1887

Abstract

We describe the mutagenesis of the IRS1-US5 region of the human cytomegalovirus genome, demonstrating the potential of the guanosine phosphoribosyl transferase () gene as a selectable marker for insertion and deletion mutagenesis of high passage (AD169, Towne) as well as low passage (Toledo) strains of virus. Despite evidence suggesting that the US3 gene product may play a regulatory role, disruption of this gene with a insert had no effect on growth of any of these strains of virus in resting or dividing human fibroblasts, or in human thymus plus liver implants in SCID-hu mice. Transcripts of the gene, under control of the herpes simplex virus thymidine kinase promoter adjacent to the US3 enhancer in the viral genome, accumulated with delayed early (β) kinetics. Mutants with deletions in the IRS1 and US3-US5 regions were isolated by back-selection against with the drug 6-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyl transferase deficient) skin fibroblasts immortalized with human papillomavirus oncogenes. Thus, we demonstrate a dependable method for insertion and deletion mutagenesis that can be applied to any region of the viral genome.

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1995-09-01
2024-04-26
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References

  1. Alford C. A., Britt W. J. 1993; Cytomegalovirus. In The Human Herpesviruses pp 227–255 Edited by Roizman B., Whitley R. J., Lopez C. New York: Raven Press;
    [Google Scholar]
  2. Bolovan C. A., Sawtell N. M., Thompson R. L. 1994; ICP34.5 mutants of herpes simplex type 1 strain 17syn+ are attenuated for neurovirulence in mice and for replication in confluent primary mouse embryo cell cultures. Journal of Virology 68:48–55
    [Google Scholar]
  3. Brown J. M., Kaneshima H., Mocarski E. S. 1995; Dramatic interstrain differences in the replication of human cytomegalovirus in SCID–hu mice. Journal of Infectious Diseases 171:1599–1603
    [Google Scholar]
  4. Browne H., Churcher M., Minson T. 1992; Construction and characterization of a human cytomegalovirus mutant with the UL18 (class I homologue) gene deleted. Journal of Virology 66:6784–6787
    [Google Scholar]
  5. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A. I., Kouzarides T., Martignetti J. A., Preddie E., Satchwell S. C., Tomlinson P., Weston K. M., Barrell B. G. 1990; Analysis of the protein coding content of the sequence of human cytomegalovirus strain AD169. Current Topics in Microbiology and Immunology 154:125–170
    [Google Scholar]
  6. Chen C. A., Okayama H. 1987; High-efficiency transformation of mammalian cells by plasmid DNA. Molecular and Cellular Biology 7:2745–2752
    [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. 1979; Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299
    [Google Scholar]
  8. Colberg-Poley A. M., Santomenna L. D., Harlow P. P., Benfield P. A., Tenney D. J. 1992; Human cytomegalovirus US3 and UL36–38 immediate-early proteins regulate gene ex pression. Journal of Virology 66:95–105
    [Google Scholar]
  9. Compton T. 1993; An immortalized human fibroblast cell line is permissive for human cytomegalovirus infection. Journal of Virology 67:3644–3648
    [Google Scholar]
  10. Falkner F. G., Moss B. 1990; Transient dominant selection of recombinant vaccinia viruses. Journal of Virology 64:3108–3111
    [Google Scholar]
  11. Feinberg A. P., Vogelstein B. 1984; A technique for radio labeling DNA restriction endonuclease fragments to high specific activity. Annals of Biochemistry 137:266–267
    [Google Scholar]
  12. Fleckenstein B., Muller I., Collins J. 1982; Cloning of the complete human cytomegalovirus genome in cosmids. Gene 18:39–46
    [Google Scholar]
  13. Halpert C. L., Demers G. W., Galloway D. A. 1991; The E7 gene of human papillomavirus 16 is sufficient for immortalization of human epithelial cells. Journal of Virology 65:473–478
    [Google Scholar]
  14. Jones T. R., Muzithras V. P. 1992; A cluster of dispensable genes within the human cytomegalovirus genome short component: IRS1, US1 through US5, and the US6 family. Journal of Virology 66:2541–2516
    [Google Scholar]
  15. Jones T. R., Muzithras V. P., Gluzman Y. 1991; Replacement mutagenesis of the human cytomegalovirus genome: US10 and US 11 gene products are nonessential. Journal of Virology 65:5860–5872
    [Google Scholar]
  16. Kaye J., Browne H., Stoffel M., Minson T. 1992; The UL16 gene of human cytomegalovirus encodes a glycoprotein that is dispensable for growth in vitro. Journal of Virology 66:6606–6615
    [Google Scholar]
  17. Kollert-Jons A., Bogner E., Radsak K. 1991; A 15-kilobase-pair region of the human cytomegalovirus genome which includes US1 through US13 is dispensable for growth in cell culture. Journal of Virology 65:5184–5199
    [Google Scholar]
  18. McCune J. M., Namikawa R., Kaneshima H., Shultz L. D., Lieberman M., Weissman I. L. 1988; The SCID-hu mouse: murine model for the analysis of human hematolymphoid differen tiation and function. Science 241:1632–1639
    [Google Scholar]
  19. Malloy S. R., Nunn W. D. 1981; Selection for loss of tetracycline resistance by Escherichia coli . Journal of Bacteriology 145:1110–1112
    [Google Scholar]
  20. Maniatis T., Fristch E. F., Sambrook J. 1982 Molecular Cloning-. A Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Marshall D. R., Reilly J. D., Liu X., Silva R. F. 1993; Selection of Marek’s disease virus recombinants expressing the Escherichia coli gpt gene. Virology 195:638–648
    [Google Scholar]
  22. Masse M., Karlin S., Schachtel G. A., Mocarski E. S. 1992; Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region. Proceedings of the National Academy of Sciences, USA 89:5246–5250
    [Google Scholar]
  23. Miller J. H. 1972 Experiments in Molecular Genetics New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Mocarski E. S. 1993; Cytomegalovirus biology and replication. In The Human Herpesviruses pp 173–226 Edited by Roizman B., Whitley R. J., Lopez C. New York: Raven Press;
    [Google Scholar]
  25. Mocarski E. S., Bonyhadi M., Salimi S., McCune J. M., Kaneshima H. 1993; Human cytomegalovirus in a SCID–hu mouse: thymic epithelial cells are prominent targets of viral replication. Proceedings of the National Academy of Sciences, USA 90:104–108
    [Google Scholar]
  26. Mulligan R. C., Berg P. 1981; Selection for animal cells that express the Escherichia coli gene coding for xanthine–guanine phosphoribosyltransferase. Proceedings of the National Academy of Sciences, USA 78:2072–2076
    [Google Scholar]
  27. Pari G. S., Kacica M. A., Anders D. G. 1993; Open reading frames UL44, IRS1/TRS1, and UL36–38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. Journal of Virology 67:2575–2582
    [Google Scholar]
  28. Post L. E., Roizman B. 1981; A generalized technique for the deletion of specific genes in large genomes: α gene 22 of herpes simplex virus 1 is not essential for growth. Cell 25:227–232
    [Google Scholar]
  29. Post L. E., Conley A. J., Mocarski E. S., Roizman B. 1980; Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences as Bam HI fragments. Proceedings of the National Academy of Sciences, USA 77:4201–1205
    [Google Scholar]
  30. Preston V. G., Darling A., McDougall I. M. 1988; The herpes simplex virus type 1 temperature-sensitive mutant ts 1222 has a single base pair deletion in the small subunit of ribonucleotide reductase. Virology 167:458–467
    [Google Scholar]
  31. Ripalti A., Mocarski E. S. 1991; The products of human cytomegalovirus genes UL1–UL7, including gp48, are dispensable for growth in cell culture. In Progress in Cytomegalovirus Research Proceedings of the Third International Cytomegalovirus Workshop pp 57–60 Edited by Landini M. P. Amsterdam: Elsevier Science Publishers;
    [Google Scholar]
  32. Roizman B., Sears A. M. 1993; Herpes simplex viruses and their replication. In The Human Herpesviruses pp 11–68 Edited by Roizman B., Whitley R. J., Lopez C. New York: Raven Press;
    [Google Scholar]
  33. Spaete R. R., Mocarski E. S. 1985; The a sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes. Journal of Virology 54:817–824
    [Google Scholar]
  34. Spaete R. R., Mocarski E. S. 1987; Insertion and deletion mutagenesis of the human cytomegalovirus genome. Proceedings of the National Academy of Sciences, USA 84:7213–7217
    [Google Scholar]
  35. Stasiak P. C., Mocarski E. S. 1992; Transactivation of the cytomegalovirus ICP36 gene promoter requires the a gene product TRS1 in addition to IE1 and IE2. Journal of Virology 66:1050–1058
    [Google Scholar]
  36. Takekoshi M., Ihara S., Tanaka S., Maeda-Takekoshi F., Watanabe Y. 1987; A new human cytomegalovirus isolate has an invertible subsegment within its L component producing eight genome isomers. Journal of General Virology 68:765–776
    [Google Scholar]
  37. Takekoshi M., Maeda-Takekoshi F., Ihara S., Sakuma S., Watanabe Y. 1991; Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter. Gene 101:209–213
    [Google Scholar]
  38. Tischfield J., Schafer I. A., Dickerman L. H., Trill J., Mulivor R. A., Greene A. E., Coriell L. L. 1979; Lesch-Nyhan syndrome. Cytogenetics and Cell Genetics 24:199–200
    [Google Scholar]
  39. Vieira J., Messing J. 1991; New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194
    [Google Scholar]
  40. Vieira J., Farrell H. E., Rawlinson W. D., Mocarski E. S. 1994; Genes in the Hind III fragment of the murine cyto megalovirus genome are dispensable for growth in cultured cells: insertion mutagenesis with a lacZ/gpt cassette. Journal of Virology 68:4837–1846
    [Google Scholar]
  41. Walboomers J. M., ter Schegget J. 1976; A new method for the isolation of herpes simplex virus type 2 DNA. Virology 74:256–258
    [Google Scholar]
  42. Weston K. 1988; An enhancer element in the short unique region of human cytomegalovirus regulates the production of a group of abundant immediate early transcripts. Virology 162:406–416
    [Google Scholar]
  43. Weston K., Barrell B. G. 1986; Sequence of the short unique region, short repeats, and part of the long repeats of human cytomegalovirus. Journal of Molecular Biology 192:177–208
    [Google Scholar]
  44. Wolff D., Jahn G., Plachter B. 1993; Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene. Gene 130:167–173
    [Google Scholar]
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