1887

Abstract

The gene product of the human immunodeficiency virus (HIV) is suggested to be a negative factor involved in down-regulating viral expression by a mechanism in which the correct conformation of the protein is essential. The protein expressed by vaccinia virus recombinants is phosphorylated by protein kinase C. We investigated the synthesis of the protein and its state of phosphorylation during HIV-1 infection of a T4 cell line (CEM cells). Maximum synthesis of viral proteins occurred 3 days after infection, when more than 90% of cells were producing viral proteins. The synthesis of the protein was detected in parallel with the and proteins. As expected, the protein was myristylated but not phosphorylated, and its half-life was less than 1 h. By the use of the polymerase chain reaction technique, we isolated and sequenced the gene of this HIV-1 stock. Two significant mutations were observed. Firstly, threonine, at amino acid number 15, the site of phosphorylation by protein kinase C, was mutated into an alanine, and secondly aspartic acid of the tetrapeptide WRFD, which is probably involved in GTP binding, was mutated into an asparagine. The mutated gene was expressed in a vaccinia virus system, in which it was not phosphorylated and its half-life was dramatically reduced compared to the wild-type gene product. Furthermore, down- regulation of CD4 cell surface expression was no longer affected by the mutated gene. These results emphasize that phosphorylation of the protein provides an efficient test to monitor its biological activity.

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1990-10-01
2024-03-28
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References

  1. Ahmad N., Venkatesan S. 1988; Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241:1481–1485
    [Google Scholar]
  2. Ahmad N., Maitra R. K., Venkatesan S. 1989; Rev-induced modulation of nef protein underlies temporal regulation of human immunodeficiency virus replication. Proceedings of the National Academy of Sciences U.S.A.: 866111–6115
    [Google Scholar]
  3. Alizon M., Wain-Hobson S., Montagnier L., Sonigo P. 1986; Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell 46:63–74
    [Google Scholar]
  4. Allan S., Coligan J. E., Lee T. H., Mclane M. F., Kanki P. J., Groopman J. E., Essex M. 1986; A new HTLVIII/LAV encoded antigen detected by antibodies from AIDS patients. Science 230:810–813
    [Google Scholar]
  5. Barré-Sinoussi F., Chermann J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauguet C., Axler-Blin C., Brun-Vezinet F., Rouzioux C., Rozenbaum W., Montagnier L. 1983; Isolation of T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868–871
    [Google Scholar]
  6. Cheng-Mayer C., Seto D., Tateno M., Levy J. A. 1988; Biologic features of HIV-1 that correlate with virulence in the host. Science 240:80–82
    [Google Scholar]
  7. Cheng-Mayer C., Iannello P., Shaw K., Luciw P. A., Levy J. A. 1989; Differential effects of nef on HIV replication: implications for viral pathogenesis in the host. Science 246:1629–1632
    [Google Scholar]
  8. Cullen B. R., Greene W. C. 1989; Regulatory pathways governing HIV-1 replication. Cells 58:423–426
    [Google Scholar]
  9. Franchini G., Robert-Guroff M., Ghrayeb J., Chang N. T., Wong-Staal F. 1986; Cytoplasmic localization of the HTLVIII3′ orf protein in cultures of T cells. Virology 155:593–599
    [Google Scholar]
  10. Gallo R., Wong-Staal F., Montagnier L., Haseltine W., Yoshida M. 1988; HIV/HTLV gene nomenclature. Nature; London: 333504
    [Google Scholar]
  11. Gilman A. G. 1987; G proteins: transducers of receptor-generated signals. Annual Review of Biochemistry 56:615–649
    [Google Scholar]
  12. Gould K. L., Woodgett J. R., Cooper J. A., Buss J. E., Shalloway D., Hunter T. 1985; Protein kinase C phosphory- lates pp60sreat a novel site. Cell 42:849–857
    [Google Scholar]
  13. Guy B., Kieny M. P., Rivière Y., Lepeuch C., Dott K., Girard M., Montagnier L., Lecocq J. P. 1987; HIV F/3′ orf encodes a phosphorylated G TP-binding protein resembling an oncogene product. Nature; London: 330266–269
    [Google Scholar]
  14. Guy B., Rivière Y., Dorr K., Regnault A., Kieny M. P. 1990a; Mutational analysis of the HIV nef protein. Virology in press
    [Google Scholar]
  15. Guy B., Acres B., Kieny M. P., Lecocq J. P. 1990b; DNA binding factors that bind to the negative regulatory element of the human immunodeficiency virus-1: regulation by nef. Journal of AIDS in press
    [Google Scholar]
  16. Guyader M., Emermann M., Sonigo P., Clavel F., Montagnier L., Alizon M. 1987; Genome organization and transactivation of the human immunodeficiency virus type 2. Nature; London: 326662–667
    [Google Scholar]
  17. Hammes S. R., Dixon E. P., Malim M. H., Cullen B. R., Greene W. C. 1989; Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proceedings of the National Academy of Sciences U.S.A.: 869549–9553
    [Google Scholar]
  18. Hwang Y. W., Miller D. L. 1987; A mutation that alters the nucleotide specificity of EFTu, a GTP regulatory protein. Journal of Biological Chemistry 262:13081–13085
    [Google Scholar]
  19. Kieny M. P., Lathe R., Drillien R., Spenher D., Skory S., Schmitt D., Wiktor T., Koprowski H., Lecocq J. P. 1984; Expression of rabies glycoprotein from a recombinant vaccinia virus. Nature; London: 312163–166
    [Google Scholar]
  20. Kim S., Ikeuchi K., Byrn R., Groopman J., Baltimore D. 1989; Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences U.S.A.: 869544–9548
    [Google Scholar]
  21. Lathe R., Hirth P. 1980; Cell-free synthesis of enterotoxin of E. colifrom a cloned gene. Nature; London: 284473–474
    [Google Scholar]
  22. Laurent A. G., Krust B., Rey M. A., Montagnier L., Hovanessian A. G. 1989; Cell surface expression of several species of human immunodeficiency virus type 1 major core protein. Journal of Virology 63:4074–4078
    [Google Scholar]
  23. Loche M., Mach B. 1988; Identification of HIV-infected seronegative individuals by a direct diagnostic test based on hybridization to amplified viral DNA. Lancet ii:418–421
    [Google Scholar]
  24. Lochrie M. A., Simon M. I. 1988; G protein multiplicity in eukaryotic signal transduction systems. Biochemistry 27: 14 4957–4965
    [Google Scholar]
  25. Luciw P. A., Cheng-Mayer C., Levy J. A. 1987; Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication. Proceedings of the National Academy of Sciences U.S.A.: 841434–1438
    [Google Scholar]
  26. Montagnier L., Chermann J. L., Barré-Sinoussi F., Chamaret S., Gruest J., Nugeyre M. T., Rey F., Dauguet C., Axler-Blin C., Brun-Vezinet F., Rouzioux C., Saimot A. G., Rozenbaum W., Gluckman J. C., Klatzmann D., Vilmer E., Griscelli C., Gazengel C., Brunet J. B. 1984; A new human T-lymphotropic retrovirus: characterization and possible role in lymphadenopathy and acquired immune deficiency syndrome. In Human T Cell LeukemialLymphoma Viruses pp 363–379 Gallo R. C., Essex M. E., Gross L. Edited by New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Niederman T. M. J., Thielan B. J., Ratner L. 1989; Human immunodeficiency virus type 1 negative factor is a transcriptional silencer. Proceedings of the National Academy of Sciences U.S.A.: 861128–1132
    [Google Scholar]
  28. O’Farrell P. H. 1975; High resolution two dimensional electrophoresis of proteins. Journal of Biological Chemistry 250:4007–4021
    [Google Scholar]
  29. Paulin L., Levy J. A. 1989; In vitroexpression of a functional HIV-nef gene product. Vth International Congress on AIDS, Montreal 588:
    [Google Scholar]
  30. Ratner L., Starcich B., Josephs F., Hahn B. H., Reddy E. P., Livak K. J., Petteway S. R., Pearson M. L., Haseltine W. A., Arya S. K., Wong-Staal F. 1985; Polymorphism of the 3′ open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III. Nucleic Acids Research 13:8219–8229
    [Google Scholar]
  31. Rey M. A., Krust B., Laurent A. G., Montagnier L., Hovanessian A. G. 1989; Characterization of human immunodeficiency virus type 2 envelope glycoproteins: dimerization of the glycoprotein precursor during processing. Journal of Virology 63:647–658
    [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences U.S.A.: 745463–5467
    [Google Scholar]
  33. Sprre B., Sire J., Zachar V., Rey F., Barré-Sinoussi F., Galibert F., Hampe A., Chermann J. C. 1989; Nucleotide sequence of HIV-l-NDK: a highly cytopathic strain of the human immunodeficiency virus. Gene 81:275–284
    [Google Scholar]
  34. Terwilliger E., Sodroski J. G., Rosen C. A., Haseltine W. A. 1986; Effects of mutations within the 3′ orf region of HLVIII/LAV on replication and cytopathogenicity. Journal of Virology 60:754–760
    [Google Scholar]
  35. Veronese F. D. M., Copeland T. D., Oroszlan S., Gallo R. C., Sarngadharan M. G. 1988; Biochemical and immunological analysis of human immunodeficiency virus gaggene products pl7 and p24. Journal of Virology 62:795–801
    [Google Scholar]
  36. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. 1985; Nucleotide sequence of the AIDS virus, LAV. Cell 40:9–17
    [Google Scholar]
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