Journal of Molecular Modeling

, Volume 18, Issue 10, pp 4603–4613 | Cite as

Validation of a novel secretion modification region (SMR) of HIV-1 Nef using cohort sequence analysis and molecular modeling

  • Patrick E. Campbell
  • Olexandr Isayev
  • Syed A. Ali
  • William W. Roth
  • Ming-Bo Huang
  • Michael D. Powell
  • Jerzy Leszczynski
  • Vincent C. Bond
Original Paper

Abstract

The HIV-1 accessory protein Nef plays an active role in the pathogenesis of AIDS by its numerous cellular interactions that facilitate the release of virus particles. This 27 kDa protein is required for maintenance of the viral replication in HIV, and is also known to contribute to immune evasion, blocking of apoptosis in virus-infected cells and enhancement of virus infectivity. Nef has been shown to be secreted and is present on the surface of virus-infected cells. Recent studies from our laboratory have shown that the Nef protein is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (40–100 nm diam.) that do not contain virions. We have identified three amino-terminal domains of Nef as necessary for secretion: (i) the four arginine residues (17,19,21, 22) comprising the basic region; (ii) the phosphofurin acidic cluster sequence (PACS) composed of four glutamic acid residues (61–64); (iii) a previously unknown motif spanning amino acid residues 65–69 (VGFPV) which we named the secretion modification region (SMR). In this study, we have used population-based phylogeny data and sequence analysis to characterize the conservation of the Nef SMR domain that regulates vesicle secretion. We have performed in silico computational chemistry analysis involving molecular dynamic structure modeling of mutations in the SMR motif. Sequence analysis of Nef from HIV-1-infected patients, including slow progressors (SP), long term progressors (LTP) and long term non-progressors (LTNP) demonstrated 99 % conservation of the Nef SMR motif. Computational analysis including modeling of wild-type HIV-1 Nef and V66A Nef SMR mutant using structural homology and molecular dynamics of ligand-associated interactions indicated significant structural changes in the Nef mutant, thus supporting the importance of the SMR domain for mediating Nef vesicle secretion.

Figure

Novel secretion modification region (SMR) of HIV-1 Nef

Keywords

HIV-1 protein Homology modeling Molecular dynamics Structure-function relationship 

References

  1. 1.
    Smith SM (2006) The pathogenesis of HIV infection: stupid may not be so dumb after all. Retrovirol 3:60–65CrossRefGoogle Scholar
  2. 2.
    McIntyre LB, Geczy AF, Dyer WB, Learmont JC, Sullivan JS (1999) The Sydney Blood Bank Cohort: a case–control study using a transfused HIV-1 seronegative group. Ann Epidemiol 9:436–440CrossRefGoogle Scholar
  3. 3.
    Hanna Z, Priceputu E, Hu C, Vincent P, Jolicoeur P (2006) HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice. Virol 346:40–52. doi:10.1016/j.virol.2005.10.010 CrossRefGoogle Scholar
  4. 4.
    Priceputu E, Rodrigue I, Chrobak P, Poudrier J, Mak TW, Hanna Z, Hu C, Kay DG, Jolicoeur P (2005) The Nef-mediated AIDS-like disease of CD4C/human immunodeficiency virus transgenic mice is associated with increased Fas/FasL expression on T cells and T-cell death but is not prevented in Fas-, FasL-, tumor necrosis factor receptor 1-, or interleukin-1beta-converting enzyme-deficient or Bcl2-expressing transgenic mice. J Virol 79:6377–6391CrossRefGoogle Scholar
  5. 5.
    Simard MC, Chrobak P, Kay DG, Hanna Z, Jothy S, Jolicoeur P (2002) Expression of simian immunodeficiency virus nef in immune cells of transgenic mice leads to a severe AIDS-like disease. J Virol 76:3981–3995CrossRefGoogle Scholar
  6. 6.
    Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P (1998) Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95:163–175CrossRefGoogle Scholar
  7. 7.
    Klotman PE, Notkins AL (1996) Transgenic models of human immunodeficiency virus type-1. Curr Top Microbiol Immunol 206:197–222CrossRefGoogle Scholar
  8. 8.
    Fujii Y, Otake K, Tashiro M, Adachi A (1996) Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells. FEBS Lett 393:105–108CrossRefGoogle Scholar
  9. 9.
    Fujii Y, Otake K, Tashiro M, Adachi A (1996) Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells. FEBS Lett 393:93–96CrossRefGoogle Scholar
  10. 10.
    Okada H, Takei R, Tashiro M (1997) Nef protein of HIV-1 induces apoptotic cytolysis of murine lymphoid cells independently of CD95 (Fas) and its suppression by serine/threonine protein kinase inhibitors. FEBS Lett 417:61–64CrossRefGoogle Scholar
  11. 11.
    Okada H, Takei R, Tashiro M (1997) HIV-1 Nef protein-induced apoptotic cytolysis of a broad spectrum of uninfected human blood cells independently of CD95(Fas). FEBS Lett 414:603–606CrossRefGoogle Scholar
  12. 12.
    Roeth JF, Collins KL (2006) Human immunodeficiency virus type 1 Nef: adapting to intracellular trafficking pathways. Microbiol Mol Biol Rev 70:548–563CrossRefGoogle Scholar
  13. 13.
    Fujii Y, Otake K, Fujita Y, Yamamoto N, Nagai Y, Tashiro M, Adachi A (1996) Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface. FEBS Lett 395:257–261CrossRefGoogle Scholar
  14. 14.
    Zeigler ZR, Rosenfeld CS, Andrews DF III, Nemunaitis J, Raymond JM, Shadduck RK, Kramer RE, Gryn JF, Rintels PB, Besa EC, George JN (1996) Plasma von Willebrand Factor Antigen (vWF:AG) and thrombomodulin (TM) levels in Adult Thrombotic Thrombocytopenic Purpura/Hemolytic Uremic Syndromes (TTP/HUS) and bone marrow transplant-associated thrombotic microangiopathy (BMT-TM). Am J Hematol 53:213–220CrossRefGoogle Scholar
  15. 15.
    Varin A, Manna SK, Quivy V, Decrion AZ, Van Lint C, Herbein G, Aggarwal BB (2003) Exogenous Nef protein activates NF-kappa B, AP-1, and c-Jun N-terminal kinase and stimulates HIV transcription in promonocytic cells. Role in AIDS pathogenesis. J Biol Chem 278:2219–2227CrossRefGoogle Scholar
  16. 16.
    Huang MB, Jin LL, James CO, Khan M, Powell MD, Bond VC (2004) Characterization of Nef-CXCR4 interactions important for apoptosis induction. J Virol 78:11084–11096CrossRefGoogle Scholar
  17. 17.
    James CO, Huang M-B, Khan M, Garcia-Barrio M, Powell MD, Bond VC (2004) Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 78:3099–3109CrossRefGoogle Scholar
  18. 18.
    Calenda V, Graber P, Delamarter JF, Chermann JC (1994) Involvement of HIV nef protein in abnormal hematopoiesis in AIDS: in vitro study on bone marrow progenitor cells. Eur J Haematol 52:103–107CrossRefGoogle Scholar
  19. 19.
    Federico M, Percario Z, Olivetta E, Fiorucci G, Muratori C, Micheli A, Romeo G, Affabris E (2001) HIV-1 Nef activates STAT1 in human monocytes/macrophages through the release of soluble factors. Blood 98:2752–2761CrossRefGoogle Scholar
  20. 20.
    Percario Z, Olivetta E, Fiorucci G, Mangino G, Peretti S, Romeo G, Affabris E, Federico M (2003) Human immunodeficiency virus type 1 (HIV-1) Nef activates STAT3 in primary human monocyte/macrophages through the release of soluble factors: involvement of Nef domains interacting with the cell endocytotic machinery. J Leukocyte Biol 74:821–832CrossRefGoogle Scholar
  21. 21.
    Campbell TD, Khan M, Huang M-B, Bond VC, Powell MD (2008) HIV-1 Nef protein is secreted into vesicles that can fuse with target cells and virions. Ethn Dis 18:S2–S9Google Scholar
  22. 22.
    Ali SA, Huang M-B, Campbell PE, Roth WW, Campbell T, Khan M, Newman G, Villinger F, Powell MD, Bond VC (2010) Genetic characterization of HIV type 1 Nef-induced vesicle secretion. AIDS Res Hum Retroviruses 26:173–192CrossRefGoogle Scholar
  23. 23.
    Leitner T, Foley B, Hahn B, Marx P, McCutchan F, Mellors J, Wolinsky S, Korber B, (2007) HIV Sequence Compendium 2006/2007. Los Alamos National Laboratory, NM 07–Google Scholar
  24. 24.
    van Marle G, Henry S, Todoruk T, Sullivan A, Silva C, Rourke SB, Holden J, McArthur JC, Gill MJ, Power C (2004) Human immunodeficiency virus type 1 Nef protein mediates neural cell death: a neurotoxic role for IP-10. Virol 329:302–318CrossRefGoogle Scholar
  25. 25.
    Rhodes DI, Ashton L, Solomon A, Carr A, Cooper D, Kaldor J, Deacon N (2000) Characterization of three nef-defective human immunodeficiency virus type 1 strains associated with long-term nonprogression. Australian Long-Term Nonprogressor Study Group. J Virol 74:10581–10588CrossRefGoogle Scholar
  26. 26.
    Catucci M, Venturi G, Romano L, Valensin PE, Zazzi M (2000) Analysis of the HIV-1 nef gene in five intravenous drug users with long-term nonprogressive HIV-1 infection in Italy. J Med Virol 60:294–299CrossRefGoogle Scholar
  27. 27.
    Yamada T, Iwamoto A (2000) Comparison of proviral accessory genes between long-term nonprogressors and progressors of human immunodeficiency virus type 1 infection. Arch Virol 145:1021–1027CrossRefGoogle Scholar
  28. 28.
    Combet C, Jambon M, Deleage G, Geourjon C (2002) Geno3D: automatic comparative molecular modelling of protein. Bioinforma 18:213–214CrossRefGoogle Scholar
  29. 29.
    Case DA, Cheatham TE III, Darden T, Gohlke H, Luo R, Merz KM Jr, Onufriev A, Simmerling C, Wang B, Woods RJ (2005) The Amber biomolecular simulation programs. J Comput Chem 26:1668–1688CrossRefGoogle Scholar
  30. 30.
    Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65:712–725CrossRefGoogle Scholar
  31. 31.
    Price DJ, Brooks CL III (2004) A modified TIP3P water potential for simulation with Ewald summation. J Chem Phys 121:10096–10103CrossRefGoogle Scholar
  32. 32.
    Jorgensen WL, Tirado-Rives J (2005) Potential energy functions for atomic-level simulations of water and organic and biomolecular systems. Proc Natl Acad Sci USA 102:6665–6670CrossRefGoogle Scholar
  33. 33.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612CrossRefGoogle Scholar
  34. 34.
    Petsko GA, Dagmar R (2003) Protein structure and function. Wiley-Blackwell, LondonGoogle Scholar
  35. 35.
    Lovell SC, Davis IW, Arendall WB III, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by Calpha geometry: phi, psi and Cbeta deviation. Proteins 50:437–450CrossRefGoogle Scholar
  36. 36.
    Raymond AD, Campbell-Sims TC, Khan M, Lang M, Huang MB, Bond VC, Powell MD (2011) HIV type 1 Nef is released from infected cells in CD45+ microvesicles and is present in the plasma of HIV-infected individuals. AIDS Res Hum Retroviruses 27:167–178. doi:10.1089/aid.2009.0170 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Patrick E. Campbell
    • 1
    • 4
  • Olexandr Isayev
    • 2
  • Syed A. Ali
    • 1
    • 5
  • William W. Roth
    • 1
  • Ming-Bo Huang
    • 1
  • Michael D. Powell
    • 1
  • Jerzy Leszczynski
    • 3
  • Vincent C. Bond
    • 1
  1. 1.Department of Microbiology, Immunology and BiochemistryMorehouse School of MedicineAtlantaUSA
  2. 2.Department of ChemistryCase Western Reserve UniversityClevelandUSA
  3. 3.Department of ChemistryJackson State UniversityJacksonUSA
  4. 4.University of the West IndiesSt. AugustineTrinidad and Tobago
  5. 5.Universiti Sains MalaysiaPulau PinangMalaysia

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