Nef is an important player for viral infectivity and AIDS progression, but the mechanisms involved are not completely understood. It was previously demonstrated that Nef interacts with GagPol through p6*-Protease region. Because p6* and Protease are involved in processing, we explored the effect of Nef on viral Protease activity and virion assembly. Using in vitro assays, we observed that Nef is highly capable of inhibiting Protease activity. The IC50 for nef-deficient viruses in drug susceptibility assays were 1.7- to 3.5-fold higher than the wild-type counterpart varying with the type of the Protease inhibitor used. Indicating that, in the absence of Nef, Protease is less sensitive to Protease inhibitors. We compared the protein content between wild-type and nef-deficient mature viral particles by gradient sedimentation and observed up to 2.7-fold reduction in the Integrase levels in nef-deficient mature particles. This difference in levels of Integrase correlated with the difference in infectivity levels of wild type and nef-deficient viral progeny. In addition, an overall decrease in the production of mature particles was detected in nef-deficient viruses. Collectively, our data support the hypothesis that the decreased infectivity typical of nef-deficient viruses is due to an abnormal function of the viral Protease, which is in turn associated with less mature particles being produced and the loss of Integrase content in these particles, and these results may characterize Nef as a regulator of viral Protease activity.
References
[1]
Schindler M, Münch J, Brenner M, Stahl-Hennig C, Skowronski J, et al. (2004) Comprehensive analysis of nef functions selected in simian immunodeficiency virus-infected macaques. J Virol 78: 10588–10597. doi: 10.1128/jvi.78.19.10588-10597.2004
[2]
Fackler OT, Moris A, Tibroni N, Giese SI, Glass B, et al. (2006) Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology 351: 322–339. doi: 10.1016/j.virol.2006.03.044
[3]
Dyer WB, Ogg GS, Demoitie MA, Jin X, Geczy AF, et al. (1999) Strong human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte activity in Sydney Blood Bank Cohort patients infected with nef-defective HIV type 1. J Virol 73: 436–443.
[4]
Garcia JV, Miller AD (1991) Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350: 508–511. doi: 10.1038/350508a0
[5]
Janvier K, Craig H, Le Gall S, Benarous R, Guatelli J, et al. (2001) Nef-induced CD4 downregulation: a diacidic sequence in human immunodeficiency virus type 1 Nef does not function as a protein sorting motif through direct binding to beta-COP. J Virol 75: 3971–3976. doi: 10.1128/jvi.75.8.3971-3976.2001
[6]
Geyer M, Fackler OT, Peterlin BM (2001) Structure–function relationships in HIV-1 Nef. EMBO Rep 2: 580–585. doi: 10.1093/embo-reports/kve141
[7]
Lubben NB, Sahlender DA, Motley AM, Lehner PJ, Benaroch P, et al. (2007) HIV-1 Nef-induced down-regulation of MHC class I requires AP-1 and clathrin but not PACS-1 and is impeded by AP-2. Mol Biol Cell 18: 3351–3365. doi: 10.1091/mbc.e07-03-0218
[8]
Costa LJ, Chen N, Lopes A, Aguiar RS, Tanuri A, et al. (2006) Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate egress of HIV-1. Retrovirology 3: 33.
[9]
Aiken C, Trono D (1995) Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J Virol 69: 5048–5056.
[10]
Schwartz O, Maréchal V, Danos O, Heard JM (1995) Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell. J Virol 69: 4053–4059.
[11]
Khan M, Garcia-Barrio M, Powell MD (2001) Restoration of wild-type infectivity to human immunodeficiency virus type 1 strains lacking nef by intravirion reverse transcription. J Virol 75: 12081–12087. doi: 10.1128/jvi.75.24.12081-12087.2001
[12]
Pizzato M, Helander A, Popova E, Calistri A, Zamborlini A, et al. (2007) Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef. Proc Natl Acad Sci U S A 104: 6812–6817. doi: 10.1073/pnas.0607622104
[13]
Stolp B, Abraham L, Rudolph JM, Fackler OT (2010) Lentiviral Nef proteins utilize PAK2-mediated deregulation of cofilin as a general strategy to interfere with actin remodeling. J Virol 84: 3935–3948. doi: 10.1128/jvi.02467-09
[14]
Miller MD, Warmerdam MT, Gaston I, Greene WC, Feinberg MB (1994) The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. J Exp Med 179: 101–113. doi: 10.1084/jem.179.1.101
[15]
Goldsmith MA, Warmerdam MT, Atchison RE, Miller MD, Greene WC (1995) Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef. J Virol 69: 4112–4121.
[16]
Chowers MY, Pandori MW, Spina CA, Richman DD, Guatelli JC (1995) The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation. Virology 212: 451–457. doi: 10.1006/viro.1995.1502
[17]
de Marco A, Heuser AM, Glass B, Kr？usslich HG, Müller B, et al. (2012) Role of the SP2 domain and its proteolytic cleavage in HIV-1 structural maturation and infectivity. J Virol 86: 13708–13716. doi: 10.1128/jvi.01704-12
[18]
Chiu HC, Wang FD, Chen YM, Wang CT (2006) Effects of human immunodeficiency virus type 1 transframe protein p6* mutations on viral protease-mediated Gag processing. J Gen Virol 87: 2041–2046. doi: 10.1099/vir.0.81601-0
[19]
Adamson CS, Salzwedel K, Freed EO (2009) Virus maturation as a new HIV-1 therapeutic target. Expert Opin Ther Targets 13: 895–908. doi: 10.1517/14728220903039714
[20]
Kaplan AH, Zack JA, Knigge M, Paul DA, Kempf DJ, et al. (1993) Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles. J Virol 67: 4050–4055.
[21]
Navia MA, McKeever BM (1990) A role for the aspartyl protease from the human immunodeficiency virus type 1 (HIV-1) in the orchestration of virus assembly. Ann N Y Acad Sci 616: 73–85. doi: 10.1111/j.1749-6632.1990.tb17829.x
[22]
Pettit SC, Everitt LE, Choudhury S, Dunn BM, Kaplan AH (2004) Initial cleavage of the human immunodeficiency virus type 1 GagPol precursor by its activated protease occurs by an intramolecular mechanism. J Virol 78: 8477–8485. doi: 10.1128/jvi.78.16.8477-8485.2004
[23]
Costa LJ, Zheng YH, Sabotic J, Mak J, Fackler OT, et al. (2004) Nef binds p6* in GagPol during replication of human immunodeficiency virus type 1. J Virol 78: 5311–5323. doi: 10.1128/jvi.78.10.5311-5323.2004
[24]
Ludwig C, Leiherer A, Wagner R (2008) Importance of protease cleavage sites within and flanking human immunodeficiency virus type 1 transframe protein p6* for spatiotemporal regulation of protease activation. J Virol 82: 4573–4584. doi: 10.1128/jvi.02353-07
[25]
Partin K, Zybarth G, Ehrlich L, DeCrombrugghe M, Wimmer E, et al. (1991) Deletion of sequences upstream of the proteinase improves the proteolytic processing of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 88: 4776–4780. doi: 10.1073/pnas.88.11.4776
[26]
Grandgenett DP, Goodarzi G (1994) Folding of the multidomain human immunodeficiency virus type-I integrase. Protein Sci 3: 888–897. doi: 10.1002/pro.5560030604
[27]
Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, et al. (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59: 284–291.
[28]
Schindler M, Würfl S, Benaroch P, Greenough TC, Daniels R, et al. (2003) Down-modulation of mature major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles. J Virol 77: 10548–10556. doi: 10.1128/jvi.77.19.10548-10556.2003
[29]
Schindler M, Münch J, Kutsch O, Li H, Santiago ML, et al. (2006) Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell 125: 1055–1067. doi: 10.1016/j.cell.2006.04.033
[30]
Kikukawa R, Koyanagi Y, Harada S, Kobayashi N, Hatanaka M, et al. (1986) Differential susceptibility to the acquired immunodeficiency syndrome retrovirus in cloned cells of human leukemic T-cell line Molt-4. J Virol 57: 1159–1162.
[31]
Dettenhofer M, Yu XF (1999) Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions. J Virol 73: 1460–1467.
[32]
Sanches M, Martins NH, Calazans A, Brindeiro ReM, Tanuri A, et al. (2004) Crystallization of a non-B and a B mutant HIV protease. Acta Crystallogr D Biol Crystallogr 60: 1625–1627. doi: 10.1107/s0907444904015276
[33]
Kotler M, Simm M, Zhao YS, Sova P, Chao W, et al. (1997) Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro. J Virol 71: 5774–5781.
[34]
Miller MD, Warmerdam MT, Ferrell SS, Benitez R, Greene WC (1997) Intravirion generation of the C-terminal core domain of HIV-1 Nef by the HIV-1 protease is insufficient to enhance viral infectivity. Virology 234: 215–225. doi: 10.1006/viro.1997.8641
[35]
Pandori M, Craig H, Moutouh L, Corbeil J, Guatelli J (1998) Virological importance of the protease-cleavage site in human immunodeficiency virus type 1 Nef is independent of both intravirion processing and CD4 down-regulation. Virology 251: 302–316. doi: 10.1006/viro.1998.9407
[36]
Pettit SC, Lindquist JN, Kaplan AH, Swanstrom R (2005) Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates. Retrovirology 2: 66.
[37]
Wyckoff EE, Lloyd RE, Ehrenfeld E (1992) Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity. J Virol 66: 2943–2951.
[38]
Mahalingam B, Louis JM, Hung J, Harrison RW, Weber IT (2001) Structural implications of drug-resistant mutants of HIV-1 protease: high-resolution crystal structures of the mutant protease/substrate analogue complexes. Proteins 43: 455–464. doi: 10.1002/prot.1057
[39]
Ishima R, Gong Q, Tie Y, Weber IT, Louis JM (2010) Highly conserved glycine 86 and arginine 87 residues contribute differently to the structure and activity of the mature HIV-1 protease. Proteins 78: 1015–1025. doi: 10.1002/prot.22625
[40]
Westerman KA, Ao Z, Cohen EA, Leboulch P (2007) Design of a trans protease lentiviral packaging system that produces high titer virus. Retrovirology 4: 96. doi: 10.1186/1742-4690-4-96
[41]
Garcia-Perez J, Sanchez-Palomino S, Perez-Olmeda M, Fernandez B, Alcami J (2007) A new strategy based on recombinant viruses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1. J Med Virol 79: 127–137. doi: 10.1002/jmv.20770
[42]
Covens K, Dekeersmaeker N, Schrooten Y, Weber J, Schols D, et al. (2009) Novel recombinant virus assay for measuring susceptibility of human immunodeficiency virus type 1 group M subtypes to clinically approved drugs. J Clin Microbiol 47: 2232–2242. doi: 10.1128/jcm.01739-08
[43]
Kaplan AH, Manchester M, Swanstrom R (1994) The activity of the protease of human immunodeficiency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency. J Virol 68: 6782–6786.
[44]
Adamson CS, Freed EO (2007) Human immunodeficiency virus type 1 assembly, release, and maturation. Adv Pharmacol 55: 347–387. doi: 10.1016/s1054-3589(07)55010-6
[45]
Ludwig M, Kümmel C, Schroeder-Printzen I, Ringert RH, Weidner W (1998) Evaluation of seminal plasma parameters in patients with chronic prostatitis or leukocytospermia. Andrologia 30 Suppl 141–47. doi: 10.1111/j.1439-0272.1998.tb02825.x
[46]
Gatlin J, Arrigo SJ, Schmidt MG (1998) HIV-1 protease regulation: the role of the major homology region and adjacent C-terminal capsid sequences. J Biomed Sci 5: 305–308. doi: 10.1159/000025344
[47]
Freund J, Kellner R, Konvalinka J, Wolber V, Kr？usslich HG, et al. (1994) A possible regulation of negative factor (Nef) activity of human immunodeficiency virus type 1 by the viral protease. Eur J Biochem 223: 589–593. doi: 10.1111/j.1432-1033.1994.tb19029.x
[48]
Freund J, Kellner R, Houthaeve T, Kalbitzer HR (1994) Stability and proteolytic domains of Nef protein from human immunodeficiency virus (HIV) type 1. Eur J Biochem 221: 811–819. doi: 10.1111/j.1432-1033.1994.tb18795.x
[49]
Gaedigk-Nitschko K, Sch？n A, Wachinger G, Erfle V, Kohleisen B (1995) Cleavage of recombinant and cell derived human immunodeficiency virus 1 (HIV-1) Nef protein by HIV-1 protease. FEBS Lett 357: 275–278. doi: 10.1016/0014-5793(94)01370-g
[50]
Schorr J, Kellner R, Fackler O, Freund J, Konvalinka J, et al. (1996) Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases. J Virol 70: 9051–9054.
[51]
Malbec M, Sourisseau M, Guivel-Benhassine F, Porrot F, Blanchet F, et al. (2013) HIV-1 Nef promotes the localization of Gag to the cell membrane and facilitates viral cell-to-cell transfer. Retrovirology 10: 80. doi: 10.1186/1742-4690-10-80
[52]
Pettit SC, Clemente JC, Jeung JA, Dunn BM, Kaplan AH (2005) Ordered processing of the human immunodeficiency virus type 1 GagPol precursor is influenced by the context of the embedded viral protease. J Virol 79: 10601–10607. doi: 10.1128/jvi.79.16.10601-10607.2005
[53]
Karacostas V, Wolffe EJ, Nagashima K, Gonda MA, Moss B (1993) Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. Virology 193: 661–671. doi: 10.1006/viro.1993.1174
[54]
Luukkonen BG, Feny？ EM, Schwartz S (1995) Overexpression of human immunodeficiency virus type 1 protease increases intracellular cleavage of Gag and reduces virus infectivity. Virology 206: 854–865. doi: 10.1006/viro.1995.1008
[55]
Forshey BM, Aiken C (2003) Disassembly of human immunodeficiency virus type 1 cores in vitro reveals association of Nef with the subviral ribonucleoprotein complex. J Virol 77: 4409–4414. doi: 10.1128/jvi.77.7.4409-4414.2003
[56]
Wu X, Liu H, Xiao H, Conway JA, Hehl E, et al. (1999) Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex. J Virol 73: 2126–2135.
[57]
Dobard CW, Briones MS, Chow SA (2007) Molecular mechanisms by which human immunodeficiency virus type 1 integrase stimulates the early steps of reverse transcription. J Virol 81: 10037–10046. doi: 10.1128/jvi.00519-07
