Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 May;69(5):2881–2888. doi: 10.1128/jvi.69.5.2881-2888.1995

Replication in vitro and in vivo of an equine infectious anemia virus mutant deficient in dUTPase activity.

D L Lichtenstein 1, K E Rushlow 1, R F Cook 1, M L Raabe 1, C J Swardson 1, G J Kociba 1, C J Issel 1, R C Montelaro 1
PMCID: PMC188985  PMID: 7707512

Abstract

As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equine kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells. In FEK cells, which are permissive for EIAV replication, the mutant virus replicated as well as the parental virus. In primary cultures of EBMM cells, which are primary targets of EIAV infection in vivo, the DU mutant showed delayed replication kinetics and replicated to a lower extent than did the parental virus. As the multiplicity of infection decreased, the difference between the parental and mutant viruses increased, such that at the lowest multiplicity of infection tested, there was over a 100-fold difference in virus production. The mutant virus was also much less cytopathic. The role of DU in replication in vivo was examined using a Shetland pony model of EIAV infection. Shetland ponies that were infected with the parental and mutant viruses showed transient virus RNA levels in plasma approximately 5 to 10 days postinfection. The peak virus levels in plasma (as measured by a quantitative reverse transcriptase PCR assay) were 10- to 100-fold lower in the mutant virus-infected animals than in the animals infected with the parental virus. However, ponies infected with the mutant virus mounted similar antibody responses despite the marked differences in virus replication. These studies demonstrate that EIAV DU is important for the efficient replication of the virus in macrophages in vitro and in vivo and suggests that variations in the DU sequence could markedly affect the biological and pathogenic properties of EIAV.

Full Text

The Full Text of this article is available as a PDF (475.6 KB).

Selected References

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

  1. Ball J. M., Rushlow K. E., Issel C. J., Montelaro R. C. Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies. J Virol. 1992 Feb;66(2):732–742. doi: 10.1128/jvi.66.2.732-742.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergman A. C., Björnberg O., Nord J., Nyman P. O., Rosengren A. M. The protein p30, encoded at the gag-pro junction of mouse mammary tumor virus, is a dUTPase fused with a nucleocapsid protein. Virology. 1994 Oct;204(1):420–424. doi: 10.1006/viro.1994.1547. [DOI] [PubMed] [Google Scholar]
  3. Broyles S. S. Vaccinia virus encodes a functional dUTPase. Virology. 1993 Aug;195(2):863–865. doi: 10.1006/viro.1993.1446. [DOI] [PubMed] [Google Scholar]
  4. Canman C. E., Lawrence T. S., Shewach D. S., Tang H. Y., Maybaum J. Resistance to fluorodeoxyuridine-induced DNA damage and cytotoxicity correlates with an elevation of deoxyuridine triphosphatase activity and failure to accumulate deoxyuridine triphosphate. Cancer Res. 1993 Nov 1;53(21):5219–5224. [PubMed] [Google Scholar]
  5. Curtin N. J., Harris A. L., Aherne G. W. Mechanism of cell death following thymidylate synthase inhibition: 2'-deoxyuridine-5'-triphosphate accumulation, DNA damage, and growth inhibition following exposure to CB3717 and dipyridamole. Cancer Res. 1991 May 1;51(9):2346–2352. [PubMed] [Google Scholar]
  6. Daniel M. D., Kirchhoff F., Czajak S. C., Sehgal P. K., Desrosiers R. C. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science. 1992 Dec 18;258(5090):1938–1941. doi: 10.1126/science.1470917. [DOI] [PubMed] [Google Scholar]
  7. Doignon F., Biteau N., Aigle M., Crouzet M. The complete sequence of a 6794 bp segment located on the right arm of chromosome II of Saccharomyces cerevisiae. Finding of a putative dUTPase in a yeast. Yeast. 1993 Oct;9(10):1131–1137. doi: 10.1002/yea.320091014. [DOI] [PubMed] [Google Scholar]
  8. Dorn P., DaSilva L., Martarano L., Derse D. Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins. J Virol. 1990 Apr;64(4):1616–1624. doi: 10.1128/jvi.64.4.1616-1624.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elder J. H., Lerner D. L., Hasselkus-Light C. S., Fontenot D. J., Hunter E., Luciw P. A., Montelaro R. C., Phillips T. R. Distinct subsets of retroviruses encode dUTPase. J Virol. 1992 Mar;66(3):1791–1794. doi: 10.1128/jvi.66.3.1791-1794.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fisher A. G., Ratner L., Mitsuya H., Marselle L. M., Harper M. E., Broder S., Gallo R. C., Wong-Staal F. Infectious mutants of HTLV-III with changes in the 3' region and markedly reduced cytopathic effects. Science. 1986 Aug 8;233(4764):655–659. doi: 10.1126/science.3014663. [DOI] [PubMed] [Google Scholar]
  11. Fisher F. B., Preston V. G. Isolation and characterisation of herpes simplex virus type 1 mutants which fail to induce dUTPase activity. Virology. 1986 Jan 15;148(1):190–197. doi: 10.1016/0042-6822(86)90414-9. [DOI] [PubMed] [Google Scholar]
  12. Focher F., Mazzarello P., Verri A., Hübscher U., Spadari S. Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development. Mutat Res. 1990 Mar;237(2):65–73. doi: 10.1016/0921-8734(90)90012-g. [DOI] [PubMed] [Google Scholar]
  13. GREENBERG G. R., SOMERVILLE R. L. Deoxyuridylate kinase activity and deoxyuridinetriphosphatase in Escherichia coli. Proc Natl Acad Sci U S A. 1962 Feb;48:247–257. doi: 10.1073/pnas.48.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gadsden M. H., McIntosh E. M., Game J. C., Wilson P. J., Haynes R. H. dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae. EMBO J. 1993 Nov;12(11):4425–4431. doi: 10.1002/j.1460-2075.1993.tb06127.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giroir L. E., Deutsch W. A. Drosophila deoxyuridine triphosphatase. Purification and characterization. J Biol Chem. 1987 Jan 5;262(1):130–134. [PubMed] [Google Scholar]
  16. Goulaouic H., Subra F., Mouscadet J. F., Carteau S., Auclair C. Exogenous nucleosides promote the completion of MoMLV DNA synthesis in G0-arrested Balb c/3T3 fibroblasts. Virology. 1994 Apr;200(1):87–97. doi: 10.1006/viro.1994.1166. [DOI] [PubMed] [Google Scholar]
  17. Goulian M., Bleile B. M., Dickey L. M., Grafstrom R. H., Ingraham H. A., Neynaber S. A., Peterson M. S., Tseng B. Y. Mechanism of thymineless death. Adv Exp Med Biol. 1986;195(Pt B):89–95. doi: 10.1007/978-1-4684-1248-2_15. [DOI] [PubMed] [Google Scholar]
  18. Hammes S. R., Dixon E. P., Malim M. H., Cullen B. R., Greene W. C. Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9549–9553. doi: 10.1073/pnas.86.23.9549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hokari S., Sakagishi Y., Tsukada K. Enhanced activity of deoxyuridine 5'-triphosphatase in regenerating rat liver. Biochem Biophys Res Commun. 1982 Sep 16;108(1):95–101. doi: 10.1016/0006-291x(82)91836-8. [DOI] [PubMed] [Google Scholar]
  20. Hokari S., Takizawa A., Tanaka M., Sakagishi Y. Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition. Biochem Int. 1989 Sep;19(3):453–461. [PubMed] [Google Scholar]
  21. Jamieson B. D., Aldrovandi G. M., Planelles V., Jowett J. B., Gao L., Bloch L. M., Chen I. S., Zack J. A. Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity. J Virol. 1994 Jun;68(6):3478–3485. doi: 10.1128/jvi.68.6.3478-3485.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kestler H. W., 3rd, Mori K., Silva D. P., Kodama T., King N. W., Daniel M. D., Desrosiers R. C. Nef genes of SIV. J Med Primatol. 1990;19(3-4):421–429. [PubMed] [Google Scholar]
  23. Kestler H. W., 3rd, Ringler D. J., Mori K., Panicali D. L., Sehgal P. K., Daniel M. D., Desrosiers R. C. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell. 1991 May 17;65(4):651–662. doi: 10.1016/0092-8674(91)90097-i. [DOI] [PubMed] [Google Scholar]
  24. Kim S., Ikeuchi K., Byrn R., Groopman J., Baltimore D. Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9544–9548. doi: 10.1073/pnas.86.23.9544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Köppe B., Menéndez-Arias L., Oroszlan S. Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity. J Virol. 1994 Apr;68(4):2313–2319. doi: 10.1128/jvi.68.4.2313-2319.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lirette R., Caradonna S. Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection. J Cell Biochem. 1990 Aug;43(4):339–353. doi: 10.1002/jcb.240430406. [DOI] [PubMed] [Google Scholar]
  27. Martarano L., Stephens R., Rice N., Derse D. Equine infectious anemia virus trans-regulatory protein Rev controls viral mRNA stability, accumulation, and alternative splicing. J Virol. 1994 May;68(5):3102–3111. doi: 10.1128/jvi.68.5.3102-3111.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McGeoch D. J. Protein sequence comparisons show that the 'pseudoproteases' encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family. Nucleic Acids Res. 1990 Jul 25;18(14):4105–4110. doi: 10.1093/nar/18.14.4105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mercer A. A., Fraser K. M., Stockwell P. A., Robinson A. J. A homologue of retroviral pseudoproteases in the parapoxvirus, orf virus. Virology. 1989 Oct;172(2):665–668. doi: 10.1016/0042-6822(89)90212-2. [DOI] [PubMed] [Google Scholar]
  30. Noiman S., Gazit A., Tori O., Sherman L., Miki T., Tronick S. R., Yaniv A. Identification of sequences encoding the equine infectious anemia virus tat gene. Virology. 1990 May;176(1):280–288. doi: 10.1016/0042-6822(90)90254-o. [DOI] [PubMed] [Google Scholar]
  31. O'Brien W. A., Namazi A., Kalhor H., Mao S. H., Zack J. A., Chen I. S. Kinetics of human immunodeficiency virus type 1 reverse transcription in blood mononuclear phagocytes are slowed by limitations of nucleotide precursors. J Virol. 1994 Feb;68(2):1258–1263. doi: 10.1128/jvi.68.2.1258-1263.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pardo E. G., Gutiérrez C. Cell cycle- and differentiation stage-dependent variation of dUTPase activity in higher plant cells. Exp Cell Res. 1990 Jan;186(1):90–98. doi: 10.1016/0014-4827(90)90214-u. [DOI] [PubMed] [Google Scholar]
  33. Payne S. L., Fang F. D., Liu C. P., Dhruva B. R., Rwambo P., Issel C. J., Montelaro R. C. Antigenic variation and lentivirus persistence: variations in envelope gene sequences during EIAV infection resemble changes reported for sequential isolates of HIV. Virology. 1987 Dec;161(2):321–331. doi: 10.1016/0042-6822(87)90124-3. [DOI] [PubMed] [Google Scholar]
  34. Payne S. L., Rausch J., Rushlow K., Montelaro R. C., Issel C., Flaherty M., Perry S., Sellon D., Fuller F. Characterization of infectious molecular clones of equine infectious anaemia virus. J Gen Virol. 1994 Feb;75(Pt 2):425–429. doi: 10.1099/0022-1317-75-2-425. [DOI] [PubMed] [Google Scholar]
  35. Payne S. L., Rushlow K., Dhruva B. R., Issel C. J., Montelaro R. C. Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli. Virology. 1989 Oct;172(2):609–615. doi: 10.1016/0042-6822(89)90203-1. [DOI] [PubMed] [Google Scholar]
  36. Perno C. F., Yarchoan R., Cooney D. A., Hartman N. R., Gartner S., Popovic M., Hao Z., Gerrard T. L., Wilson Y. A., Johns D. G. Inhibition of human immunodeficiency virus (HIV-1/HTLV-IIIBa-L) replication in fresh and cultured human peripheral blood monocytes/macrophages by azidothymidine and related 2',3'-dideoxynucleosides. J Exp Med. 1988 Sep 1;168(3):1111–1125. doi: 10.1084/jem.168.3.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pri-Hadash A., Hareven D., Lifschitz E. A meristem-related gene from tomato encodes a dUTPase: analysis of expression in vegetative and floral meristems. Plant Cell. 1992 Feb;4(2):149–159. doi: 10.1105/tpc.4.2.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pyles R. B., Sawtell N. M., Thompson R. L. Herpes simplex virus type 1 dUTPase mutants are attenuated for neurovirulence, neuroinvasiveness, and reactivation from latency. J Virol. 1992 Nov;66(11):6706–6713. doi: 10.1128/jvi.66.11.6706-6713.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Richards R. G., Sowers L. C., Laszlo J., Sedwick W. D. The occurrence and consequences of deoxyuridine in DNA. Adv Enzyme Regul. 1984;22:157–185. doi: 10.1016/0065-2571(84)90013-x. [DOI] [PubMed] [Google Scholar]
  40. Robinson J. E., Holton D., Liu J., McMurdo H., Murciano A., Gohd R. A novel enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HIV-1 envelope glycoproteins based on immobilization of viral glycoproteins in microtiter wells coated with concanavalin A. J Immunol Methods. 1990 Aug 28;132(1):63–71. doi: 10.1016/0022-1759(90)90399-g. [DOI] [PubMed] [Google Scholar]
  41. Shibata R., Adachi A., Sakai H., Ishimoto A., Miura T., Hayami M. Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2. J Med Primatol. 1990;19(3-4):217–225. [PubMed] [Google Scholar]
  42. Shlomai J., Kornberg A. Deoxyuridine triphosphatase of Escherichia coli. Purification, properties, and use as a reagent to reduce uracil incorporation into DNA. J Biol Chem. 1978 May 10;253(9):3305–3312. [PubMed] [Google Scholar]
  43. Spector R., Boose B. Development and regional distribution of deoxyuridine 5'-triphosphatase in rabbit brain. J Neurochem. 1983 Oct;41(4):1192–1195. doi: 10.1111/j.1471-4159.1983.tb09073.x. [DOI] [PubMed] [Google Scholar]
  44. Strahler J. R., Zhu X. X., Hora N., Wang Y. K., Andrews P. C., Roseman N. A., Neel J. V., Turka L., Hanash S. M. Maturation stage and proliferation-dependent expression of dUTPase in human T cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4991–4995. doi: 10.1073/pnas.90.11.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Swardson C. J., Kociba G. J., Perryman L. E. Effects of equine infectious anemia virus on hematopoietic progenitors in vitro. Am J Vet Res. 1992 Jul;53(7):1176–1179. [PubMed] [Google Scholar]
  46. Terai C., Carson D. A. Pyrimidine nucleotide and nucleic acid synthesis in human monocytes and macrophages. Exp Cell Res. 1991 Apr;193(2):375–381. doi: 10.1016/0014-4827(91)90110-g. [DOI] [PubMed] [Google Scholar]
  47. Terwilliger E., Sodroski J. G., Rosen C. A., Haseltine W. A. Effects of mutations within the 3' orf open reading frame region of human T-cell lymphotropic virus type III (HTLV-III/LAV) on replication and cytopathogenicity. J Virol. 1986 Nov;60(2):754–760. doi: 10.1128/jvi.60.2.754-760.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Threadgill D. S., Steagall W. K., Flaherty M. T., Fuller F. J., Perry S. T., Rushlow K. E., Le Grice S. F., Payne S. L. Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant. J Virol. 1993 May;67(5):2592–2600. doi: 10.1128/jvi.67.5.2592-2600.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tye B. K., Lehman I. R. Excision repair of uracil incorporated in DNA as a result of a defect in dUTPase. J Mol Biol. 1977 Dec 5;117(2):293–306. doi: 10.1016/0022-2836(77)90128-0. [DOI] [PubMed] [Google Scholar]
  50. Tye B. K., Nyman P. O., Lehman I. R., Hochhauser S., Weiss B. Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA. Proc Natl Acad Sci U S A. 1977 Jan;74(1):154–157. doi: 10.1073/pnas.74.1.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vertessy B. G., Zalud P., Nyman P. O., Zeppezauer M. Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase. Biochim Biophys Acta. 1994 Mar 16;1205(1):146–150. doi: 10.1016/0167-4838(94)90103-1. [DOI] [PubMed] [Google Scholar]
  52. Wagaman P. C., Hasselkus-Light C. S., Henson M., Lerner D. L., Phillips T. R., Elder J. H. Molecular cloning and characterization of deoxyuridine triphosphatase from feline immunodeficiency virus (FIV). Virology. 1993 Oct;196(2):451–457. doi: 10.1006/viro.1993.1501. [DOI] [PubMed] [Google Scholar]
  53. Williams M. V., Cheng Y. Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia. J Biol Chem. 1979 Apr 25;254(8):2897–2901. [PubMed] [Google Scholar]
  54. Williams M. V. Herpes simplex virus-induced dUTPase: target site for antiviral chemotherapy. Virology. 1988 Sep;166(1):262–264. doi: 10.1016/0042-6822(88)90171-7. [DOI] [PubMed] [Google Scholar]
  55. el-Hajj H. H., Zhang H., Weiss B. Lethality of a dut (deoxyuridine triphosphatase) mutation in Escherichia coli. J Bacteriol. 1988 Mar;170(3):1069–1075. doi: 10.1128/jb.170.3.1069-1075.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES