Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1993 Aug;67(8):4639–4650. doi: 10.1128/jvi.67.8.4639-4650.1993

Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo.

D C Shugars 1, M S Smith 1, D H Glueck 1, P V Nantermet 1, F Seillier-Moiseiwitsch 1, R Swanstrom 1
PMCID: PMC237849  PMID: 8043040

Abstract

The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function.

Full text

PDF
4639

Images in this article

4648Image
on p.4648

Selected References

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

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Anderson S., Shugars D. C., Swanstrom R., Garcia J. V. Nef from primary isolates of human immunodeficiency virus type 1 suppresses surface CD4 expression in human and mouse T cells. J Virol. 1993 Aug;67(8):4923–4931. doi: 10.1128/jvi.67.8.4923-4931.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balfe P., Simmonds P., Ludlam C. A., Bishop J. O., Brown A. J. Concurrent evolution of human immunodeficiency virus type 1 in patients infected from the same source: rate of sequence change and low frequency of inactivating mutations. J Virol. 1990 Dec;64(12):6221–6233. doi: 10.1128/jvi.64.12.6221-6233.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barker W. C., Dayhoff M. O. Viral src gene products are related to the catalytic chain of mammalian cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1982 May;79(9):2836–2839. doi: 10.1073/pnas.79.9.2836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blumberg B. M., Epstein L. G., Saito Y., Chen D., Sharer L. R., Anand R. Human immunodeficiency virus type 1 nef quasispecies in pathological tissue. J Virol. 1992 Sep;66(9):5256–5264. doi: 10.1128/jvi.66.9.5256-5264.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burch H. B., Nagy E. V., Lukes Y. G., Cai W. Y., Wartofsky L., Burman K. D. Nucleotide and amino acid homology between the human thyrotropin receptor and the HIV-1 Nef protein: identification and functional analysis. Biochem Biophys Res Commun. 1991 Nov 27;181(1):498–505. doi: 10.1016/s0006-291x(05)81447-0. [DOI] [PubMed] [Google Scholar]
  8. Burger H., Weiser B., Flaherty K., Gulla J., Nguyen P. N., Gibbs R. A. Evolution of human immunodeficiency virus type 1 nucleotide sequence diversity among close contacts. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11236–11240. doi: 10.1073/pnas.88.24.11236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cammarota G., Scheirle A., Takacs B., Doran D. M., Knorr R., Bannwarth W., Guardiola J., Sinigaglia F. Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules. Nature. 1992 Apr 30;356(6372):799–801. doi: 10.1038/356799a0. [DOI] [PubMed] [Google Scholar]
  10. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  11. Cullen B. R. Regulation of human immunodeficiency virus replication. Annu Rev Microbiol. 1991;45:219–250. doi: 10.1146/annurev.mi.45.100191.001251. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Delassus S., Cheynier R., Wain-Hobson S. Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro. J Virol. 1991 Jan;65(1):225–231. doi: 10.1128/jvi.65.1.225-231.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dever T. E., Glynias M. J., Merrick W. C. GTP-binding domain: three consensus sequence elements with distinct spacing. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1814–1818. doi: 10.1073/pnas.84.7.1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dillon P. J., Rosen C. A. A rapid method for the construction of synthetic genes using the polymerase chain reaction. Biotechniques. 1990 Sep;9(3):298–300. [PubMed] [Google Scholar]
  17. Eckert K. A., Kunkel T. A. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990 Jul 11;18(13):3739–3744. doi: 10.1093/nar/18.13.3739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fisher A. G., Ensoli B., Looney D., Rose A., Gallo R. C., Saag M. S., Shaw G. M., Hahn B. H., Wong-Staal F. Biologically diverse molecular variants within a single HIV-1 isolate. Nature. 1988 Aug 4;334(6181):444–447. doi: 10.1038/334444a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Garcia J. V., Alfano J., Miller A. D. The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4. J Virol. 1993 Mar;67(3):1511–1516. doi: 10.1128/jvi.67.3.1511-1516.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Garcia J. V., Miller A. D. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature. 1991 Apr 11;350(6318):508–511. doi: 10.1038/350508a0. [DOI] [PubMed] [Google Scholar]
  22. Gaynor R. Cellular transcription factors involved in the regulation of HIV-1 gene expression. AIDS. 1992 Apr;6(4):347–363. doi: 10.1097/00002030-199204000-00001. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Hahn B. H., Gonda M. A., Shaw G. M., Popovic M., Hoxie J. A., Gallo R. C., Wong-Staal F. Genomic diversity of the acquired immune deficiency syndrome virus HTLV-III: different viruses exhibit greatest divergence in their envelope genes. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4813–4817. doi: 10.1073/pnas.82.14.4813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Harris M., Hislop S., Patsilinacos P., Neil J. C. In vivo derived HIV-1 nef gene products are heterogeneous and lack detectable nucleotide binding activity. AIDS Res Hum Retroviruses. 1992 May;8(5):537–543. doi: 10.1089/aid.1992.8.537. [DOI] [PubMed] [Google Scholar]
  27. Hovanessian A. G. On the HIV nef gene product. Res Virol. 1992 Jan-Feb;143(1):31–34. doi: 10.1016/s0923-2516(06)80074-9. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Kaplan J. M., Mardon G., Bishop J. M., Varmus H. E. The first seven amino acids encoded by the v-src oncogene act as a myristylation signal: lysine 7 is a critical determinant. Mol Cell Biol. 1988 Jun;8(6):2435–2441. doi: 10.1128/mcb.8.6.2435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kennelly P. J., Krebs E. G. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem. 1991 Aug 25;266(24):15555–15558. [PubMed] [Google Scholar]
  31. 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]
  32. Kienzle N., Bachmann M., Müller W. E., Müller-Lantzsch N. Expression and cellular localization of the Nef protein from human immunodeficiency virus-1 in stably transfected B-cells. Arch Virol. 1992;124(1-2):123–132. doi: 10.1007/BF01314630. [DOI] [PubMed] [Google Scholar]
  33. König R., Huang L. Y., Germain R. N. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. Nature. 1992 Apr 30;356(6372):796–798. doi: 10.1038/356796a0. [DOI] [PubMed] [Google Scholar]
  34. LaRosa G. J., Davide J. P., Weinhold K., Waterbury J. A., Profy A. T., Lewis J. A., Langlois A. J., Dreesman G. R., Boswell R. N., Shadduck P. Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science. 1990 Aug 24;249(4971):932–935. doi: 10.1126/science.2392685. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Lochrie M. A., Simon M. I. G protein multiplicity in eukaryotic signal transduction systems. Biochemistry. 1988 Jul 12;27(14):4957–4965. doi: 10.1021/bi00414a001. [DOI] [PubMed] [Google Scholar]
  37. Luria S., Chambers I., Berg P. Expression of the type 1 human immunodeficiency virus Nef protein in T cells prevents antigen receptor-mediated induction of interleukin 2 mRNA. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5326–5330. doi: 10.1073/pnas.88.12.5326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Martins L. P., Chenciner N., Asjö B., Meyerhans A., Wain-Hobson S. Independent fluctuation of human immunodeficiency virus type 1 rev and gp41 quasispecies in vivo. J Virol. 1991 Aug;65(8):4502–4507. doi: 10.1128/jvi.65.8.4502-4507.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Maurer B., Flügel R. M. The 3'-orf protein of human immunodeficiency virus 2 shows sequence homology with the bel3 gene of the human spumaretrovirus. FEBS Lett. 1987 Oct 5;222(2):286–288. doi: 10.1016/0014-5793(87)80387-3. [DOI] [PubMed] [Google Scholar]
  40. McNearney T., Hornickova Z., Markham R., Birdwell A., Arens M., Saah A., Ratner L. Relationship of human immunodeficiency virus type 1 sequence heterogeneity to stage of disease. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10247–10251. doi: 10.1073/pnas.89.21.10247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Meyerhans A., Cheynier R., Albert J., Seth M., Kwok S., Sninsky J., Morfeldt-Månson L., Asjö B., Wain-Hobson S. Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell. 1989 Sep 8;58(5):901–910. doi: 10.1016/0092-8674(89)90942-2. [DOI] [PubMed] [Google Scholar]
  42. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  44. Munis J. R., Kornbluth R. S., Guatelli J. C., Richman D. D. Ordered appearance of human immunodeficiency virus type 1 nucleic acids following high multiplicity infection of macrophages. J Gen Virol. 1992 Aug;73(Pt 8):1899–1906. doi: 10.1099/0022-1317-73-8-1899. [DOI] [PubMed] [Google Scholar]
  45. Niederman T. M., Garcia J. V., Hastings W. R., Luria S., Ratner L. Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells. J Virol. 1992 Oct;66(10):6213–6219. doi: 10.1128/jvi.66.10.6213-6219.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
  47. Ratner L., Starcich B., Josephs S. F., Hahn B. H., Reddy E. P., Livak K. J., Petteway S. R., Jr, Pearson M. L., Haseltine W. A., Arya S. K. 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 Res. 1985 Nov 25;13(22):8219–8229. doi: 10.1093/nar/13.22.8219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Robert-Guroff M., Popovic M., Gartner S., Markham P., Gallo R. C., Reitz M. S. Structure and expression of tat-, rev-, and nef-specific transcripts of human immunodeficiency virus type 1 in infected lymphocytes and macrophages. J Virol. 1990 Jul;64(7):3391–3398. doi: 10.1128/jvi.64.7.3391-3398.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Saag M. S., Hahn B. H., Gibbons J., Li Y., Parks E. S., Parks W. P., Shaw G. M. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature. 1988 Aug 4;334(6181):440–444. doi: 10.1038/334440a0. [DOI] [PubMed] [Google Scholar]
  50. Samuel K. P., Hodge D. R., Chen Y. M., Papas T. S. Nef proteins of the human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV) are structurally similar to leucine zipper transcriptional activation factors. AIDS Res Hum Retroviruses. 1991 Aug;7(8):697–706. doi: 10.1089/aid.1991.7.697. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  54. Simmonds P., Balfe P., Peutherer J. F., Ludlam C. A., Bishop J. O., Brown A. J. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol. 1990 Feb;64(2):864–872. doi: 10.1128/jvi.64.2.864-872.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Staden R. Methods to define and locate patterns of motifs in sequences. Comput Appl Biosci. 1988 Mar;4(1):53–60. doi: 10.1093/bioinformatics/4.1.53. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Tsunetsugu-Yokota Y., Matsuda S., Maekawa M., Saito T., Takemori T., Takebe Y. Constitutive expression of the nef gene suppresses human immunodeficiency virus type 1 (HIV-1) replication in monocytic cell lines. Virology. 1992 Dec;191(2):960–963. doi: 10.1016/0042-6822(92)90272-q. [DOI] [PubMed] [Google Scholar]
  58. Vartanian J. P., Meyerhans A., Asjö B., Wain-Hobson S. Selection, recombination, and G----A hypermutation of human immunodeficiency virus type 1 genomes. J Virol. 1991 Apr;65(4):1779–1788. doi: 10.1128/jvi.65.4.1779-1788.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vega M. A., Guigó R., Smith T. F. Autoimmune response in AIDS. Nature. 1990 May 3;345(6270):26–26. doi: 10.1038/345026a0. [DOI] [PubMed] [Google Scholar]
  60. Werner T., Ferroni S., Saermark T., Brack-Werner R., Banati R. B., Mager R., Steinaa L., Kreutzberg G. W., Erfle V. HIV-1 Nef protein exhibits structural and functional similarity to scorpion peptides interacting with K+ channels. AIDS. 1991 Nov;5(11):1301–1308. doi: 10.1097/00002030-199111000-00003. [DOI] [PubMed] [Google Scholar]
  61. Yu G., Felsted R. L. Effect of myristoylation on p27 nef subcellular distribution and suppression of HIV-LTR transcription. Virology. 1992 Mar;187(1):46–55. doi: 10.1016/0042-6822(92)90293-x. [DOI] [PubMed] [Google Scholar]
  62. Zweig M., Samuel K. P., Showalter S. D., Bladen S. V., DuBois G. C., Lautenberger J. A., Hodge D. R., Papas T. S. Heterogeneity of Nef proteins in cells infected with human immunodeficiency virus type 1. Virology. 1990 Nov;179(1):504–507. doi: 10.1016/0042-6822(90)90325-l. [DOI] [PubMed] [Google Scholar]

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

RESOURCES