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. 1996 Mar;70(3):1365–1374. doi: 10.1128/jvi.70.3.1365-1374.1996

Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism.

V J Cavanaugh 1, R M Stenberg 1, T L Staley 1, H W Virgin 4th 1, M R MacDonald 1, S Paetzold 1, H E Farrell 1, W D Rawlinson 1, A E Campbell 1
PMCID: PMC189955  PMID: 8627652

Abstract

Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.

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Selected References

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  1. Bale J. F., Jr, O'Neil M. E. Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice. J Virol. 1989 Jun;63(6):2667–2673. doi: 10.1128/jvi.63.6.2667-2673.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bancroft G. J., Collins H. L., Sigola L. B., Cross C. E. Modulation of murine macrophage behavior in vivo and in vitro. Methods Cell Biol. 1994;45:129–146. doi: 10.1016/s0091-679x(08)61849-x. [DOI] [PubMed] [Google Scholar]
  3. Bankier A. T., Weston K. M., Barrell B. G. Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. Methods Enzymol. 1987;155:51–93. doi: 10.1016/0076-6879(87)55009-1. [DOI] [PubMed] [Google Scholar]
  4. Brautigam A. R., Dutko F. J., Olding L. B., Oldstone M. B. Pathogenesis of murine cytomegalovirus infection: the macrophage as a permissive cell for cytomegalovirus infection, replication and latency. J Gen Virol. 1979 Aug;44(2):349–359. doi: 10.1099/0022-1317-44-2-349. [DOI] [PubMed] [Google Scholar]
  5. Bühler B., Keil G. M., Weiland F., Koszinowski U. H. Characterization of the murine cytomegalovirus early transcription unit e1 that is induced by immediate-early proteins. J Virol. 1990 May;64(5):1907–1919. doi: 10.1128/jvi.64.5.1907-1919.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Campbell A. E., Slater J. S., Futch W. S. Murine cytomegalovirus-induced suppression of antigen-specific cytotoxic T lymphocyte maturation. Virology. 1989 Nov;173(1):268–275. doi: 10.1016/0042-6822(89)90243-2. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Collins T. M., Quirk M. R., Jordan M. C. Biphasic viremia and viral gene expression in leukocytes during acute cytomegalovirus infection of mice. J Virol. 1994 Oct;68(10):6305–6311. doi: 10.1128/jvi.68.10.6305-6311.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dankner W. M., McCutchan J. A., Richman D. D., Hirata K., Spector S. A. Localization of human cytomegalovirus in peripheral blood leukocytes by in situ hybridization. J Infect Dis. 1990 Jan;161(1):31–36. doi: 10.1093/infdis/161.1.31. [DOI] [PubMed] [Google Scholar]
  11. Davison A. J. Experience in shotgun sequencing a 134 kilobase pair DNA molecule. DNA Seq. 1991;1(6):389–394. doi: 10.3109/10425179109020794. [DOI] [PubMed] [Google Scholar]
  12. Dear S., Staden R. A sequence assembly and editing program for efficient management of large projects. Nucleic Acids Res. 1991 Jul 25;19(14):3907–3911. doi: 10.1093/nar/19.14.3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dear S., Staden R. A standard file format for data from DNA sequencing instruments. DNA Seq. 1992;3(2):107–110. doi: 10.3109/10425179209034003. [DOI] [PubMed] [Google Scholar]
  14. Elliott R., Clark C., Jaquish D., Spector D. H. Transcription analysis and sequence of the putative murine cytomegalovirus DNA polymerase gene. Virology. 1991 Nov;185(1):169–186. doi: 10.1016/0042-6822(91)90765-4. [DOI] [PubMed] [Google Scholar]
  15. Fiala M., Mosca J. D., Barry P., Luciw P. A., Vinters H. V. Multi-step pathogenesis of AIDS--role of cytomegalovirus. Res Immunol. 1991 Feb;142(2):87–95. doi: 10.1016/0923-2494(91)90016-c. [DOI] [PubMed] [Google Scholar]
  16. Ginsberg H. S., Lundholm-Beauchamp U., Horswood R. L., Pernis B., Wold W. S., Chanock R. M., Prince G. A. Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc Natl Acad Sci U S A. 1989 May;86(10):3823–3827. doi: 10.1073/pnas.86.10.3823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gooding L. R. Virus proteins that counteract host immune defenses. Cell. 1992 Oct 2;71(1):5–7. doi: 10.1016/0092-8674(92)90259-f. [DOI] [PubMed] [Google Scholar]
  18. Heise M. T., Virgin H. W., 4th The T-cell-independent role of gamma interferon and tumor necrosis factor alpha in macrophage activation during murine cytomegalovirus and herpes simplex virus infections. J Virol. 1995 Feb;69(2):904–909. doi: 10.1128/jvi.69.2.904-909.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ho M. Observations from transplantation contributing to the understanding of pathogenesis of CMV infection. Transplant Proc. 1991 Jun;23(3 Suppl 3):104-8, discussion 108-9. [PubMed] [Google Scholar]
  20. Ibanez C. E., Schrier R., Ghazal P., Wiley C., Nelson J. A. Human cytomegalovirus productively infects primary differentiated macrophages. J Virol. 1991 Dec;65(12):6581–6588. doi: 10.1128/jvi.65.12.6581-6588.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones T. R., Hanson L. K., Sun L., Slater J. S., Stenberg R. M., Campbell A. E. Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains. J Virol. 1995 Aug;69(8):4830–4841. doi: 10.1128/jvi.69.8.4830-4841.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jones T. R., Muzithras V. P. A cluster of dispensable genes within the human cytomegalovirus genome short component: IRS1, US1 through US5, and the US6 family. J Virol. 1992 Apr;66(4):2541–2546. doi: 10.1128/jvi.66.4.2541-2546.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones T. R., Muzithras V. P., Gluzman Y. Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential. J Virol. 1991 Nov;65(11):5860–5872. doi: 10.1128/jvi.65.11.5860-5872.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Keil G. M., Ebeling-Keil A., Koszinowski U. H. Sequence and structural organization of murine cytomegalovirus immediate-early gene 1. J Virol. 1987 Jun;61(6):1901–1908. doi: 10.1128/jvi.61.6.1901-1908.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kerry J. A., Sehgal A., Barlow S. W., Cavanaugh V. J., Fish K., Nelson J. A., Stenberg R. M. Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region. J Virol. 1995 Jun;69(6):3868–3872. doi: 10.1128/jvi.69.6.3868-3872.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kondo K., Kaneshima H., Mocarski E. S. Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11879–11883. doi: 10.1073/pnas.91.25.11879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koszinowski U. H., Del Val M., Reddehase M. J. Cellular and molecular basis of the protective immune response to cytomegalovirus infection. Curr Top Microbiol Immunol. 1990;154:189–220. doi: 10.1007/978-3-642-74980-3_8. [DOI] [PubMed] [Google Scholar]
  28. Lagenaur L. A., Manning W. C., Vieira J., Martens C. L., Mocarski E. S. Structure and function of the murine cytomegalovirus sgg1 gene: a determinant of viral growth in salivary gland acinar cells. J Virol. 1994 Dec;68(12):7717–7727. doi: 10.1128/jvi.68.12.7717-7727.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lewis M. A., Slater J. S., Leverone L. I., Campbell A. E. Enhancement of interleukin-1 activity by murine cytomegalovirus infection of a macrophage cell line. Virology. 1990 Oct;178(2):452–460. doi: 10.1016/0042-6822(90)90342-o. [DOI] [PubMed] [Google Scholar]
  30. Liu B., Stinski M. F. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J Virol. 1992 Jul;66(7):4434–4444. doi: 10.1128/jvi.66.7.4434-4444.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Manning W. C., Stoddart C. A., Lagenaur L. A., Abenes G. B., Mocarski E. S. Cytomegalovirus determinant of replication in salivary glands. J Virol. 1992 Jun;66(6):3794–3802. doi: 10.1128/jvi.66.6.3794-3802.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mauel J., Defendi V. Infection and transformation of mouse peritoneal macrophages by simian virus 40. J Exp Med. 1971 Aug 1;134(2):335–350. doi: 10.1084/jem.134.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meignier B., Longnecker R., Mavromara-Nazos P., Sears A. E., Roizman B. Virulence of and establishment of latency by genetically engineered deletion mutants of herpes simplex virus 1. Virology. 1988 Jan;162(1):251–254. doi: 10.1016/0042-6822(88)90417-5. [DOI] [PubMed] [Google Scholar]
  35. Messerle M., Bühler B., Keil G. M., Koszinowski U. H. Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3. J Virol. 1992 Jan;66(1):27–36. doi: 10.1128/jvi.66.1.27-36.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Messerle M., Keil G. M., Schneider K., Koszinowski U. H. Characterization of the murine cytomegalovirus genes encoding the major DNA binding protein and the ICP18.5 homolog. Virology. 1992 Nov;191(1):355–367. doi: 10.1016/0042-6822(92)90198-x. [DOI] [PubMed] [Google Scholar]
  37. Mocarski E. S., Jr, Abenes G. B., Manning W. C., Sambucetti L. C., Cherrington J. M. Molecular genetic analysis of cytomegalovirus gene regulation in growth, persistence and latency. Curr Top Microbiol Immunol. 1990;154:47–74. doi: 10.1007/978-3-642-74980-3_3. [DOI] [PubMed] [Google Scholar]
  38. Nicholas J., Martin M. E. Nucleotide sequence analysis of a 38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding human cytomegalovirus immediate-early gene homologs and transactivating functions. J Virol. 1994 Feb;68(2):597–610. doi: 10.1128/jvi.68.2.597-610.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Osborn J. E., Walker D. L. Virulence and attenuation of murine cytomegalovirus. Infect Immun. 1971 Feb;3(2):228–236. doi: 10.1128/iai.3.2.228-236.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pollock J. L., Virgin H. W., 4th Latency, without persistence, of murine cytomegalovirus in the spleen and kidney. J Virol. 1995 Mar;69(3):1762–1768. doi: 10.1128/jvi.69.3.1762-1768.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rapp M., Lucin P., Messerle M., Loh L. C., Koszinowski U. H. Expression of the murine cytomegalovirus glycoprotein H by recombinant vaccinia virus. J Gen Virol. 1994 Jan;75(Pt 1):183–188. doi: 10.1099/0022-1317-75-1-183. [DOI] [PubMed] [Google Scholar]
  42. Rapp M., Messerle M., Bühler B., Tannheimer M., Keil G. M., Koszinowski U. H. Identification of the murine cytomegalovirus glycoprotein B gene and its expression by recombinant vaccinia virus. J Virol. 1992 Jul;66(7):4399–4406. doi: 10.1128/jvi.66.7.4399-4406.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Saltzman R. L., Quirk M. R., Jordan M. C. Disseminated cytomegalovirus infection. Molecular analysis of virus and leukocyte interactions in viremia. J Clin Invest. 1988 Jan;81(1):75–81. doi: 10.1172/JCI113313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schrier R. D., Nelson J. A., Oldstone M. B. Detection of human cytomegalovirus in peripheral blood lymphocytes in a natural infection. Science. 1985 Nov 29;230(4729):1048–1051. doi: 10.1126/science.2997930. [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. Smith V., Craxton M., Bankier A. T., Brown C. M., Rawlinson W. D., Chee M. S., Barrell B. G. Preparation and fluorescent sequencing of M13 clones: microtiter methods. Methods Enzymol. 1993;218:173–187. doi: 10.1016/0076-6879(93)18015-5. [DOI] [PubMed] [Google Scholar]
  47. Staczek J. Animal cytomegaloviruses. Microbiol Rev. 1990 Sep;54(3):247–265. doi: 10.1128/mr.54.3.247-265.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Staden R. The current status and portability of our sequence handling software. Nucleic Acids Res. 1986 Jan 10;14(1):217–231. doi: 10.1093/nar/14.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Stoddart C. A., Cardin R. D., Boname J. M., Manning W. C., Abenes G. B., Mocarski E. S. Peripheral blood mononuclear phagocytes mediate dissemination of murine cytomegalovirus. J Virol. 1994 Oct;68(10):6243–6253. doi: 10.1128/jvi.68.10.6243-6253.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Taylor-Wiedeman J., Sissons J. G., Borysiewicz L. K., Sinclair J. H. Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol. 1991 Sep;72(Pt 9):2059–2064. doi: 10.1099/0022-1317-72-9-2059. [DOI] [PubMed] [Google Scholar]
  52. Thäle R., Lucin P., Schneider K., Eggers M., Koszinowski U. H. Identification and expression of a murine cytomegalovirus early gene coding for an Fc receptor. J Virol. 1994 Dec;68(12):7757–7765. doi: 10.1128/jvi.68.12.7757-7765.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tufariello J., Cho S., Horwitz M. S. The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model. J Virol. 1994 Jan;68(1):453–462. doi: 10.1128/jvi.68.1.453-462.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Upton C., Macen J. L., Schreiber M., McFadden G. Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence. Virology. 1991 Sep;184(1):370–382. doi: 10.1016/0042-6822(91)90853-4. [DOI] [PubMed] [Google Scholar]
  55. Vieira J., Farrell H. E., Rawlinson W. D., Mocarski E. S. Genes in the HindIII J fragment of the murine cytomegalovirus genome are dispensable for growth in cultured cells: insertion mutagenesis with a lacZ/gpt cassette. J Virol. 1994 Aug;68(8):4837–4846. doi: 10.1128/jvi.68.8.4837-4846.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Welsh R. M., Brubaker J. O., Vargas-Cortes M., O'Donnell C. L. Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK cell-dependent control of virus infections occur independently of T and B cell function. J Exp Med. 1991 May 1;173(5):1053–1063. doi: 10.1084/jem.173.5.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Xu J., Scalzo A. A., Lyons P. A., Farrell H. E., Rawlinson W. D., Shellam G. R. Identification, sequencing and expression of the glycoprotein L gene of murine cytomegalovirus. J Gen Virol. 1994 Nov;75(Pt 11):3235–3240. doi: 10.1099/0022-1317-75-11-3235. [DOI] [PubMed] [Google Scholar]
  59. York I. A., Roop C., Andrews D. W., Riddell S. R., Graham F. L., Johnson D. C. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell. 1994 May 20;77(4):525–535. doi: 10.1016/0092-8674(94)90215-1. [DOI] [PubMed] [Google Scholar]

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