Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1983 Apr;46(1):1–14. doi: 10.1128/jvi.46.1.1-14.1983

Organization and expression of the immediate early genes of human cytomegalovirus.

M F Stinski, D R Thomsen, R M Stenberg, L C Goldstein
PMCID: PMC255088  PMID: 6298447

Abstract

The immediate early genes of human cytomegalovirus were characterized according to map location, RNA transcripts, and translation products. Three regions in the large unique component (0.709 to 0.751 map units) were transcribed in the presence of an inhibitor of protein synthesis (anisomycin). A single size class of polyadenylated mRNA, 1.95 kilobases (kb), was transcribed abundantly relative to the other size classes. The predominant 1.95-kb viral RNA was transcribed from right to left on the prototype arrangement of the viral genome and spanned a region of approximately 2.8 kb (0.739 to 0.751 map units). This mRNA codes for a 75,000-dalton protein that represents the predominant immediate early protein detected in infected cells. Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant immediate early protein is synthesized as a protein of 75,000 daltons, but is presumably modified in vivo, resulting in a broad banding pattern ranging from 75,000 to 68,000 daltons. A different immediate early viral gene (0.732 to 0.739 map units) is transcribed from left to right at relatively low levels. The 3' ends of the above viral RNAs terminate at approximately 230 base pairs apart in the region of approximately 0.739 map units. Five RNA size classes ranging from 2.25 to 1.10 kb were detected, but the 1.75-kb and 1.40-kb RNA size classes were more abundant from this region. At least four minor proteins are coded by these mRNAs, with apparent molecular weights ranging from 56,000 to 16,500. Last, a 1.95-kb mRNA was transcribed from a third region (0.709 to 0.728 map units). This viral mRNA was present at relatively low concentration and coded for a minor protein of 68,000 daltons. Since immediate early gene expression of human cytomegalovirus is dominated by the synthesis of an mRNA from the region of 0.739 to 0.751 map units that codes for the predominant immediate early protein found in the infected cell, we hypothesize that this protein is the major regulatory protein influencing the switch from restricted to extensive transcription.

Full text

PDF
1

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson K. P., Costa R. H., Holland L. E., Wagner E. K. Characterization of herpes simplex virus type 1 RNA present in the absence of de novo protein synthesis. J Virol. 1980 Apr;34(1):9–27. doi: 10.1128/jvi.34.1.9-27.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson K. P., Stringer J. R., Holland L. E., Wagner E. K. Isolation and localization of herpes simplex virus type 1 mRNA. J Virol. 1979 Jun;30(3):805–820. doi: 10.1128/jvi.30.3.805-820.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  4. Bishop D. H., Claybrook J. R., Spiegelman S. Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J Mol Biol. 1967 Jun 28;26(3):373–387. doi: 10.1016/0022-2836(67)90310-5. [DOI] [PubMed] [Google Scholar]
  5. Blanton R. A., Tevethia M. J. Immunoprecipitation of virus-specific immediate-early and early polypeptides from cells lytically infected with human cytomegalovirus strain AD 169. Virology. 1981 Jul 15;112(1):262–273. doi: 10.1016/0042-6822(81)90631-0. [DOI] [PubMed] [Google Scholar]
  6. Bächi B., Arber W. Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI restriction fragments of bacteriophage P1 DNA. Mol Gen Genet. 1977 Jun 24;153(3):311–324. doi: 10.1007/BF00431596. [DOI] [PubMed] [Google Scholar]
  7. Cameron J. M., Preston C. M. Comparison of the immediate early polypeptides of human cytomegalovirus isolates. J Gen Virol. 1981 Jun;54(Pt 2):421–424. doi: 10.1099/0022-1317-54-2-421. [DOI] [PubMed] [Google Scholar]
  8. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  9. Chua C. C., Carter T. H., St Jeor S. Transcription of the human cytomegalovirus genome in productively infected cells. J Gen Virol. 1981 Sep;56(Pt 1):1–11. doi: 10.1099/0022-1317-56-1-1. [DOI] [PubMed] [Google Scholar]
  10. Clements J. B., McLauchlan J., McGeoch D. J. Orientation of herpes simplex virus type 1 immediate early mRNA's. Nucleic Acids Res. 1979 Sep 11;7(1):77–91. doi: 10.1093/nar/7.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Easton A. J., Clements J. B. Temporal regulation of herpes simplex virus type 2 transcription and characterization of virus immediate early mRNA's. Nucleic Acids Res. 1980 Jun 25;8(12):2627–2645. doi: 10.1093/nar/8.12.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feldman L. T., Demarchi J. M., Ben-Porat T., Kaplan A. S. Control of abundance of immediate-early mRNA in herpesvirus (pseudorabies)-infected cells. Virology. 1982 Jan 15;116(1):250–263. doi: 10.1016/0042-6822(82)90417-2. [DOI] [PubMed] [Google Scholar]
  14. Feldman L., Rixon F. J., Jean J. H., Ben-Porat T., Kaplan A. S. Transcription of the genome of pseudorabies virus (A herpesvirus) is strictly controlled. Virology. 1979 Sep;97(2):316–327. doi: 10.1016/0042-6822(79)90343-x. [DOI] [PubMed] [Google Scholar]
  15. Furukawa T., Fioretti A., Plotkin S. Growth characteristics of cytomegalovirus in human fibroblasts with demonstration of protein synthesis early in viral replication. J Virol. 1973 Jun;11(6):991–997. doi: 10.1128/jvi.11.6.991-997.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gibson W. Immediate-early proteins of human cytomegalovirus strains AD 169, Davis, and Towne differ in electrophoretic mobility. Virology. 1981 Jul 15;112(1):350–354. doi: 10.1016/0042-6822(81)90641-3. [DOI] [PubMed] [Google Scholar]
  17. Goldstein L. C., McDougall J., Hackman R., Meyers J. D., Thomas E. D., Nowinski R. C. Monoclonal antibodies to cytomegalovirus: rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus pneumonia. Infect Immun. 1982 Oct;38(1):273–281. doi: 10.1128/iai.38.1.273-281.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jean J. H., Ben-Porat T., Kaplan A. S. Early functions of the genome of herpesvirus. 3. Inhibition of the transcription of the viral genome in cells treated with cycloheximide early during the infective process. Virology. 1974 Jun;59(2):516–523. doi: 10.1016/0042-6822(74)90461-9. [DOI] [PubMed] [Google Scholar]
  19. Jones P. C., Hayward G. S., Roizman B. Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA. J Virol. 1977 Jan;21(1):268–276. doi: 10.1128/jvi.21.1.268-276.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jones P. C., Roizman B. Regulation of herpesvirus macromolecular synthesis. VIII. The transcription program consists of three phases during which both extent of transcription and accumulation of RNA in the cytoplasm are regulated. J Virol. 1979 Aug;31(2):299–314. doi: 10.1128/jvi.31.2.299-314.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kamata T., Tanaka S., Watanabe Y. Human cytomegalovirus-induced chromatin factors responsible for changes in template activity and structure of infected cell chromatin. Virology. 1978 Oct 15;90(2):197–208. doi: 10.1016/0042-6822(78)90303-3. [DOI] [PubMed] [Google Scholar]
  22. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Leung W. C., Dimock K., Smiley J. R., Bacchetti S. Herpes simplex virus thymidine kinase transcripts are absent from both nucleus and cytoplasm during infection in the presence of cycloheximide. J Virol. 1980 Nov;36(2):361–365. doi: 10.1128/jvi.36.2.361-365.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mackem S., Roizman B. Regulation of herpesvirus macromolecular synthesis: transcription-initiation sites and domains of alpha genes. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7122–7126. doi: 10.1073/pnas.77.12.7122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McGrogan M., Spector D. J., Goldenberg C. J., Halbert D., Raskas H. J. Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution. Nucleic Acids Res. 1979 Feb;6(2):593–607. doi: 10.1093/nar/6.2.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McLauchlan J., Clements J. B. A 3' co-terminus of two early herpes simplex virus type 1 mRNAs. Nucleic Acids Res. 1982 Jan 22;10(2):501–512. doi: 10.1093/nar/10.2.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Michelson-Fiske S., Horodniceanu F., Guillon J. C. Immediate early antigens in human cytomegalovirus infected cells. Nature. 1977 Dec 15;270(5638):615–617. doi: 10.1038/270615a0. [DOI] [PubMed] [Google Scholar]
  29. Noyes B. E., Stark G. R. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell. 1975 Jul;5(3):301–310. doi: 10.1016/0092-8674(75)90105-1. [DOI] [PubMed] [Google Scholar]
  30. Palmiter R. D. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undergraded polysomes and messenger ribonucleic acid. Biochemistry. 1974 Aug 13;13(17):3606–3615. doi: 10.1021/bi00714a032. [DOI] [PubMed] [Google Scholar]
  31. Parris D. S., Courtney R. J., Schaffer P. A. Temperature-sensitive mutants of herpes simplex virus type 1 defective in transcriptional and post-transcriptional functions required for viral DNA synthesis. Virology. 1978 Oct 15;90(2):177–186. doi: 10.1016/0042-6822(78)90301-x. [DOI] [PubMed] [Google Scholar]
  32. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  33. Preston C. M. Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant tsK. J Virol. 1979 Jan;29(1):275–284. doi: 10.1128/jvi.29.1.275-284.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rakusanova T., Ben-Porat T., Himeno M., Kaplan A. S. Early functions of the genome of herpesvirus. I. Characterization of the RNA synthesized in cycloheximide-treated, infected cells. Virology. 1971 Dec;46(3):877–889. doi: 10.1016/0042-6822(71)90088-2. [DOI] [PubMed] [Google Scholar]
  35. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  36. Smith H. O., Birnstiel M. L. A simple method for DNA restriction site mapping. Nucleic Acids Res. 1976 Sep;3(9):2387–2398. doi: 10.1093/nar/3.9.2387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  38. Stark G. R., Williams J. G. Quantitative analysis of specific labelled RNA'S using DNA covalently linked to diazobenzyloxymethyl-paper. Nucleic Acids Res. 1979 Jan;6(1):195–203. doi: 10.1093/nar/6.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Stinski M. F. Synthesis of proteins and glycoproteins in cells infected with human cytomegalovirus. J Virol. 1977 Sep;23(3):751–767. doi: 10.1128/jvi.23.3.751-767.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stinski M. F., Thomsen D. R., Rodriguez J. E. Synthesis of human cytomegalovirus-specified RNA and protein in interferon-treated cells at early times after infection. J Gen Virol. 1982 Jun;60(Pt 2):261–270. doi: 10.1099/0022-1317-60-2-261. [DOI] [PubMed] [Google Scholar]
  42. Tanaka S., Otsuka M., Ihara S., Maeda F., Watanabe Y. Induction of pre-early nuclear antigen(s) in HEL cells infected with human cytomegalovirus. Microbiol Immunol. 1979;23(4):263–271. doi: 10.1111/j.1348-0421.1979.tb00462.x. [DOI] [PubMed] [Google Scholar]
  43. Thomas M., White R. L., Davis R. W. Hybridization of RNA to double-stranded DNA: formation of R-loops. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2294–2298. doi: 10.1073/pnas.73.7.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thomsen D. R., Stinski M. F. Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments. Gene. 1981 Dec;16(1-3):207–216. doi: 10.1016/0378-1119(81)90077-9. [DOI] [PubMed] [Google Scholar]
  45. Tseng B. Y., Goulian M. Initiator RNA of discontinuous DNA synthesis in human lymphocytes. Cell. 1977 Oct;12(2):483–489. doi: 10.1016/0092-8674(77)90124-6. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Wathen M. W., Thomsen D. R., Stinski M. F. Temporal regulation of human cytomegalovirus transcription at immediate early and early times after infection. J Virol. 1981 May;38(2):446–459. doi: 10.1128/jvi.38.2.446-459.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Watson R. J., Clements J. B. A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis. Nature. 1980 May 29;285(5763):329–330. doi: 10.1038/285329a0. [DOI] [PubMed] [Google Scholar]
  49. Watson R. J., Clements J. B. Characterization of transcription-deficient temperature-sensitive mutants of herpes simplex virus type 1. Virology. 1978 Dec;91(2):364–379. doi: 10.1016/0042-6822(78)90384-7. [DOI] [PubMed] [Google Scholar]
  50. Watson R. J., Preston C. M., Clements J. B. Separation and characterization of herpes simplex virus type 1 immediate-early mRNA's. J Virol. 1979 Jul;31(1):42–52. doi: 10.1128/jvi.31.1.42-52.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wentworth B. B., French L. Plaque assay of cytomegalovirus strains of human origin. Proc Soc Exp Biol Med. 1970 Nov;135(2):253–258. doi: 10.3181/00379727-135-35031. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES