor to sequences in the first intron. Nucleic Acids Res. 1988 May 11;16(9):3771–3786. doi: 10.1093/nar/16.9.3771. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jansen-Durr P., Mondésert G., Kédinger C. Replication-dependent activation of the adenovirus major late promoter is mediated by the increased binding of a transcription factor to sequences in the first intron. J Virol. 1989 Dec;63(12):5124–5132. doi: 10.1128/jvi.63.12.5124-5132.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiménez-García L. F., Spector D. L. In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell. 1993 Apr 9;73(1):47–59. doi: 10.1016/0092-8674(93)90159-n. [DOI] [PubMed] [Google Scholar]
- Kalland K. H., Langhoff E., Bos H. J., Göttlinger H., Haseltine W. A. Rex-dependent nucleolar accumulation of HTLV-I mRNAs. New Biol. 1991 Apr;3(4):389–397. [PubMed] [Google Scholar]
- Kedinger C., Gissinger F., Chambon P. Animal DNA-dependent RNA polymerases. Molecular structures and immunological properties of calf-thymus enzyme AI and of calf-thymus and rat-liver enzymes B. Eur J Biochem. 1974 May 15;44(2):421–436. doi: 10.1111/j.1432-1033.1974.tb03500.x. [DOI] [PubMed] [Google Scholar]
- Krippl B., Ferguson B., Rosenberg M., Westphal H. Functions of purified E1A protein microinjected into mammalian cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6988–6992. doi: 10.1073/pnas.81.22.6988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Leong K., Lee W., Berk A. J. High-level transcription from the adenovirus major late promoter requires downstream binding sites for late-phase-specific factors. J Virol. 1990 Jan;64(1):51–60. doi: 10.1128/jvi.64.1.51-60.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis J. B., Esche H., Smart J. E., Stillman B. W., Harter M. L., Mathews M. B. Organization and expression of the left third of the genome of adenovirus. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):493–508. doi: 10.1101/sqb.1980.044.01.052. [DOI] [PubMed] [Google Scholar]
- Logan J., Shenk T. In vivo identification of sequence elements required for normal function of the adenovirus major late transcriptional control region. Nucleic Acids Res. 1986 Aug 11;14(15):6327–6335. [PMC free article] [PubMed] [Google Scholar]
- Lutz Y., Jacob M., Fuchs J. P. The distribution of two hnRNP-associated proteins defined by a monoclonal antibody is altered in heat-shocked HeLa cells. Exp Cell Res. 1988 Mar;175(1):109–124. doi: 10.1016/0014-4827(88)90259-5. [DOI] [PubMed] [Google Scholar]
- MacLean C. A., Rixon F. J., Marsden H. S. The products of gene US11 of herpes simplex virus type 1 are DNA-binding and localize to the nucleoli of infected cells. J Gen Virol. 1987 Jul;68(Pt 7):1921–1937. doi: 10.1099/0022-1317-68-7-1921. [DOI] [PubMed] [Google Scholar]
- Mansour S. L., Grodzicker T., Tjian R. Downstream sequences affect transcription initiation from the adenovirus major late promoter. Mol Cell Biol. 1986 Jul;6(7):2684–2694. doi: 10.1128/mcb.6.7.2684. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mason B. B., Davis A. R., Bhat B. M., Chengalvala M., Lubeck M. D., Zandle G., Kostek B., Cholodofsky S., Dheer S., Molnar-Kimber K. Adenovirus vaccine vectors expressing hepatitis B surface antigen: importance of regulatory elements in the adenovirus major late intron. Virology. 1990 Aug;177(2):452–461. doi: 10.1016/0042-6822(90)90509-p. [DOI] [PubMed] [Google Scholar]
- Mondesert G., Tribouley C., Kedinger C. Identification of a novel downstream binding protein implicated in late-phase-specific activation of the adenovirus major late promotor. Nucleic Acids Res. 1992 Aug 11;20(15):3881–3889. doi: 10.1093/nar/20.15.3881. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mondésert G., Kédinger C. Cooperation between upstream and downstream elements of the adenovirus major late promoter for maximal late phase-specific transcription. Nucleic Acids Res. 1991 Jun 25;19(12):3221–3228. doi: 10.1093/nar/19.12.3221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nosaka T., Takamatsu T., Miyazaki Y., Sano K., Sato A., Kubota S., Sakurai M., Ariumi Y., Nakai M., Fujita S. Cytotoxic activity of rev protein of human immunodeficiency virus type 1 by nucleolar dysfunction. Exp Cell Res. 1993 Nov;209(1):89–102. doi: 10.1006/excr.1993.1289. [DOI] [PubMed] [Google Scholar]
- Puvion-Dutilleul F. Localization of viral-specific 21 kDa protein in nucleoli of herpes simplex infected cells. Eur J Cell Biol. 1987 Jun;43(3):487–498. [PubMed] [Google Scholar]
- Puvion-Dutilleul F., Puvion E. Sites of transcription of adenovirus type 5 genomes in relation to early viral DNA replication in infected HeLa cells. A high resolution in situ hybridization and autoradiographical study. Biol Cell. 1991;71(1-2):135–147. doi: 10.1016/0248-4900(91)90060-z. [DOI] [PubMed] [Google Scholar]
- Puvion-Dutilleul F., Roussev R., Puvion E. Distribution of viral RNA molecules during the adenovirus type 5 infectious cycle in HeLa cells. J Struct Biol. 1992 May-Jun;108(3):209–220. doi: 10.1016/1047-8477(92)90021-2. [DOI] [PubMed] [Google Scholar]
- Reach M., Xu L. X., Young C. S. Transcription from the adenovirus major late promoter uses redundant activating elements. EMBO J. 1991 Nov;10(11):3439–3446. doi: 10.1002/j.1460-2075.1991.tb04908.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roller R. J., Roizman B. The herpes simplex virus 1 RNA binding protein US11 is a virion component and associates with ribosomal 60S subunits. J Virol. 1992 Jun;66(6):3624–3632. doi: 10.1128/jvi.66.6.3624-3632.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rost B., Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. doi: 10.1002/prot.340190108. [DOI] [PubMed] [Google Scholar]
- Scheer U., Rose K. M. Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1431–1435. doi: 10.1073/pnas.81.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sharp P. A., Manley J., Fire A., Gefter M. Regulation of adenovirus mRNA synthesis. Ann N Y Acad Sci. 1980;354:1–15. doi: 10.1111/j.1749-6632.1980.tb27954.x. [DOI] [PubMed] [Google Scholar]
- Shaw A. R., Ziff E. B. Transcripts from the adenovirus-2 major late promoter yield a single early family of 3' coterminal mRNAs and five late families. Cell. 1980 Dec;22(3):905–916. doi: 10.1016/0092-8674(80)90568-1. [DOI] [PubMed] [Google Scholar]
- Spector D. L. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. doi: 10.1146/annurev.cb.09.110193.001405. [DOI] [PubMed] [Google Scholar]
- Tribouley C., Lutz P., Staub A., Kedinger C. The product of the adenovirus intermediate gene IVa2 is a transcriptional activator of the major late promoter. J Virol. 1994 Jul;68(7):4450–4457. doi: 10.1128/jvi.68.7.4450-4457.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winter N., D'Halluin J. C. Regulation of the biosynthesis of subgroup C adenovirus protein IVa2. J Virol. 1991 Oct;65(10):5250–5259. doi: 10.1128/jvi.65.10.5250-5259.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de StGroth S. F., Scheidegger D. Production of monoclonal antibodies: strategy and tactics. J Immunol Methods. 1980;35(1-2):1–21. doi: 10.1016/0022-1759(80)90146-5. [DOI] [PubMed] [Google Scholar]
