Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2004 Feb 6;134(1):91–102. doi: 10.1016/0042-6822(84)90275-7

Physical mapping of human cytomegalovirus genes: Identification of DNA sequences coding for a virion phosphoprotein of 71 kDa and a viral 65-kDa polypeptide

Barbara Nowak ∗, Agnes Gmeiner ∗, Peter Sarnow 1, Arnold J Levine †,2, Bernhard Fleckenstein ∗
PMCID: PMC7131772  PMID: 6324477

Abstract

Polyadenylated RNA was isolated from fibroblast cultures infected with human cytomegalovirus (HCMV) strain AD169 during the late phase of viral replication. The RNA was selected by hybridization to a series of cosmid clones containing the entire viral genome in partially overlapping segments. Translation of this RNA in a reticulocyte cell-free system allowed the mapping of virus specific polypeptides. Nine polypeptides synthesized in vitro comigrated with major virion structural proteins. An in vitro-translated protein of 71 kDa was precipitated by a monoclonal antibody directed against the phosphorylated internal envelope protein of 71 kDa. The map coordinates of viral DNA coding for this phosphoprotein were localized by hybrid selection with subcloned DNA fragments, and the direction of transcription was determined by hybrid selection with single-stranded DNA cloned in bacteriophage vector M13mp9. An in vitro translation with size-fractionated RNA, combined with immunoprecipitation and Northern blot analyses, indicated that an mRNA of 4 kb encodes the 71-kDa phosphoprotein. An mRNA of the same size, map coordinates, and orientation was translated into an abundant 65-kDa polypeptide which had the same size as the major structural phosphoprotein of HCMV.

References

  1. Aviv H., Leder P. Vol. 69. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose; pp. 1408–1412. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bodemer W., Summers W.C., Niederman J.C. Detection of virus-specific antigen in EB-(P3HR-1) Virus superinfected Raji cells by immunoprecipitation. Virology. 1980;103:340–349. doi: 10.1016/0042-6822(80)90192-0. [DOI] [PubMed] [Google Scholar]
  3. Bonner W.M., Laskey R.A. A film detection method for tritium labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 1974;46:83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  4. Chang A.C.Y., Cohen S.N. Construction and characterization at amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 1978;134:1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Copeland N.G., Cooper G.M. Transfection by exogenous and endogenous murine retrovirus DNAs. Cell. 1979;16:347–356. doi: 10.1016/0092-8674(79)90011-4. [DOI] [PubMed] [Google Scholar]
  6. DeMarchi J.M. Human cytomegalovirus DNA: Restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs. Virology. 1981;114:23–38. doi: 10.1016/0042-6822(81)90249-x. [DOI] [PubMed] [Google Scholar]
  7. Ebeling A., Keil G., Nowak B., Fleckenstein B., Berthelot N., Sheldrick P. Genome structure and virion polypeptides of the primate herpesvirus Herpesvirus aotus Types 1 and 3: Comparison with human cytomegalovirus. J. Virol. 1983;45:715–726. doi: 10.1128/jvi.45.2.715-726.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fleckenstein B., Muller I., Collins J. Cloning of the complete human ctyomegalovirus genome in cosmids. Gene. 1982;18:39–46. doi: 10.1016/0378-1119(82)90054-3. [DOI] [PubMed] [Google Scholar]
  9. Geelen J.L.M.C., Walig C., Wertheim P., Van Der Noordaa J. Human cytomegalovirus DNA, I. Molecular weight and infectivity. J. Virol. 1978;26:813–816. doi: 10.1128/jvi.26.3.813-816.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Graham F.L., Van Der Eb A.J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973;52:456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  11. Jahn G., Knust E., Schmolla H., Sarre T., Nelson J.A., McDougall J.K., Fleckenstein B. 1984. Predominant immediate early transcripts of human cytomegalovirus strain AD169. Submitted for publication. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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;115:1617–1624. [PubMed] [Google Scholar]
  13. Knust E., Schirm S., Dietrich W., Bodemer W., Kole E., Fleckenstein B. Cloning of Herpesvirus saimira DNA fragments representing the entire L-region of the genome. Gene. 1984 doi: 10.1016/0378-1119(83)90232-9. in press. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. La Femina R.L., Hayward G.S. Structural organization of the DNA molecules from human cytomegalovirus. In: Fields B.N., Jaenisch R., editors. Animal Virus Genetics. Academic Press; New York: 1980. pp. 39–55. [Google Scholar]
  16. Liu C.-P., Slate D.L., Gravel R., Ruddle F.H. Vol. 76. 1979. Biological detection of specific mRNA molecules by microinjection; pp. 4503–4506. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McDonough S.H., Specpor D.H. Transcription in human fibroblasts permissively infected by human cytomegalovirus strain AD169. Virology. 1983;125:31–46. doi: 10.1016/0042-6822(83)90061-2. [DOI] [PubMed] [Google Scholar]
  18. McMaster G.K., Carmichael G.G. Vol. 74. 1977. Analysis of single and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange; pp. 4835–4838. (Proc. Natl. Acad Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Messing J., Crea R., Seeburg P.H. A system for shotgun DNA sequencing. Nucl. Acids Res. 1981;9:309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J. An integrative strategy of DNA sequencing and experiments beyond. In: Hollaender A., Setlow J., editors. Vol. 4. Plenum; New York: 1982. (Genetic Engineering, Principals and Methods). [Google Scholar]
  21. Morse L.S., Pereira L., Roizman B., Schaffer P.A. Anatomy of HSV DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 × HSV-2 recombinants. J. Virol. 1978;26:389–410. doi: 10.1128/jvi.26.2.389-410.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nowak B., Sullivan C.A., Sarnow P., Thomas R., Nicolas J.-C., Fleckenstein B., Levine A.J. Characterization of monoclonal antibodies and polyclonal immune sera directed against human CMV virion proteins. Virology. 1984;132:325–338. doi: 10.1016/0042-6822(84)90039-4. [DOI] [PubMed] [Google Scholar]
  23. Oram J.D., Downing R.G., Akrigg A., Dollery A.A., Duggleby C.J., Wilkinson G.W.G., Greenaway P.J. Use of recombinant plasmids to investigate the structure of the human cytomegalovirus genome. J. Gen. Virol. 1982;59:111–129. doi: 10.1099/0022-1317-59-1-111. [DOI] [PubMed] [Google Scholar]
  24. Pelham H.R.B., Jackson R.J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 1976;67:247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  25. Ricciardi R.P., Miller J.S., Roberts B.E. Vol. 76. 1979. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation; pp. 4927–4931. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ross S.R., Flint S.J., Levine A.J. Identification of the adenovirus early proteins and their genomic map position. Virology. 1980;100:419–432. doi: 10.1016/0042-6822(80)90533-4. [DOI] [PubMed] [Google Scholar]
  27. Sarnow P., Ho Y.S., Williams J., Levine A.J. Adenovirus Elb-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell. 1982;28:387–394. doi: 10.1016/0092-8674(82)90356-7. [DOI] [PubMed] [Google Scholar]
  28. Sarnow P., Sullivan C.A., Levine A.J. A monoclonal antibody detecting the adenovirus type 5 Elb-58 kDa tumor antigen: Characterization of the Elb-58 kDa tumor antigen in adenovirus infected and transformed cells. Virology. 1982;120:510–517. doi: 10.1016/0042-6822(82)90054-x. [DOI] [PubMed] [Google Scholar]
  29. Siddell S.G., Wege H., Barthel A., Ter Meulen B. Coronavirus JHM:Cell-free synthesis of structural protein p60. J. Virol. 1980;33:10–117. doi: 10.1128/jvi.33.1.10-17.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Southern E.M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 1975;98:503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  31. Stinski M.F., Thomsen D.R., Stenberg R.M., Goldstein L.C. Organization and expression of the immediate early genes of human cytomegalovirus. J. Virol. 1983;46:1–14. doi: 10.1128/jvi.46.1.1-14.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tamashiro J.C., Hock L.J., Spector D.H. Construction of a cloned library of the EcoRI fragments from the cutomegalovirus strain AD169. J. Virol. 1982;42:547–557. doi: 10.1128/jvi.42.2.547-557.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thomas P.S. Vol. 77. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitro-cellulose; pp. 5201–5205. (Proc. Nat. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thomsen D.R., Stinski M.F. Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments. Gene. 1981;16:207–216. doi: 10.1016/0378-1119(81)90077-9. [DOI] [PubMed] [Google Scholar]
  35. 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;41:462–477. doi: 10.1128/jvi.41.2.462-477.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Virology are provided here courtesy of Elsevier

RESOURCES