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. 1991 Nov;65(11):5860–5872. doi: 10.1128/jvi.65.11.5860-5872.1991

Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential.

T R Jones 1, V P Muzithras 1, Y Gluzman 1
PMCID: PMC250248  PMID: 1656074

Abstract

The US6 gene family, located within the unique short region (US) of the human cytomegalovirus (HCMV) genome, contains six open reading frames (US6 through US11) which may encode glycoproteins, such as gcII (D. Gretch, B. Kari, R. Gehrz, and M. Stinski, J. Virol. 62:1956-1962, 1988). By homologous recombination, several different recombinant HCMV were created which contain a marker gene, beta-glucuronidase, inserted within this gene family. It was demonstrated that beta-glucuronidase has utility as a marker gene for the identification of recombinants in this herpesvirus system, without the occurrence of deletions in other regions of the viral genome. DNA and RNA blot analyses attested to the fidelity of the recombination. Immunoprecipitation experiments using monospecific polyclonal antisera indicated that the US10 and/or US11 gene products were not expressed in the recombinants, as predicted. These results, along with single-cycle growth analyses, indicated that the US10 and US11 gene products are nonessential for virus replication and growth in tissue culture. HCMV recombinants expressing beta-glucuronidase seemed to be genetically stable.

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Selected References

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

  1. Anders D. G., Gibson W. Location, transcript analysis, and partial nucleotide sequence of the cytomegalovirus gene encoding an early DNA-binding protein with similarities to ICP8 of herpes simplex virus type 1. J Virol. 1988 Apr;62(4):1364–1372. doi: 10.1128/jvi.62.4.1364-1372.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersson M., Päbo S., Nilsson T., Peterson P. A. Impaired intracellular transport of class I MHC antigens as a possible means for adenoviruses to evade immune surveillance. Cell. 1985 Nov;43(1):215–222. doi: 10.1016/0092-8674(85)90026-1. [DOI] [PubMed] [Google Scholar]
  3. Boldogh I., AbuBakar S., Deng C. Z., Albrecht T. Transcriptional activation of cellular oncogenes fos, jun, and myc by human cytomegalovirus. J Virol. 1991 Mar;65(3):1568–1571. doi: 10.1128/jvi.65.3.1568-1571.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burgert H. G., Kvist S. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell. 1985 Jul;41(3):987–997. doi: 10.1016/s0092-8674(85)80079-9. [DOI] [PubMed] [Google Scholar]
  5. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  6. Chou J., Kern E. R., Whitley R. J., Roizman B. Mapping of herpes simplex virus-1 neurovirulence to gamma 134.5, a gene nonessential for growth in culture. Science. 1990 Nov 30;250(4985):1262–1266. doi: 10.1126/science.2173860. [DOI] [PubMed] [Google Scholar]
  7. Colberg-Poley A. M., Santomenna L. D. Selective induction of chromosomal gene expression by human cytomegalovirus. Virology. 1988 Sep;166(1):217–228. doi: 10.1016/0042-6822(88)90163-8. [DOI] [PubMed] [Google Scholar]
  8. DeLuca N. A., Courtney M. A., Schaffer P. A. Temperature-sensitive mutants in herpes simplex virus type 1 ICP4 permissive for early gene expression. J Virol. 1984 Dec;52(3):767–776. doi: 10.1128/jvi.52.3.767-776.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gao M., Knipe D. M. Potential role for herpes simplex virus ICP8 DNA replication protein in stimulation of late gene expression. J Virol. 1991 May;65(5):2666–2675. doi: 10.1128/jvi.65.5.2666-2675.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goldstein D. J., Weller S. K. An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis. J Virol. 1988 Aug;62(8):2970–2977. doi: 10.1128/jvi.62.8.2970-2977.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldstein D. J., Weller S. K. Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant. J Virol. 1988 Jan;62(1):196–205. doi: 10.1128/jvi.62.1.196-205.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gooding L. R., Elmore L. W., Tollefson A. E., Brady H. A., Wold W. S. A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell. 1988 May 6;53(3):341–346. doi: 10.1016/0092-8674(88)90154-7. [DOI] [PubMed] [Google Scholar]
  13. Greenaway P. J., Wilkinson G. W. Nucleotide sequence of the most abundantly transcribed early gene of human cytomegalovirus strain AD169. Virus Res. 1987 Feb;7(1):17–31. doi: 10.1016/0168-1702(87)90055-4. [DOI] [PubMed] [Google Scholar]
  14. Gretch D. R., Kari B., Gehrz R. C., Stinski M. F. A multigene family encodes the human cytomegalovirus glycoprotein complex gcII (gp47-52 complex). J Virol. 1988 Jun;62(6):1956–1962. doi: 10.1128/jvi.62.6.1956-1962.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gretch D. R., Kari B., Rasmussen L., Gehrz R. C., Stinski M. F. Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus. J Virol. 1988 Mar;62(3):875–881. doi: 10.1128/jvi.62.3.875-881.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holland L. E., Sandri-Goldin R. M., Goldin A. L., Glorioso J. C., Levine M. Transcriptional and genetic analyses of the herpes simplex virus type 1 genome: coordinates 0.29 to 0.45. J Virol. 1984 Mar;49(3):947–959. doi: 10.1128/jvi.49.3.947-959.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holland T. C., Homa F. L., Marlin S. D., Levine M., Glorioso J. Herpes simplex virus type 1 glycoprotein C-negative mutants exhibit multiple phenotypes, including secretion of truncated glycoproteins. J Virol. 1984 Nov;52(2):566–574. doi: 10.1128/jvi.52.2.566-574.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ihara S., Hirai K., Watanabe Y. Temperature-sensitive mutants of human cytomegalovirus: isolation and partial characterization of DNA-minus mutants. Virology. 1978 Jan;84(1):218–221. doi: 10.1016/0042-6822(78)90238-6. [DOI] [PubMed] [Google Scholar]
  19. Isom H. C. Stimulation of ornithine decarboxylase by human cytomegalovirus. J Gen Virol. 1979 Feb;42(2):265–278. doi: 10.1099/0022-1317-42-2-265. [DOI] [PubMed] [Google Scholar]
  20. Jefferson R. A., Burgess S. M., Hirsh D. beta-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8447–8451. doi: 10.1073/pnas.83.22.8447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jefferson R. A., Klass M., Wolf N., Hirsh D. Expression of chimeric genes in Caenorhabditis elegans. J Mol Biol. 1987 Jan 5;193(1):41–46. doi: 10.1016/0022-2836(87)90624-3. [DOI] [PubMed] [Google Scholar]
  22. Jenkins F. J., Casadaban M. J., Roizman B. Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4773–4777. doi: 10.1073/pnas.82.14.4773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones T. R., Muzithras V. P. Fine mapping of transcripts expressed from the US6 gene family of human cytomegalovirus strain AD169. J Virol. 1991 Apr;65(4):2024–2036. doi: 10.1128/jvi.65.4.2024-2036.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kari B., Goertz R., Gehrz R. Characterization of cytomegalovirus glycoproteins in a family of complexes designated gC-II with murine monoclonal antibodies. Arch Virol. 1990;112(1-2):55–65. doi: 10.1007/BF01348985. [DOI] [PubMed] [Google Scholar]
  25. Kemble G. W., McCormick A. L., Pereira L., Mocarski E. S. A cytomegalovirus protein with properties of herpes simplex virus ICP8: partial purification of the polypeptide and map position of the gene. J Virol. 1987 Oct;61(10):3143–3151. doi: 10.1128/jvi.61.10.3143-3151.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ligas M. W., Johnson D. C. A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells. J Virol. 1988 May;62(5):1486–1494. doi: 10.1128/jvi.62.5.1486-1494.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Longnecker R., Roizman B. Clustering of genes dispensable for growth in culture in the S component of the HSV-1 genome. Science. 1987 May 1;236(4801):573–576. doi: 10.1126/science.3033823. [DOI] [PubMed] [Google Scholar]
  28. Longnecker R., Roizman B. Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene. J Virol. 1986 May;58(2):583–591. doi: 10.1128/jvi.58.2.583-591.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Luckow V. A., Summers M. D. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1989 May;170(1):31–39. doi: 10.1016/0042-6822(89)90348-6. [DOI] [PubMed] [Google Scholar]
  30. Manning W. C., Mocarski E. S. Insertional mutagenesis of the murine cytomegalovirus genome: one prominent alpha gene (ie2) is dispensable for growth. Virology. 1988 Dec;167(2):477–484. [PubMed] [Google Scholar]
  31. McCarthy A. M., McMahan L., Schaffer P. A. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J Virol. 1989 Jan;63(1):18–27. doi: 10.1128/jvi.63.1.18-27.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McDonough S. H., Staprans S. I., Spector D. H. Analysis of the major transcripts encoded by the long repeat of human cytomegalovirus strain AD169. J Virol. 1985 Mar;53(3):711–718. doi: 10.1128/jvi.53.3.711-718.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McKnight S. L. The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res. 1980 Dec 20;8(24):5949–5964. doi: 10.1093/nar/8.24.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oram J. D., Downing R. G., Akrigg A., Dollery A. A., Duggleby C. J., Wilkinson G. W., Greenaway P. J. Use of recombinant plasmids to investigate the structure of the human cytomegalovirus genome. J Gen Virol. 1982 Mar;59(Pt 1):111–129. doi: 10.1099/0022-1317-59-1-111. [DOI] [PubMed] [Google Scholar]
  35. Paigen K. Acid hydrolases as models of genetic control. Annu Rev Genet. 1979;13:417–466. doi: 10.1146/annurev.ge.13.120179.002221. [DOI] [PubMed] [Google Scholar]
  36. Parris D. S., Dixon R. A., Schaffer P. A. Physical mapping of herpes simplex virus type 1 ts mutants by marker rescue: correlation of the physical and genetic maps. Virology. 1980 Jan 30;100(2):275–287. doi: 10.1016/0042-6822(80)90519-x. [DOI] [PubMed] [Google Scholar]
  37. Post L. E., Mackem S., Roizman B. Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters. Cell. 1981 May;24(2):555–565. doi: 10.1016/0092-8674(81)90346-9. [DOI] [PubMed] [Google Scholar]
  38. Post L. E., Roizman B. A generalized technique for deletion of specific genes in large genomes: alpha gene 22 of herpes simplex virus 1 is not essential for growth. Cell. 1981 Jul;25(1):227–232. doi: 10.1016/0092-8674(81)90247-6. [DOI] [PubMed] [Google Scholar]
  39. Radsak K., Kaiser C. J., Haustein D., Rapp F. Polypeptide synthesis in human fibroblasts infected with DNA-negative mutants of cytomegalovirus. Intervirology. 1988;29(2):101–107. doi: 10.1159/000150034. [DOI] [PubMed] [Google Scholar]
  40. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  41. Sacks W. R., Greene C. C., Aschman D. P., Schaffer P. A. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J Virol. 1985 Sep;55(3):796–805. doi: 10.1128/jvi.55.3.796-805.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Santomenna L. D., Colberg-Poley A. M. Induction of cellular hsp70 expression by human cytomegalovirus. J Virol. 1990 May;64(5):2033–2040. doi: 10.1128/jvi.64.5.2033-2040.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schaffer P. A., Aron G. M., Biswal N., Benyesh-Melnick M. Temperature-sensitive mutants of herpes simplex virus type 1: isolation, complementation and partial characterization. Virology. 1973 Mar;52(1):57–71. doi: 10.1016/0042-6822(73)90398-x. [DOI] [PubMed] [Google Scholar]
  44. Schranz P., Neidhardt H., Schröder C. H., Kaerner H. C. A viable HSV-1 mutant deleted in two nonessential major glycoproteins. Virology. 1989 May;170(1):273–276. doi: 10.1016/0042-6822(89)90377-2. [DOI] [PubMed] [Google Scholar]
  45. Sears A. E., Halliburton I. W., Meignier B., Silver S., Roizman B. Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice. J Virol. 1985 Aug;55(2):338–346. doi: 10.1128/jvi.55.2.338-346.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sharp J. A., Wagner M. J., Summers W. C. Transcription of herpes simplex virus genes in vivo: overlap of a late promoter with the 3' end of the early thymidine kinase gene. J Virol. 1983 Jan;45(1):10–17. doi: 10.1128/jvi.45.1.10-17.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Smiley J. R. Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus. Nature. 1980 May 29;285(5763):333–335. doi: 10.1038/285333a0. [DOI] [PubMed] [Google Scholar]
  48. Smith J. D., De Harven E. Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication. J Virol. 1973 Oct;12(4):919–930. doi: 10.1128/jvi.12.4.919-930.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Spaete R. R., Mocarski E. S. Insertion and deletion mutagenesis of the human cytomegalovirus genome. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7213–7217. doi: 10.1073/pnas.84.20.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Spaete R. R., Mocarski E. S. The alpha sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes. J Virol. 1985 Jun;54(3):817–824. doi: 10.1128/jvi.54.3.817-824.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stenberg R. M., Depto A. S., Fortney J., Nelson J. A. Regulated expression of early and late RNAs and proteins from the human cytomegalovirus immediate-early gene region. J Virol. 1989 Jun;63(6):2699–2708. doi: 10.1128/jvi.63.6.2699-2708.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stinski M. F. Human cytomegalovirus: glycoproteins associated with virions and dense bodies. J Virol. 1976 Aug;19(2):594–609. doi: 10.1128/jvi.19.2.594-609.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Stow E. C., Stow N. D. Complementation of a herpes simplex virus type 1 Vmw110 deletion mutant by human cytomegalovirus. J Gen Virol. 1989 Mar;70(Pt 3):695–704. doi: 10.1099/0022-1317-70-3-695. [DOI] [PubMed] [Google Scholar]
  55. Weber P. C., Levine M., Glorioso J. C. Rapid identification of nonessential genes of herpes simplex virus type 1 by Tn5 mutagenesis. Science. 1987 May 1;236(4801):576–579. doi: 10.1126/science.3033824. [DOI] [PubMed] [Google Scholar]
  56. Weller S. K., Aschman D. P., Sacks W. R., Coen D. M., Schaffer P. A. Genetic analysis of temperature-sensitive mutants of HSV-1: the combined use of complementation and physical mapping for cistron assignment. Virology. 1983 Oct 30;130(2):290–305. doi: 10.1016/0042-6822(83)90084-3. [DOI] [PubMed] [Google Scholar]
  57. Weller S. K., Lee K. J., Sabourin D. J., Schaffer P. A. Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein. J Virol. 1983 Jan;45(1):354–366. doi: 10.1128/jvi.45.1.354-366.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Weston K., Barrell B. G. Sequence of the short unique region, short repeats, and part of the long repeats of human cytomegalovirus. J Mol Biol. 1986 Nov 20;192(2):177–208. doi: 10.1016/0022-2836(86)90359-1. [DOI] [PubMed] [Google Scholar]
  59. Yamanishi K., Rapp F. Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus. Virology. 1979 Apr 15;94(1):237–241. doi: 10.1016/0042-6822(79)90457-4. [DOI] [PubMed] [Google Scholar]
  60. Yamanishi K., Rapp F. Temperature-sensitive mutants of human cytomegalovirus. J Virol. 1977 Oct;24(1):416–418. doi: 10.1128/jvi.24.1.416-418.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]

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