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. 1997 Jun;71(6):4372–4377. doi: 10.1128/jvi.71.6.4372-4377.1997

The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix.

S Swingler 1, P Gallay 1, D Camaur 1, J Song 1, A Abo 1, D Trono 1
PMCID: PMC191654  PMID: 9151826

Abstract

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.

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Selected References

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  1. Aiken C., Konner J., Landau N. R., Lenburg M. E., Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell. 1994 Mar 11;76(5):853–864. doi: 10.1016/0092-8674(94)90360-3. [DOI] [PubMed] [Google Scholar]
  2. Aiken C., Krause L., Chen Y. L., Trono D. Mutational analysis of HIV-1 Nef: identification of two mutants that are temperature-sensitive for CD4 downregulation. Virology. 1996 Mar 1;217(1):293–300. doi: 10.1006/viro.1996.0116. [DOI] [PubMed] [Google Scholar]
  3. Aiken C., Trono D. Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J Virol. 1995 Aug;69(8):5048–5056. doi: 10.1128/jvi.69.8.5048-5056.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baur A. S., Sawai E. T., Dazin P., Fantl W. J., Cheng-Mayer C., Peterlin B. M. HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. Immunity. 1994 Aug;1(5):373–384. doi: 10.1016/1074-7613(94)90068-x. [DOI] [PubMed] [Google Scholar]
  5. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  6. Bukrinskaya A. G., Ghorpade A., Heinzinger N. K., Smithgall T. E., Lewis R. E., Stevenson M. Phosphorylation-dependent human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):367–371. doi: 10.1073/pnas.93.1.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bukrinsky M. I., Haggerty S., Dempsey M. P., Sharova N., Adzhubel A., Spitz L., Lewis P., Goldfarb D., Emerman M., Stevenson M. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature. 1993 Oct 14;365(6447):666–669. doi: 10.1038/365666a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burnette B., Yu G., Felsted R. L. Phosphorylation of HIV-1 gag proteins by protein kinase C. J Biol Chem. 1993 Apr 25;268(12):8698–8703. [PubMed] [Google Scholar]
  9. Chowers M. Y., Pandori M. W., Spina C. A., Richman D. D., Guatelli J. C. The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation. Virology. 1995 Oct 1;212(2):451–457. doi: 10.1006/viro.1995.1502. [DOI] [PubMed] [Google Scholar]
  10. Chowers M. Y., Spina C. A., Kwoh T. J., Fitch N. J., Richman D. D., Guatelli J. C. Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. J Virol. 1994 May;68(5):2906–2914. doi: 10.1128/jvi.68.5.2906-2914.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deacon N. J., Tsykin A., Solomon A., Smith K., Ludford-Menting M., Hooker D. J., McPhee D. A., Greenway A. L., Ellett A., Chatfield C. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science. 1995 Nov 10;270(5238):988–991. doi: 10.1126/science.270.5238.988. [DOI] [PubMed] [Google Scholar]
  12. Dorfman T., Mammano F., Haseltine W. A., Göttlinger H. G. Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J Virol. 1994 Mar;68(3):1689–1696. doi: 10.1128/jvi.68.3.1689-1696.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Freed E. O., Martin M. A. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J Virol. 1995 Mar;69(3):1984–1989. doi: 10.1128/jvi.69.3.1984-1989.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Freed E. O., Orenstein J. M., Buckler-White A. J., Martin M. A. Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J Virol. 1994 Aug;68(8):5311–5320. doi: 10.1128/jvi.68.8.5311-5320.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fäcke M., Janetzko A., Shoeman R. L., Kräusslich H. G. A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum. J Virol. 1993 Aug;67(8):4972–4980. doi: 10.1128/jvi.67.8.4972-4980.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gallay P., Stitt V., Mundy C., Oettinger M., Trono D. Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import. J Virol. 1996 Feb;70(2):1027–1032. doi: 10.1128/jvi.70.2.1027-1032.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gallay P., Swingler S., Aiken C., Trono D. HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell. 1995 Feb 10;80(3):379–388. doi: 10.1016/0092-8674(95)90488-3. [DOI] [PubMed] [Google Scholar]
  19. Gallay P., Swingler S., Song J., Bushman F., Trono D. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell. 1995 Nov 17;83(4):569–576. doi: 10.1016/0092-8674(95)90097-7. [DOI] [PubMed] [Google Scholar]
  20. Gallina A., Mantoan G., Rindi G., Milanesi G. Influence of MA internal sequences, but not of the myristylated N-terminus sequence, on the budding site of HIV-1 Gag protein. Biochem Biophys Res Commun. 1994 Nov 15;204(3):1031–1038. doi: 10.1006/bbrc.1994.2566. [DOI] [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. 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]
  23. Kaminchik J., Margalit R., Yaish S., Drummer H., Amit B., Sarver N., Gorecki M., Panet A. Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix. AIDS Res Hum Retroviruses. 1994 Aug;10(8):1003–1010. doi: 10.1089/aid.1994.10.1003. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Kim S. Y., Byrn R., Groopman J., Baltimore D. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression. J Virol. 1989 Sep;63(9):3708–3713. doi: 10.1128/jvi.63.9.3708-3713.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kirchhoff F., Greenough T. C., Brettler D. B., Sullivan J. L., Desrosiers R. C. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med. 1995 Jan 26;332(4):228–232. doi: 10.1056/NEJM199501263320405. [DOI] [PubMed] [Google Scholar]
  27. Klotman M. E., Kim S., Buchbinder A., DeRossi A., Baltimore D., Wong-Staal F. Kinetics of expression of multiply spliced RNA in early human immunodeficiency virus type 1 infection of lymphocytes and monocytes. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):5011–5015. doi: 10.1073/pnas.88.11.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lu X., Wu X., Plemenitas A., Yu H., Sawai E. T., Abo A., Peterlin B. M. CDC42 and Rac1 are implicated in the activation of the Nef-associated kinase and replication of HIV-1. Curr Biol. 1996 Dec 1;6(12):1677–1684. doi: 10.1016/s0960-9822(02)70792-6. [DOI] [PubMed] [Google Scholar]
  29. Mammano F., Kondo E., Sodroski J., Bukovsky A., Göttlinger H. G. Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains. J Virol. 1995 Jun;69(6):3824–3830. doi: 10.1128/jvi.69.6.3824-3830.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mangasarian A., Foti M., Aiken C., Chin D., Carpentier J. L., Trono D. The HIV-1 Nef protein acts as a connector with sorting pathways in the Golgi and at the plasma membrane. Immunity. 1997 Jan;6(1):67–77. doi: 10.1016/s1074-7613(00)80243-5. [DOI] [PubMed] [Google Scholar]
  31. Martin G. A., Bollag G., McCormick F., Abo A. A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20. EMBO J. 1995 May 1;14(9):1970–1978. doi: 10.1002/j.1460-2075.1995.tb07189.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miller M. D., Warmerdam M. T., Gaston I., Greene W. C., Feinberg M. B. The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. J Exp Med. 1994 Jan 1;179(1):101–113. doi: 10.1084/jem.179.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Niederman T. M., Hastings W. R., Ratner L. Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix. Virology. 1993 Nov;197(1):420–425. doi: 10.1006/viro.1993.1605. [DOI] [PubMed] [Google Scholar]
  35. Nunn M. F., Marsh J. W. Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family. J Virol. 1996 Sep;70(9):6157–6161. doi: 10.1128/jvi.70.9.6157-6161.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rhee S. S., Marsh J. W. Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4. J Virol. 1994 Aug;68(8):5156–5163. doi: 10.1128/jvi.68.8.5156-5163.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saksela K., Cheng G., Baltimore D. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 1995 Feb 1;14(3):484–491. doi: 10.1002/j.1460-2075.1995.tb07024.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sawai E. T., Baur A. S., Peterlin B. M., Levy J. A., Cheng-Mayer C. A conserved domain and membrane targeting of Nef from HIV and SIV are required for association with a cellular serine kinase activity. J Biol Chem. 1995 Jun 23;270(25):15307–15314. doi: 10.1074/jbc.270.25.15307. [DOI] [PubMed] [Google Scholar]
  39. Sawai E. T., Baur A., Struble H., Peterlin B. M., Levy J. A., Cheng-Mayer C. Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1539–1543. doi: 10.1073/pnas.91.4.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sawai E. T., Khan I. H., Montbriand P. M., Peterlin B. M., Cheng-Mayer C., Luciw P. A. Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques. Curr Biol. 1996 Nov 1;6(11):1519–1527. doi: 10.1016/s0960-9822(96)00757-9. [DOI] [PubMed] [Google Scholar]
  41. Schwartz O., Dautry-Varsat A., Goud B., Maréchal V., Subtil A., Heard J. M., Danos O. Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes. J Virol. 1995 Jan;69(1):528–533. doi: 10.1128/jvi.69.1.528-533.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schwartz O., Maréchal V., Danos O., Heard J. M. Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell. J Virol. 1995 Jul;69(7):4053–4059. doi: 10.1128/jvi.69.7.4053-4059.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schwartz O., Maréchal V., Le Gall S., Lemonnier F., Heard J. M. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med. 1996 Mar;2(3):338–342. doi: 10.1038/nm0396-338. [DOI] [PubMed] [Google Scholar]
  44. Skowronski J., Parks D., Mariani R. Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO J. 1993 Feb;12(2):703–713. doi: 10.1002/j.1460-2075.1993.tb05704.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Spina C. A., Kwoh T. J., Chowers M. Y., Guatelli J. C., Richman D. D. The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. J Exp Med. 1994 Jan 1;179(1):115–123. doi: 10.1084/jem.179.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Subbramanian R. A., Cohen E. A. Molecular biology of the human immunodeficiency virus accessory proteins. J Virol. 1994 Nov;68(11):6831–6835. doi: 10.1128/jvi.68.11.6831-6835.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Trono D., Feinberg M. B., Baltimore D. HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell. 1989 Oct 6;59(1):113–120. doi: 10.1016/0092-8674(89)90874-x. [DOI] [PubMed] [Google Scholar]
  48. Trono D. HIV accessory proteins: leading roles for the supporting cast. Cell. 1995 Jul 28;82(2):189–192. doi: 10.1016/0092-8674(95)90306-2. [DOI] [PubMed] [Google Scholar]
  49. Veronese F. D., Copeland T. D., Oroszlan S., Gallo R. C., Sarngadharan M. G. Biochemical and immunological analysis of human immunodeficiency virus gag gene products p17 and p24. J Virol. 1988 Mar;62(3):795–801. doi: 10.1128/jvi.62.3.795-801.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yu G., Shen F. S., Sturch S., Aquino A., Glazer R. I., Felsted R. L. Regulation of HIV-1 gag protein subcellular targeting by protein kinase C. J Biol Chem. 1995 Mar 3;270(9):4792–4796. doi: 10.1074/jbc.270.9.4792. [DOI] [PubMed] [Google Scholar]
  51. Yu X., Yu Q. C., Lee T. H., Essex M. The C terminus of human immunodeficiency virus type 1 matrix protein is involved in early steps of the virus life cycle. J Virol. 1992 Sep;66(9):5667–5670. doi: 10.1128/jvi.66.9.5667-5670.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yu X., Yuan X., Matsuda Z., Lee T. H., Essex M. The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions. J Virol. 1992 Aug;66(8):4966–4971. doi: 10.1128/jvi.66.8.4966-4971.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yuan X., Yu X., Lee T. H., Essex M. Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor. J Virol. 1993 Nov;67(11):6387–6394. doi: 10.1128/jvi.67.11.6387-6394.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhou W., Parent L. J., Wills J. W., Resh M. D. Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol. 1994 Apr;68(4):2556–2569. doi: 10.1128/jvi.68.4.2556-2569.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. von Schwedler U., Kornbluth R. S., Trono D. The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6992–6996. doi: 10.1073/pnas.91.15.6992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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