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. 1992 Jul 15;89(14):6634–6638. doi: 10.1073/pnas.89.14.6634

Mutational analysis of the human immunodeficiency virus type 1 Eli Nef function.

E Zazopoulos 1, W A Haseltine 1
PMCID: PMC49556  PMID: 1631166

Abstract

The studies presented here define an internally consistent experimental system that permits systematic analysis of the effect of nef on the rate of the human immunodeficiency virus type 1 (HIV-1) replication in a CD4+ tumor T-cell line and in primary peripheral blood mononuclear cells. The parental full-length Nef protein, derived from the Eli strain of HIV-1, accelerates virus replication in both cell types. Mutations that destabilize or alter the intracellular location of the protein affect the ability of the Nef protein to accelerate virus replication. A set of mutants was made in amino acids proposed to be required for Nef function, including threonine and serine residues proposed to be targets for phosphorylation, and in sequences thought to resemble the G-1, G-3, and G-4 sites of the family of G proteins. In most cases alterations of the critical amino acids yield stable Nef proteins of parental phenotype. These results challenge the existing theories for the mechanism of Nef function. The results also identify two residues in the carboxyl half of the protein that are important for Nef function.

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Selected References

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  1. Ahmad N., Venkatesan S. Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science. 1988 Sep 16;241(4872):1481–1485. doi: 10.1126/science.3262235. [DOI] [PubMed] [Google Scholar]
  2. Alizon M., Wain-Hobson S., Montagnier L., Sonigo P. Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell. 1986 Jul 4;46(1):63–74. doi: 10.1016/0092-8674(86)90860-3. [DOI] [PubMed] [Google Scholar]
  3. Allan J. S., Coligan J. E., Lee T. H., McLane M. F., Kanki P. J., Groopman J. E., Essex M. A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. Science. 1985 Nov 15;230(4727):810–813. doi: 10.1126/science.2997921. [DOI] [PubMed] [Google Scholar]
  4. Bachelerie F., Alcami J., Hazan U., Israël N., Goud B., Arenzana-Seisdedos F., Virelizier J. L. Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity. J Virol. 1990 Jun;64(6):3059–3062. doi: 10.1128/jvi.64.6.3059-3062.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Backer J. M., Mendola C. E., Fairhurst J. L., Kovesdi I. The HIV-1 nef protein does not have guanine nucleotide binding, GTPase, or autophosphorylating activities. AIDS Res Hum Retroviruses. 1991 Dec;7(12):1015–1020. doi: 10.1089/aid.1991.7.1015. [DOI] [PubMed] [Google Scholar]
  6. Cheng-Mayer C., Iannello P., Shaw K., Luciw P. A., Levy J. A. Differential effects of nef on HIV replication: implications for viral pathogenesis in the host. Science. 1989 Dec 22;246(4937):1629–1632. doi: 10.1126/science.2531920. [DOI] [PubMed] [Google Scholar]
  7. Colombini S., Arya S. K., Reitz M. S., Jagodzinski L., Beaver B., Wong-Staal F. Structure of simian immunodeficiency virus regulatory genes. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4813–4817. doi: 10.1073/pnas.86.13.4813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cullen B. R. Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 1987;152:684–704. doi: 10.1016/0076-6879(87)52074-2. [DOI] [PubMed] [Google Scholar]
  9. Dayton A. I., Sodroski J. G., Rosen C. A., Goh W. C., Haseltine W. A. The trans-activator gene of the human T cell lymphotropic virus type III is required for replication. Cell. 1986 Mar 28;44(6):941–947. doi: 10.1016/0092-8674(86)90017-6. [DOI] [PubMed] [Google Scholar]
  10. Franchini G., Robert-Guroff M., Ghrayeb J., Chang N. T., Wong-Staal F. Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells. Virology. 1986 Dec;155(2):593–599. doi: 10.1016/0042-6822(86)90219-9. [DOI] [PubMed] [Google Scholar]
  11. Guy B., Kieny M. P., Riviere Y., Le Peuch C., Dott K., Girard M., Montagnier L., Lecocq J. P. HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature. 1987 Nov 19;330(6145):266–269. doi: 10.1038/330266a0. [DOI] [PubMed] [Google Scholar]
  12. Guy B., Rivière Y., Dott K., Regnault A., Kieny M. P. Mutational analysis of the HIV nef protein. Virology. 1990 Jun;176(2):413–425. doi: 10.1016/0042-6822(90)90011-f. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Kaminchik J., Bashan N., Itach A., Sarver N., Gorecki M., Panet A. Genetic characterization of human immunodeficiency virus type 1 nef gene products translated in vitro and expressed in mammalian cells. J Virol. 1991 Feb;65(2):583–588. doi: 10.1128/jvi.65.2.583-588.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaminchik J., Bashan N., Pinchasi D., Amit B., Sarver N., Johnston M. I., Fischer M., Yavin Z., Gorecki M., Panet A. Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product. J Virol. 1990 Jul;64(7):3447–3454. doi: 10.1128/jvi.64.7.3447-3454.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Laurent A. G., Hovanessian A. G., Rivière Y., Krust B., Regnault A., Montagnier L., Findeli A., Kieny M. P., Guy B. Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells. J Gen Virol. 1990 Oct;71(Pt 10):2273–2281. doi: 10.1099/0022-1317-71-10-2273. [DOI] [PubMed] [Google Scholar]
  19. Luciw P. A., Cheng-Mayer C., Levy J. A. Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1434–1438. doi: 10.1073/pnas.84.5.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matsuura Y., Maekawa M., Hattori S., Ikegami N., Hayashi A., Yamazaki S., Morita C., Takebe Y. Purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus. Virology. 1991 Oct;184(2):580–586. doi: 10.1016/0042-6822(91)90428-e. [DOI] [PubMed] [Google Scholar]
  21. Nebreda A. R., Bryan T., Segade F., Wingfield P., Venkatesan S., Santos E. Biochemical and biological comparison of HIV-1 NEF and ras gene products. Virology. 1991 Jul;183(1):151–159. doi: 10.1016/0042-6822(91)90128-x. [DOI] [PubMed] [Google Scholar]
  22. Niederman T. M., Thielan B. J., Ratner L. Human immunodeficiency virus type 1 negative factor is a transcriptional silencer. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1128–1132. doi: 10.1073/pnas.86.4.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Obaru K., DeClue J. E., Haseltine W. A. An amino-terminal amino acid affects the electrophoretic mobility of the HIV-1 nef protein. J Acquir Immune Defic Syndr. 1992;5(3):308–312. [PubMed] [Google Scholar]
  24. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  25. Rho H. M., Poiesz B., Ruscetti F. W., Gallo R. C. Characterization of the reverse transcriptase from a new retrovirus (HTLV) produced by a human cutaneous T-cell lymphoma cell line. Virology. 1981 Jul 15;112(1):355–360. doi: 10.1016/0042-6822(81)90642-5. [DOI] [PubMed] [Google Scholar]
  26. Rosen C. A., Sodroski J. G., Haseltine W. A. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell. 1985 Jul;41(3):813–823. doi: 10.1016/s0092-8674(85)80062-3. [DOI] [PubMed] [Google Scholar]
  27. Samuel K. P., Seth A., Konopka A., Lautenberger J. A., Papas T. S. The 3'-orf protein of human immunodeficiency virus shows structural homology with the phosphorylation domain of human interleukin-2 receptor and the ATP-binding site of the protein kinase family. FEBS Lett. 1987 Jun 22;218(1):81–86. doi: 10.1016/0014-5793(87)81023-2. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shibata R., Miura T., Hayami M., Ogawa K., Sakai H., Kiyomasu T., Ishimoto A., Adachi A. Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM). J Virol. 1990 Feb;64(2):742–747. doi: 10.1128/jvi.64.2.742-747.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Terwilliger E. F., Langhoff E., Gabuzda D., Zazopoulos E., Haseltine W. A. Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10971–10975. doi: 10.1073/pnas.88.23.10971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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