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Volume 64, Issue 2

Replicative Forms of Human Cytomegalovirus DNA with Joined Termini Are Found in Permissively Infected Human Cells But Not in Non-permissive Balb/c-3T3 Mouse Cells Free

Abstract

SUMMARY

Balb/c-3T3 mouse cells were found to be highly restricted non-permissive hosts for human cytomegalovirus (HCMV) strain Towne. These cells did not produce infectious progeny virions, did not permit virus DNA replication, and allowed expression of only a single, major, virus-specific, immediate-early polypeptide. Virus DNA synthesis was examined by three different experimental approaches. In infected Balb/c-3T3 cells, no P-labelled newly synthesized DNA was found at the virus density in CsCl gradients and no virus-specific fragments were detected after cleavage with restriction enzymes. Similarly, hybridization experiments revealed no net increase in total virus DNA over the amount of input virus-specific DNA sequences. In contrast, infected permissive human fibroblast cells synthesized P-labelled virus-specific DNA fragments and accumulated greatly increased amounts of total hybridizing virus DNA. Experiments with a cloned HI L-S joint fragment probe provided evidence for the formation of either circular or concatemeric replicative forms of HCMV DNA in which all half-molar terminal fragments were missing and the proportion of quarter-molar joint fragments increased. These forms were abundant in the first 48 h after infection of permissive human cells and mature linear monomeric forms accumulated thereafter. No detectable joining of the termini of input virus DNA occurred in either non-permissive Balb/c-3T3 cells or in human fibroblast cells in the presence of phosphonoacetic acid. In the infected Balb/c-3T3 cells a single major protein corresponding to the 68K immediate-early polypeptide could be detected within 2 h after cycloheximide reversal. Few, if any, other virus proteins were synthesized at later times or in the absence of inhibitors. The 68K protein was overproduced in Balb/c-3T3 cells to such an extent that it became a major component of the nuclear fraction and could be readily detected by direct staining procedures in polyacrylamide gels.

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Keyword(s): hybridization , immediate-early protein , phosphonoacetate and replication
© Journal of General Virology 1983
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1983-02-01
2024-04-19
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References

  1. Ben-Porat T., Kaplan A. S., Stehn B., Rubenstein A. S. 1976; Concatemeric forms of intracellulose herpesvirus DNA. Virology 69:547–560
     [Google Scholar]
  2. Brown W. M., Vinograd J. 1974; Restriction endonuclease cleavage maps of animal mitochondrial DNAs. Proceedings of the National Academy of Sciences of the United States of America 71:4617–4621
     [Google Scholar]
  3. Ciufo D. M., Hayward G. S. 1981; Tandem repeat defective DNA from the L-segment of the HSV genome. In Herpesvirus DNA: Recent Studies on the Internal Organization and Replication of the Viral Genome pp 107–128 Edited by Becker Y. The Hague: Martinus Nijhoff;
     [Google Scholar]
  4. Demarchi J. M., Kaplan A. S. 1977; Physiological state of human embryonic lung cells affects their response to human cytomegalovirus. Journal of Virology 23:126–132
     [Google Scholar]
  5. Demarchi J. M., Blankenship M. L., Brown G. S., Kaplan A. S. 1978; Size and complexity of human cytomegalovirus DNA. Virology 89:643–646
     [Google Scholar]
  6. Desrosiers R. C., Mulder C., Fleckenstein B. 1979; Methylation of herpesvirus DNA in lymphoid tumor cell lines. Proceedings of the National Academy of Sciences of the United States of America 76:3839–3843
     [Google Scholar]
  7. Furukawa T., Tanaka S., Plotkin S. A. 1975; Stimulation of macromolecular synthesis in guinea pig cells by human CMV. Proceedings of the Society for Experimental Biology and Medicine 148:211–214
     [Google Scholar]
  8. Gadler H., Wahren B. 1981; Association between viral and cellular DNA during latent cytomegalovirus infection in vitro. In International Workshop on Herpesviruses p 160 Edited by Kaplan A. S. LaPlaca M., Rapp F., Roizman B. Bologna, Italy: Esculapio Publishing Co;
     [Google Scholar]
  9. Geder L. 1976; Evidence for early nuclear antigen in cytomegalovirus-infected cells. Journal of General Virology 32:315–319
     [Google Scholar]
  10. Geelen J. L. M. C., Weststrate M. W. 1981; Organization of the human cytomegalovirus genome. In Herpesvirus DNA: Recent Studies on the Internal Organization and Replication of the Viral Genome pp 325–343 Edited by Becker Y. The Hague: Martinus Nijhoff;
     [Google Scholar]
  11. Geelen J. L. M. C., Walig C., Wertheim P., Van Der Noordaa J. 1978; Human cytomegalovirus DNA. I. Molecular weight and infectivity. Journal of Virology 26:813–816
     [Google Scholar]
  12. Gergely L., Cziigledy J., Vaczi L. 1980; Early nuclear antigen as DNA-binding protein in cytomegalovirus-infected cells. Intervirology 13:352–356
     [Google Scholar]
  13. Gibson W. 1981; Immediate-early proteins of human cytomegalovirus strains AD 169, Davis and Towne differ in electrophoretic mobility. Virology 112:350–354
     [Google Scholar]
  14. Gibson W., Murphy T. L., Roby C. 1981; Cytomegalovirus-infected cells contain a DNA-binding protein. Virology 111:251–262
     [Google Scholar]
  15. Hayward G. S., Frenkel N., Roizman B. 1975a; Anatomy of herpes simplex virus DNA: strain differences and heterogeneity in the locations of restriction endonuclease cleavage sites. Proceedings of the National Academy of Sciences of the United States of America 72:1768–1772
     [Google Scholar]
  16. Hayward G. S., Jacob R. J., Wadsworth S. C., Roizman B. 1975b; Anatomy of herpes simplex virus DNA: evidence for four populations of molecules that differ in the relative orientations of their long and short components. Proceedings of the National Academy of Sciences of the United States of America 72:4243–4247
     [Google Scholar]
  17. Jacob R. J., Roizman B. 1977; Anatomy of herpes simplex virus DNA. VIII. Properties of the duplicating DNA. Journal of Virology 23:394–411
     [Google Scholar]
  18. Jacob R. J., Morse L. S., Roizman B. 1979; Anatomy of herpes simplex virus DNA. XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA. Journal of Virology 29:448–457
     [Google Scholar]
  19. Jean J. H., Yoshimura N., Furukawa T., Plotkin S. A. 1978; Intracellular forms of the parental human cytomegalovirus genome at early stages of the infective process. Virology 86:281–286
     [Google Scholar]
  20. Jongeneel C. V., Bachenheimer S. L. 1981; Structure of replicating herpes simplex virus DNA. Journal of Virology 39:656–660
     [Google Scholar]
  21. Kilpatrick B. A., Huang E. S. 1977; Human cytomegalovirus genome: partial denaturation map and organization of genome sequences. Journal of Virology 24:261–276
     [Google Scholar]
  22. Knowles W. A. 1976; In vitro cultivation of human cytomegalovirus in thyroid epithelial cells. Archives of Virology 50:119–124
     [Google Scholar]
  23. Ladin B. F., Blankenship M. L., Ben-Porat T. 1980; Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation. Journal of Virology 32:1151–1164
     [Google Scholar]
  24. Lafemina R., Hayward G. S. 1980; Structural organization of the DNA molecules from human cytomegalovirus. In Animal Cell Genetics pp 39–55 Edited by Jaenisch R., Fields B., Fox C. F. ICN-UCLA Symposium Academic Press;
     [Google Scholar]
  25. Lindahl T., Adams A., Bjursel G., Bornkam G. W., Kaschka-Dierich C., Jehn U. 1976; Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. Journal of Molecular Biology 102:511–530
     [Google Scholar]
  26. Michelson S., Horodntceanu F., Kress M., Tardy-Punit M. 1979; Human cytomegalovirus induced immediate-early antigens: analysis in sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation. Virology 32:259–267
     [Google Scholar]
  27. Mocarski E. S., Stinski M. F. 1979; Persistence of the cytomegalovirus genome in human cells. Journal of Virology 31:761–775
     [Google Scholar]
  28. Mocarski E. S., Post L. E., Roizman B. 1980; Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversion. Cell 22:243–255
     [Google Scholar]
  29. Reynolds D. W. 1978; Development of early nuclear antigen in cytomegalovirus infected cells in the presence of RNA and protein synthesis inhibitors. Journal of General Virology 40:475–480
     [Google Scholar]
  30. St. Jeor S. C., Albrecht B., Funk F. D., Rapp F. 1974; Stimulation of cellular DNA synthesis by human cytomegalovirus. Journal of Virology 13:353–362
     [Google Scholar]
  31. Smiley J., Fong B. S., Leung W. 1981; Construction of a double-jointed herpes simplex viral DNA molecule: inverted repeats are required for segment inversion and direct repeats promote deletion. Virology 113:345–362
     [Google Scholar]
  32. Southern E. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503–518
     [Google Scholar]
  33. Stinski M. F. 1978; Sequences of protein synthesis in cells infected by human cytomegalovirus: early and late virus induced polypeptides. Journal of Virology 26:686–701
     [Google Scholar]
  34. Stinski M. F., Mocarski E. S., Thomsen D. R. 1979; DNA of human cytomegalovirus: size heterogeneity and defectiveness resulting from serial undiluted passage. Journal of Virology 31:231–239
     [Google Scholar]
  35. Tanaka S., Furukawa T., Plotkin S. A. 1975; Stimulation of cellular DNA synthesis by human cytomegalovirus. Journal of Virology 15:297–304
     [Google Scholar]
  36. Tanaka S., Otsuka M., Thara S., Maeda F., Watanabe Y. 1979; Introduction of pre-early nuclear antigen(s) in HEL cells infected with human cytomegalovirus. Microbiology and Immunology 23:263–271
     [Google Scholar]
  37. Vonka V., Anisimova E., Macek M. 1976; Replication of cytomegalovirus in human epithelioid diploid cell line. Archives of Virology 52:283–296
     [Google Scholar]
  38. Waner J. L., Weller T. H. 1974; Behavior of human cytomegalovirus in cell cultures of bovine and simian origin. Proceedings of the Society for Experimental Biology and Medicine 145:379–384
     [Google Scholar]
  39. Wathen M. W., Stinski M. F. 1982; Temporal patterns of human cytomegalovirus transcription: mapping the viral RNA synthesized at immediate-early, early and late times after infection. Journal of Virology 41:462–477
     [Google Scholar]
  40. Wathen M. W., Thomsen D. R., Stinski M. F. 1981; Temporal regulation of human cytomegalovirus transcription at immediate-early and early times after infection. Journal of Virology 38:446–459
     [Google Scholar]
  41. Watson R. J., Clements J. B. 1980; A herpes simplex type 1 function continuously required for early and late virus synthesis. Nature, London 285:329–330
     [Google Scholar]
  42. Weller T. H. 1970; Cytomegalovirus: the difficult years. Journal of Infectious Diseases 122:532–539
     [Google Scholar]
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Replicative Forms of Human Cytomegalovirus DNA with Joined Termini Are Found in Permissively Infected Human Cells But Not in Non-permissive Balb/c-3T3 Mouse Cells
J. Gen. Virol. 64, 373 (1983); https://doi.org/10.1099/0022-1317-64-2-373
/content/journal/jgv/10.1099/0022-1317-64-2-373
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