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. 1996 Jul;70(7):4394–4410. doi: 10.1128/jvi.70.7.4394-4410.1996

Disruption of M-T5, a novel myxoma virus gene member of poxvirus host range superfamily, results in dramatic attenuation of myxomatosis in infected European rabbits.

K Mossman 1, S F Lee 1, M Barry 1, L Boshkov 1, G McFadden 1
PMCID: PMC190373  PMID: 8676463

Abstract

Myxoma virus is a pathogenic poxvirus that induces a lethal myxomatosis disease profile in European rabbits, which is characterized by fulminating lesions at the primary site of inoculation, rapid dissemination to secondary internal organs and peripheral external sites, and supervening gram-negative bacterial infection. Here we describe the role of a novel myxoma virus protein encoded by the M-T5 open reading frame during pathogenesis. The myxoma virus M-T5 protein possesses no significant sequence homology to nonviral proteins but is a member of a larger poxviral superfamily designated host range proteins. An M-T5- mutant virus was constructed by disruption of both copies of the M-T5 gene followed by insertion of the selectable marker p7.5Ecogpt. Although the M-T5- deletion mutant replicated with wild-type kinetics in rabbit fibroblasts, infection of a rabbit CD4+ T-cell line (RL5) with the myxoma virus M-T5- mutant virus resulted in the rapid and complete cessation of both host and viral protein synthesis, accompanied by the manifestation of all the classical features of programmed cell death. Infection of primary rabbit peripheral mononuclear cells with the myxoma virus M-T5-mutant virus resulted in the apoptotic death of nonadherent lymphocytes but not adherent monocytes. Within the European rabbit, disruption of the M-T5 open reading frame caused a dramatic attenuation of the rapidly lethal myxomatosis infection, and none of the infected rabbits displayed any of the characteristic features of myxomatosis. The two most significant histological observations in rabbits infected with the M-T5-mutant virus were (i) the lack of progression of the infection past the primary site of inoculation, coupled with the establishment of a rapid and effective inflammatory reaction, and (ii) the inability of the virus to initiate a cellular reaction within secondary immune organs. We conclude that M-T5 functions as a critical virulence factor by allowing productive infection of immune cells such as peripheral lymphocytes, thus facilitating virus dissemination to secondary tissue sites via the lymphatic channels.

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Selected References

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  1. Ali A. N., Turner P. C., Brooks M. A., Moyer R. W. The SPI-1 gene of rabbitpox virus determines host range and is required for hemorrhagic pock formation. Virology. 1994 Jul;202(1):305–314. doi: 10.1006/viro.1994.1347. [DOI] [PubMed] [Google Scholar]
  2. Boyle D. B., Coupar B. E. A dominant selectable marker for the construction of recombinant poxviruses. Gene. 1988 May 15;65(1):123–128. doi: 10.1016/0378-1119(88)90424-6. [DOI] [PubMed] [Google Scholar]
  3. Buller R. M., Palumbo G. J. Poxvirus pathogenesis. Microbiol Rev. 1991 Mar;55(1):80–122. doi: 10.1128/mr.55.1.80-122.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen W., Drillien R., Spehner D., Buller R. M. In vitro and in vivo study of the ectromelia virus homolog of the vaccinia virus K1L host range gene. Virology. 1993 Oct;196(2):682–693. doi: 10.1006/viro.1993.1525. [DOI] [PubMed] [Google Scholar]
  5. Chou J., Chen J. J., Gross M., Roizman B. Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10516–10520. doi: 10.1073/pnas.92.23.10516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chou J., Roizman B. The gamma 1(34.5) gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programed cell death in neuronal cells. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3266–3270. doi: 10.1073/pnas.89.8.3266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clem R. J., Fechheimer M., Miller L. K. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science. 1991 Nov 29;254(5036):1388–1390. doi: 10.1126/science.1962198. [DOI] [PubMed] [Google Scholar]
  8. Drillien R., Spehner D., Kirn A. Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis. J Virol. 1978 Dec;28(3):843–850. doi: 10.1128/jvi.28.3.843-850.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Falkner F. G., Moss B. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J Virol. 1988 Jun;62(6):1849–1854. doi: 10.1128/jvi.62.6.1849-1854.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gavrieli Y., Sherman Y., Ben-Sasson S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992 Nov;119(3):493–501. doi: 10.1083/jcb.119.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goebel S. J., Johnson G. P., Perkus M. E., Davis S. W., Winslow J. P., Paoletti E. The complete DNA sequence of vaccinia virus. Virology. 1990 Nov;179(1):247-66, 517-63. doi: 10.1016/0042-6822(90)90294-2. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Graham K. A., Opgenorth A., Upton C., McFadden G. Myxoma virus M11L ORF encodes a protein for which cell surface localization is critical in manifestation of viral virulence. Virology. 1992 Nov;191(1):112–124. doi: 10.1016/0042-6822(92)90172-l. [DOI] [PubMed] [Google Scholar]
  14. Gregory C. D., Dive C., Henderson S., Smith C. A., Williams G. T., Gordon J., Rickinson A. B. Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis. Nature. 1991 Feb 14;349(6310):612–614. doi: 10.1038/349612a0. [DOI] [PubMed] [Google Scholar]
  15. Howard S. T., Chan Y. S., Smith G. L. Vaccinia virus homologues of the Shope fibroma virus inverted terminal repeat proteins and a discontinuous ORF related to the tumor necrosis factor receptor family. Virology. 1991 Feb;180(2):633–647. doi: 10.1016/0042-6822(91)90077-o. [DOI] [PubMed] [Google Scholar]
  16. Hruby D. E., Lynn D. L., Condit R. C., Kates J. R. Cellular differences in the molecular mechanisms of vaccinia virus host range restriction. J Gen Virol. 1980 Apr;47(2):485–488. doi: 10.1099/0022-1317-47-2-485. [DOI] [PubMed] [Google Scholar]
  17. Ink B. S., Gilbert C. S., Evan G. I. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes. J Virol. 1995 Feb;69(2):661–668. doi: 10.1128/jvi.69.2.661-668.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaschka-Dierich C., Werner F. J., Bauer I., Fleckenstein B. Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells. J Virol. 1982 Oct;44(1):295–310. doi: 10.1128/jvi.44.1.295-310.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kerr J. F., Wyllie A. H., Currie A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug;26(4):239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lambert S., Yu H., Prchal J. T., Lawler J., Ruff P., Speicher D., Cheung M. C., Kan Y. W., Palek J. cDNA sequence for human erythrocyte ankyrin. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1730–1734. doi: 10.1073/pnas.87.5.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lux S. E., John K. M., Bennett V. Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins. Nature. 1990 Mar 1;344(6261):36–42. doi: 10.1038/344036a0. [DOI] [PubMed] [Google Scholar]
  22. Macen J. L., Graham K. A., Lee S. F., Schreiber M., Boshkov L. K., McFadden G. Expression of the myxoma virus tumor necrosis factor receptor homologue and M11L genes is required to prevent virus-induced apoptosis in infected rabbit T lymphocytes. Virology. 1996 Apr 1;218(1):232–237. doi: 10.1006/viro.1996.0183. [DOI] [PubMed] [Google Scholar]
  23. Macen J. L., Upton C., Nation N., McFadden G. SERP1, a serine proteinase inhibitor encoded by myxoma virus, is a secreted glycoprotein that interferes with inflammation. Virology. 1993 Aug;195(2):348–363. doi: 10.1006/viro.1993.1385. [DOI] [PubMed] [Google Scholar]
  24. Massung R. F., Liu L. I., Qi J., Knight J. C., Yuran T. E., Kerlavage A. R., Parsons J. M., Venter J. C., Esposito J. J. Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975. Virology. 1994 Jun;201(2):215–240. doi: 10.1006/viro.1994.1288. [DOI] [PubMed] [Google Scholar]
  25. McFadden G., Graham K., Ellison K., Barry M., Macen J., Schreiber M., Mossman K., Nash P., Lalani A., Everett H. Interruption of cytokine networks by poxviruses: lessons from myxoma virus. J Leukoc Biol. 1995 May;57(5):731–738. doi: 10.1002/jlb.57.5.731. [DOI] [PubMed] [Google Scholar]
  26. McFadden G., Kane K. How DNA viruses perturb functional MHC expression to alter immune recognition. Adv Cancer Res. 1994;63:117–209. doi: 10.1016/s0065-230x(08)60400-5. [DOI] [PubMed] [Google Scholar]
  27. Mossman K., Nation P., Macen J., Garbutt M., Lucas A., McFadden G. Myxoma virus M-T7, a secreted homolog of the interferon-gamma receptor, is a critical virulence factor for the development of myxomatosis in European rabbits. Virology. 1996 Jan 1;215(1):17–30. doi: 10.1006/viro.1996.0003. [DOI] [PubMed] [Google Scholar]
  28. Oguiura N., Spehner D., Drillien R. Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines. J Gen Virol. 1993 Jul;74(Pt 7):1409–1413. doi: 10.1099/0022-1317-74-7-1409. [DOI] [PubMed] [Google Scholar]
  29. Opgenorth A., Graham K., Nation N., Strayer D., McFadden G. Deletion analysis of two tandemly arranged virulence genes in myxoma virus, M11L and myxoma growth factor. J Virol. 1992 Aug;66(8):4720–4731. doi: 10.1128/jvi.66.8.4720-4731.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Opgenorth A., Strayer D., Upton C., McFadden G. Deletion of the growth factor gene related to EGF and TGF alpha reduces virulence of malignant rabbit fibroma virus. Virology. 1992 Jan;186(1):175–191. doi: 10.1016/0042-6822(92)90072-w. [DOI] [PubMed] [Google Scholar]
  31. Perkus M. E., Goebel S. J., Davis S. W., Johnson G. P., Limbach K., Norton E. K., Paoletti E. Vaccinia virus host range genes. Virology. 1990 Nov;179(1):276–286. doi: 10.1016/0042-6822(90)90296-4. [DOI] [PubMed] [Google Scholar]
  32. Russell R. J., Robbins S. J. Cloning and molecular characterization of the myxoma virus genome. Virology. 1989 May;170(1):147–159. doi: 10.1016/0042-6822(89)90362-0. [DOI] [PubMed] [Google Scholar]
  33. Shchelkunov S. N., Blinov V. M., Sandakhchiev L. S. Ankyrin-like proteins of variola and vaccinia viruses. FEBS Lett. 1993 Mar 15;319(1-2):163–165. doi: 10.1016/0014-5793(93)80059-4. [DOI] [PubMed] [Google Scholar]
  34. Smith G. L. Virus strategies for evasion of the host response to infection. Trends Microbiol. 1994 Mar;2(3):81–88. doi: 10.1016/0966-842x(94)90539-8. [DOI] [PubMed] [Google Scholar]
  35. Spehner D., Gillard S., Drillien R., Kirn A. A cowpox virus gene required for multiplication in Chinese hamster ovary cells. J Virol. 1988 Apr;62(4):1297–1304. doi: 10.1128/jvi.62.4.1297-1304.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spriggs M. K. Poxvirus-encoded soluble cytokine receptors. Virus Res. 1994 Jul;33(1):1–10. doi: 10.1016/0168-1702(94)90013-2. [DOI] [PubMed] [Google Scholar]
  37. Sutter G., Ramsey-Ewing A., Rosales R., Moss B. Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant. J Virol. 1994 Jul;68(7):4109–4116. doi: 10.1128/jvi.68.7.4109-4116.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tewari M., Telford W. G., Miller R. A., Dixit V. M. CrmA, a poxvirus-encoded serpin, inhibits cytotoxic T-lymphocyte-mediated apoptosis. J Biol Chem. 1995 Sep 29;270(39):22705–22708. doi: 10.1074/jbc.270.39.22705. [DOI] [PubMed] [Google Scholar]
  39. Traktman P. Poxviruses: an emerging portrait of biological strategy. Cell. 1990 Aug 24;62(4):621–626. doi: 10.1016/0092-8674(90)90106-o. [DOI] [PubMed] [Google Scholar]
  40. Turner P. C., Moyer R. W. The molecular pathogenesis of poxviruses. Curr Top Microbiol Immunol. 1990;163:125–151. doi: 10.1007/978-3-642-75605-4_5. [DOI] [PubMed] [Google Scholar]
  41. Upton C., DeLange A. M., McFadden G. Tumorigenic poxviruses: genomic organization and DNA sequence of the telomeric region of the Shope fibroma virus genome. Virology. 1987 Sep;160(1):20–30. doi: 10.1016/0042-6822(87)90039-0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Upton C., McFadden G. Tumorigenic poxviruses: analysis of viral DNA sequences implicated in the tumorigenicity of Shope fibroma virus and malignant rabbit virus. Virology. 1986 Jul 30;152(2):308–321. doi: 10.1016/0042-6822(86)90134-0. [DOI] [PubMed] [Google Scholar]
  44. Upton C., Mossman K., McFadden G. Encoding of a homolog of the IFN-gamma receptor by myxoma virus. Science. 1992 Nov 20;258(5086):1369–1372. doi: 10.1126/science.1455233. [DOI] [PubMed] [Google Scholar]
  45. Walker N. I., Harmon B. V., Gobé G. C., Kerr J. F. Patterns of cell death. Methods Achiev Exp Pathol. 1988;13:18–54. [PubMed] [Google Scholar]
  46. White E., Sabbatini P., Debbas M., Wold W. S., Kusher D. I., Gooding L. R. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha. Mol Cell Biol. 1992 Jun;12(6):2570–2580. doi: 10.1128/mcb.12.6.2570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wyllie A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980 Apr 10;284(5756):555–556. doi: 10.1038/284555a0. [DOI] [PubMed] [Google Scholar]

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