Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1973 Dec;12(6):1473–1481. doi: 10.1128/jvi.12.6.1473-1481.1973

Human Cytomegalovirus I. Purification and Characterization of Viral DNA

Eng-Shang Huang 1, Sheng-Tu Chen 1, Joseph S Pagano 1
PMCID: PMC356790  PMID: 4128379

Abstract

Two human strains (AD-169 and C87) and one simian strain (GR2757) of cytomegalovirus (CMV) have been purified from the extracellular fluids of virus-infected cultures by sedimentation through a sucrose gradient followed by brief centrifugation in a preformed gradient of CsCl. Enveloped virus particles were located in the density region, 1.219 g/cm3, and nucleocapsids at 1.263 g/cm3. Purified viral DNA, both human and simian, sedimented in the region of 55S in neutral sucrose gradients with herpes simplex type I DNA as a marker; the molecular weight of the CMV DNA was estimated as approximately 108. The density of the viral DNA determined by analytical ultracentrifugation was 1.716 g/cm3 for the human strains and 1.710 g/cm3 for the simian strain. Tritiated viral complementary RNA synthesized in vitro with Escherichia coli transcriptase has been used for detection and localization of viral genome in membrane hybridization and in situ cytohybridization. Newly synthesized viral DNA appeared 24 h after infection and localized at two acrocentric areas; later the viral DNA distributed in a band resembling intranuclear inclusions 48 to 70 h after infection. Total DNA synthesis began to increase 24 h after infection and reached its peak at 70 h; RNA synthesis increased at 13 h, and reached its peak at 24 to 33 h. The viral DNA was also labeled with 3H-TTP by repair-synthesis in vitro with Kornberg's enzyme in order to analyze the purity of the DNA and for detection of viral DNA by DNA-DNA reassociation kinetics.

Full text

PDF
1473

Images in this article

1479Image
on p.1479

Selected References

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

  1. BENYESH-MELNICK M., ROSENBERG H. S., WATSON B. VIRUSES IN CELL CULTURES OF KIDNEYS OF CHILDREN WITH CONGENITAL HEART MALFORMATIONS AND OTHER DISEASES. Proc Soc Exp Biol Med. 1964 Nov;117:452–459. doi: 10.3181/00379727-117-29607. [DOI] [PubMed] [Google Scholar]
  2. Burgess R. R. A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6160–6167. [PubMed] [Google Scholar]
  3. CRAWFORD L. V., LEE A. J. DISCUSSION AND PRELIMINARY REPORTS. THE NUCLEIC ACID OF HUMAN CYTOMEGALOVIRUS. Virology. 1964 May;23:105–107. doi: 10.1016/s0042-6822(64)80014-3. [DOI] [PubMed] [Google Scholar]
  4. Chambers R. W., Rose J. A., Rabson A. S., Bond H. E., Hall W. T. Propagation and purification of high-titer human cytomegalovirus. Appl Microbiol. 1971 Nov;22(5):914–918. doi: 10.1128/am.22.5.914-918.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dreesman G. R., Benyesh-Melnick M. Spectrum of human cytomegalovirus complement-fixing antigens. J Immunol. 1967 Dec;99(6):1106–1114. [PubMed] [Google Scholar]
  6. Freifelder D. Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J Mol Biol. 1970 Dec 28;54(3):567–577. doi: 10.1016/0022-2836(70)90127-0. [DOI] [PubMed] [Google Scholar]
  7. Frenkel N., Roizman B., Cassai E., Nahmias A. A DNA fragment of Herpes simplex 2 and its transcription in human cervical cancer tissue. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3784–3789. doi: 10.1073/pnas.69.12.3784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gelb L. D., Kohne D. E., Martin M. A. Quantitation of Simian virus 40 sequences in African green monkey, mouse and virus-transformed cell genomes. J Mol Biol. 1971 Apr 14;57(1):129–145. doi: 10.1016/0022-2836(71)90123-9. [DOI] [PubMed] [Google Scholar]
  9. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  10. Graham B. J., Ludwig H., Bronson D. L., Benyesh-Melnick M., Biswal N. Physicochemical properties of the DNA of herpes viruses. Biochim Biophys Acta. 1972 Jan 18;259(1):13–23. doi: 10.1016/0005-2787(72)90469-8. [DOI] [PubMed] [Google Scholar]
  11. Haines H. G., Von Essen R., Benyesh-Melnick M., Melnick J. L. Preparation of specific antisera to cytomegaloviruses in goats. Proc Soc Exp Biol Med. 1971 Dec;138(3):846–849. doi: 10.3181/00379727-138-36003. [DOI] [PubMed] [Google Scholar]
  12. Jovin T. M., Englund P. T., Bertsch L. L. Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):2996–3008. [PubMed] [Google Scholar]
  13. Kieff E. D., Bachenheimer S. L., Roizman B. Size, composition, and structure of the deoxyribonucleic acid of herpes simplex virus subtypes 1 and 2. J Virol. 1971 Aug;8(2):125–132. doi: 10.1128/jvi.8.2.125-132.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lang D. J., Kummer J. F. Demonstration of cytomegalovirus in semen. N Engl J Med. 1972 Oct 12;287(15):756–758. doi: 10.1056/NEJM197210122871508. [DOI] [PubMed] [Google Scholar]
  15. Nonoyama M., Pagano J. S. Detection of Epstein-Barr viral genome in nonproductive cells. Nat New Biol. 1971 Sep 22;233(38):103–106. doi: 10.1038/newbio233103a0. [DOI] [PubMed] [Google Scholar]
  16. Nonoyama M., Pagano J. S. Homology between Epstein-Barr virus DNA and viral DNA from Burkitt's lymphoma and nasopharyngeal carcinoma determined by DNA-DNA reassociation kinetics. Nature. 1973 Mar 2;242(5392):44–47. doi: 10.1038/242044a0. [DOI] [PubMed] [Google Scholar]
  17. Plummer G., Goodheart C. R., Henson D., Bowling C. P. A comparative study of the DNA density and behavior in tissue cultures of fourteen different herpesviruses. Virology. 1969 Sep;39(1):134–137. doi: 10.1016/0042-6822(69)90355-9. [DOI] [PubMed] [Google Scholar]
  18. Probstmeyer F., Benyesh-Melnick M., McCombs R. M. Comparison of human cytomegalovirus obtained by glycine extraction and by sonic disruption of infected cells. J Virol. 1967 Feb;1(1):241–243. doi: 10.1128/jvi.1.1.241-243.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. RICHARDSON C. C., SCHILDKRAUT C. L., APOSHIAN H. V., KORNBERG A. ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. XIV. FURTHER PURIFICATION AND PROPERTIES OF DEOXYRIBONUCLEIC ACID POLYMERASE OF ESCHERICHIA COLI. J Biol Chem. 1964 Jan;239:222–232. [PubMed] [Google Scholar]
  20. ROWE W. P., HARTLEY J. W., WATERMAN S., TURNER H. C., HUEBNER R. J. Cytopathogenic agent resembling human salivary gland virus recovered from tissue cultures of human adenoids. Proc Soc Exp Biol Med. 1956 Jun;92(2):418–424. [PubMed] [Google Scholar]
  21. Spring S. B., Roizman B. Herpes simplex virus products in productive and abortive infection. I. Stabilization with formaldehyde and preliminary analyses by isopycnic centrifugation in CsCl. J Virol. 1967 Apr;1(2):294–301. doi: 10.1128/jvi.1.2.294-301.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stulberg C. S., Zuelzer W. W., Page R. H., Taylor P. E., Brough Cytomegalovirus infections with reference to isolations from lymph nodes and blood. Proc Soc Exp Biol Med. 1966 Dec;123(3):976–982. doi: 10.3181/00379727-123-31651. [DOI] [PubMed] [Google Scholar]
  23. WELLER T. H., HANSHAW J. B., SCOTT D. E. Serologic differentiation of viruses responsible for cytomegalic inclusion disease. Virology. 1960 Sep;12:130–132. doi: 10.1016/0042-6822(60)90156-2. [DOI] [PubMed] [Google Scholar]
  24. WELLER T. H., HANSHAW J. B. Virologic and clinical observations on cytomegalic inclusion disease. N Engl J Med. 1962 Jun 14;266:1233–1244. doi: 10.1056/NEJM196206142662401. [DOI] [PubMed] [Google Scholar]
  25. Weller T. H. Review. Cytomegaloviruses: the difficult years. J Infect Dis. 1970 Dec;122(6):532–539. doi: 10.1093/infdis/122.6.532. [DOI] [PubMed] [Google Scholar]
  26. Weller T. H. The cytomegaloviruses: ubiquitous agents with protean clinical manifestations. I. N Engl J Med. 1971 Jul 22;285(4):203–214. doi: 10.1056/NEJM197107222850406. [DOI] [PubMed] [Google Scholar]

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

RESOURCES