In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis

Retrovirology

J Victor Garcia¹  John L Foster¹  John F Krisko¹  Paul W Denton¹  Wei Zou¹  Richard L Watkins¹ 

[1] Division of Infectious Diseases, Center for AIDS Research, 2042 Genetic Medicine, University of North Carolina, Campus Box 7042, Chapel Hill, NC 27599-7042, USA

关键词: Mutation;    BLT humanized mice;    Pathogenesis;    Replication;    Nef;    HIV-1;   

Others  :  806717
DOI  :  10.1186/1742-4690-10-125

 received in 2013-08-16, accepted in 2013-10-23,  发布年份 2013

【 摘 要 】

Background

The HIV-1 accessory protein, Nef, is decisive for progression to AIDS. In vitro characterization of the protein has described many Nef activities of unknown in vivo significance including CD4 downregulation and a number of activities that depend on Nef interacting with host SH3 domain proteins. Here, we use the BLT humanized mouse model of HIV-1 infection to assess their impact on viral replication and pathogenesis and the selection pressure to restore these activities using enforced in vivo evolution.

Results

We followed the evolution of HIV-1_LAI (LAI) with a frame-shifted nef (LAINeffs) during infection of BLT mice. LAINeffs was rapidly replaced in blood by virus with short deletions in nef that restored the open reading frame (LAINeffs∆-1 and LAINeffs∆-13). Subsequently, LAINeffs∆-1 was often replaced by wild type LAI. Unexpectedly, LAINeffs∆-1 and LAINeffs∆-13 Nefs were specifically defective for CD4 downregulation activity. Viruses with these mutant nefs were used to infect BLT mice. LAINeffs∆-1 and LAINeffs∆-13 exhibited three-fold reduced viral replication (compared to LAI) and a 50% reduction of systemic CD4⁺ T cells (>90% for LAI) demonstrating the importance of CD4 downregulation. These results also demonstrate that functions other than CD4 downregulation enhanced viral replication and pathogenesis of LAINeffs∆-1 and LAINeffs∆-13 compared to LAINeffs. To gain insight into the nature of these activities, we constructed the double mutant P72A/P75A. Multiple Nef activities can be negated by mutating the SH3 domain binding site (P72Q73V74P75L76R77) to P72A/P75A and this mutation does not affect CD4 downregulation. Virus with nef mutated to P72A/P75A closely resembled the wild-type virus in vivo as viral replication and pathogenesis was not significantly altered. Unlike LAINeffs described above, the P72A/P75A mutation had a very weak tendency to revert to wild type sequence.

Conclusions

The in vivo phenotype of Nef is significantly dependent on CD4 downregulation but minimally on the numerous Nef activities that require an intact SH3 domain binding motif. These results suggest that CD4 downregulation plus one or more unknown Nef activities contribute to enhanced viral replication and pathogenesis and are suitable targets for anti-HIV therapy. Enforced evolution studies in BLT mice will greatly facilitate identification of these critical activities.

【 授权许可】

   
2013 Watkins et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

【 参考文献 】

• [1]Calugi G, Montella F, Favalli C, Benedetto A: Entire genome of a strain of human immunodeficiency virus type 1 with a deletion of nef that was recovered 20 years after primary infection: large pool of proviruses with deletions of env. J Virol 2006, 80:11892-11896.
• [2]Gorry PR, McPhee DA, Verity E, Dyer WB, Wesselingh SL, Learmont J, Sullivan JS, Roche M, Zaunders JJ, Gabuzda D, et al.: Pathogenicity and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology 2007, 4:66. BioMed Central Full Text
• [3]Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL, Desrosiers RC: Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med 1995, 332:228-232.
• [4]Kondo M, Shima T, Nishizawa M, Sudo K, Iwamuro S, Okabe T, Takebe Y, Imai M: Identification of attenuated variants of HIV-1 circulating recombinant form 01_AE that are associated with slow disease progression due to gross genetic alterations in the nef/long terminal repeat sequences. J Infect Dis 2005, 192:56-61.
• [5]Abraham L, Fackler OT: HIV-1 Nef: a multifaceted modulator of T cell receptor signaling. Cell Commun Signal 2012, 10:39. BioMed Central Full Text
• [6]Arhel NJ, Kirchhoff F: Implications of Nef: host cell interactions in viral persistence and progression to AIDS. Curr Top Microbiol Immunol 2009, 339:147-175.
• [7]Foster JL, Denial SJ, Temple BR, Garcia JV: Mechanisms of HIV-1 Nef function and intracellular signaling. J Neuroimmune Pharmacol 2011, 6:230-246.
• [8]Kirchhoff F: Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses. Cell Host Microbe 2010, 8:55-67.
• [9]Laguette N, Bregnard C, Benichou S, Basmaciogullari S: Human immunodeficiency virus (HIV) type-1, HIV-2 and simian immunodeficiency virus Nef proteins. Mol Aspects Med 2010, 31:418-433.
• [10]Kirchhoff F, Schindler M, Specht A, Arhel N, Munch J: Role of Nef in primate lentiviral immunopathogenesis. Cell Mol Life Sci 2008, 65:2621-2636.
• [11]Mwimanzi P, Markle TJ, Ogata Y, Martin E, Tokunaga M, Mahiti M, Kuang XT, Walker BD, Brockman MA, Brumme ZL, Ueno T: Dynamic range of Nef functions in chronic HIV-1 infection. Virology 2013, 439:74-80.
• [12]Heigele A, Schindler M, Gnanadurai CW, Leonard JA, Collins KL, Kirchhoff F: Down-modulation of CD8alphabeta is a fundamental activity of primate lentiviral Nef proteins. J Virol 2012, 86:36-48.
• [13]Hrecka K, Swigut T, Schindler M, Kirchhoff F, Skowronski J: Nef proteins from diverse groups of primate lentiviruses downmodulate CXCR4 to inhibit migration to the chemokine stromal derived factor 1. J Virol 2005, 79:10650-10659.
• [14]Kirchhoff F, Schindler M, Bailer N, Renkema GH, Saksela K, Knoop V, Muller-Trutwin MC, Santiago ML, Bibollet-Ruche F, Dittmar MT, et al.: Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors. J Virol 2004, 78:6864-6874.
• [15]Munch J, Rajan D, Schindler M, Specht A, Rucker E, Novembre FJ, Nerrienet E, Muller-Trutwin MC, Peeters M, Hahn BH, Kirchhoff F: Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses. J Virol 2007, 81:13852-13864.
• [16]Schindler M, Wurfl S, Benaroch P, Greenough TC, Daniels R, Easterbrook P, Brenner M, Munch J, Kirchhoff F: 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 2003, 77:10548-10556.
• [17]Specht A, DeGottardi MQ, Schindler M, Hahn B, Evans DT, Kirchhoff F: Selective downmodulation of HLA-A and -B by Nef alleles from different groups of primate lentiviruses. Virology 2008, 373:229-237.
• [18]Fackler OT, Moris A, Tibroni N, Giese SI, Glass B, Schwartz O, Krausslich HG: Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology 2006, 351:322-339.
• [19]Glushakova S, Munch J, Carl S, Greenough TC, Sullivan JL, Margolis L, Kirchhoff F: CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4(+) T-cell depletion in human lymphoid tissue ex vivo. J Virol 2001, 75:10113-10117.
• [20]Lundquist CA, Tobiume M, Zhou J, Unutmaz D, Aiken C: Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. J Virol 2002, 76:4625-4633.
• [21]Kuo LS, Baugh LL, Denial SJ, Watkins RL, Liu M, Garcia JV, Foster JL: Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix. Retrovirology 2012, 9:47. BioMed Central Full Text
• [22]Manninen A, Hiipakka M, Vihinen M, Lu W, Mayer BJ, Saksela K: SH3-domain binding function of HIV-1 Nef is required for association with a PAK-related kinase. Virology 1998, 250:273-282.
• [23]Olivieri KC, Mukerji J, Gabuzda D: Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2. Retrovirology 2011, 8:64. BioMed Central Full Text
• [24]O’Neill E, Kuo LS, Krisko JF, Tomchick DR, Garcia JV, Foster JL: Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef. J Virol 2006, 80:1311-1320.
• [25]Pizzato M, Helander A, Popova E, Calistri A, Zamborlini A, Palu G, Gottlinger HG: Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef. Proc Natl Acad Sci USA 2007, 104:6812-6817.
• [26]Baur AS, Sawai ET, Dazin P, Fantl WJ, Cheng-Mayer C, Peterlin BM: HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. Immunity 1994, 1:373-384.
• [27]Hanna Z, Weng X, Kay DG, Poudrier J, Lowell C, Jolicoeur P: The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck. J Virol 2001, 75:9378-9392.
• [28]Muthumani K, Choo AY, Hwang DS, Premkumar A, Dayes NS, Harris C, Green DR, Wadsworth SA, Siekierka JJ, Weiner DB: HIV-1 Nef-induced FasL induction and bystander killing requires p38 MAPK activation. Blood 2005, 106:2059-2068.
• [29]Muthumani K, Choo AY, Shedlock DJ, Laddy DJ, Sundaram SG, Hirao L, Wu L, Thieu KP, Chung CW, Lankaraman KM, et al.: Human immunodeficiency virus type 1 Nef induces programmed death 1 expression through a p38 mitogen-activated protein kinase-dependent mechanism. J Virol 2008, 82:11536-11544.
• [30]Stove V, Naessens E, Stove C, Swigut T, Plum J, Verhasselt B: Signaling but not trafficking function of HIV-1 protein Nef is essential for Nef-induced defects in human intrathymic T-cell development. Blood 2003, 102:2925-2932.
• [31]Trible RP, Emert-Sedlak L, Smithgall TE: HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction. J Biol Chem 2006, 281:27029-27038.
• [32]Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G: HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 2002, 111:853-866.
• [33]Greenberg ME, Iafrate AJ, Skowronski J: The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. Embo J 1998, 17:2777-2789.
• [34]Hung CH, Thomas L, Ruby CE, Atkins KM, Morris NP, Knight ZA, Scholz I, Barklis E, Weinberg AD, Shokat KM, Thomas G: HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I. Cell Host Microbe 2007, 1:121-133.
• [35]Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y: Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef. Nat Struct Mol Biol 2012, 19:701-706.
• [36]Xu W, Santini PA, Sullivan JS, He B, Shan M, Ball SC, Dyer WB, Ketas TJ, Chadburn A, Cohen-Gould L, et al.: HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits. Nat Immunol 2009, 10:1008-1017.
• [37]Goldsmith MA, Warmerdam MT, Atchison RE, Miller MD, Greene WC: Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef. J Virol 1995, 69:4112-4121.
• [38]Mangasarian A, Piguet V, Wang JK, Chen YL, Trono D: Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal alpha helix and proline repeat of Nef selectively regulate MHC-I trafficking. J Virol 1999, 73:1964-1973.
• [39]Brainard DM, Seung E, Frahm N, Cariappa A, Bailey CC, Hart WK, Shin HS, Brooks SF, Knight HL, Eichbaum Q, et al.: Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice. J Virol 2009, 83:7305-7321.
• [40]Denton PW, Estes JD, Sun Z, Othieno FA, Wei BL, Wege AK, Powell DA, Payne D, Haase AT, Garcia JV: Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice. PLoS Med 2008, 5:e16.
• [41]Dudek TE, No DC, Seung E, Vrbanac VD, Fadda L, Bhoumik P, Boutwell CL, Power KA, Gladden AD, Battis L, et al.: Rapid evolution of HIV-1 to functional CD8+ T cell responses in humanized BLT mice. Sci Transl Med 2012, 4:143ra198.
• [42]Long BR, Stoddart CA: Alpha interferon and HIV infection cause activation of human T cells in NSG-BLT mice. J Virol 2012, 86:3327-3336.
• [43]Murooka TT, Deruaz M, Marangoni F, Vrbanac VD, Seung E, von Andrian UH, Tager AM, Luster AD, Mempel TR: HIV-infected T cells are migratory vehicles for viral dissemination. Nature 2012, 490:283-287.
• [44]Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, Garcia JV: Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4 + CD8+ thymocytes. Retrovirology 2012, 9:44. BioMed Central Full Text
• [45]Arganaraz ER, Schindler M, Kirchhoff F, Cortes MJ, Lama J: Enhanced CD4 down-modulation by late stage HIV-1 nef alleles is associated with increased Env incorporation and viral replication. J Biol Chem 2003, 278:33912-33919.
• [46]Carl S, Greenough TC, Krumbiegel M, Greenberg M, Skowronski J, Sullivan JL, Kirchhoff F: Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS. J Virol 2001, 75:3657-3665.
• [47]Kirchhoff F, Easterbrook PJ, Douglas N, Troop M, Greenough TC, Weber J, Carl S, Sullivan JL, Daniels RS: Sequence variations in human immunodeficiency virus type 1 Nef are associated with different stages of disease. J Virol 1999, 73:5497-5508.
• [48]Lewis MJ, Balamurugan A, Ohno A, Kilpatrick S, Ng HL, Yang OO: Functional adaptation of Nef to the immune milieu of HIV-1 infection in vivo. J Immunol 2008, 180:4075-4081.
• [49]Michael NL, Chang G, d’Arcy LA, Tseng CJ, Birx DL, Sheppard HW: Functional characterization of human immunodeficiency virus type 1 nef genes in patients with divergent rates of disease progression. J Virol 1995, 69:6758-6769.
• [50]Chateau M, Swanson MD, Garcia JV: Inefficient vaginal transmission of tenofovir-resistant HIV-1. J Virol 2012, 87:1274-1277.
• [51]Krisko JF, Martinez-Torres F, Foster JL, Garcia JV: HIV restriction by APOBEC3 in humanized mice. PLoS Pathog 2013, 9:e1003242.
• [52]Garcia JV, Miller AD: Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 1991, 350:508-511.
• [53]Schwartz O, Marechal V, Le Gall S, Lemonnier F, Heard JM: Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med 1996, 2:338-342.
• [54]Arora VK, Molina RP, Foster JL, Blakemore JL, Chernoff J, Fredericksen BL, Garcia JV: Lentivirus Nef specifically activates Pak2. J Virol 2000, 74:11081-11087.
• [55]Sawai ET, Baur A, Struble H, Peterlin BM, Levy JA, Cheng-Mayer C: Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proc Natl Acad Sci USA 1994, 91:1539-1543.
• [56]Lundquist CA, Zhou J, Aiken C: Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication. J Virol 2004, 78:6287-6296.
• [57]Sun Z, Denton PW, Estes JD, Othieno FA, Wei BL, Wege AK, Melkus MW, Padgett-Thomas A, Zupancic M, Haase AT, Garcia JV: Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1. J Exp Med 2007, 204:705-714.
• [58]Schindler M, Munch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F, Muller-Trutwin MC, Novembre FJ, Peeters M, Courgnaud V, et al.: Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell 2006, 125:1055-1067.
• [59]Schindler M, Schmokel J, Specht A, Li H, Munch J, Khalid M, Sodora DL, Hahn BH, Silvestri G, Kirchhoff F: Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4 + T cells in natural SIV infection. PLoS Pathog 2008, 4:e1000107.
• [60]Casartelli N, Giolo G, Neri F, Haller C, Potesta M, Rossi P, Fackler OT, Doria M: The Pro78 residue regulates the capacity of the human immunodeficiency virus type 1 Nef protein to inhibit recycling of major histocompatibility complex class I molecules in an SH3-independent manner. J Gen Virol 2006, 87:2291-2296.
• [61]Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P: Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 1998, 95:163-175.
• [62]Kouwenhoven A, Minassian VD, Marsh JW: HIV-1 Nef mediates Pak phosphorylation of Mek1 Serine298 and elicits an active phospho-state of Pak2. Curr HIV Res 2013, 11:198-209.
• [63]Pan X, Rudolph JM, Abraham L, Habermann A, Haller C, Krijnse-Locker J, Fackler OT: HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-golgi network-associated Lck signaling. Blood 2012, 119:786-797.
• [64]Ali SA, Huang MB, Campbell PE, Roth WW, Campbell T, Khan M, Newman G, Villinger F, Powell MD, Bond VC: Genetic characterization of HIV type 1 Nef-induced vesicle secretion. AIDS Res Hum Retroviruses 2010, 26:173-192.
• [65]Dinkins C, Arko-Mensah J, Deretic V: Autophagy and HIV. Semin Cell Dev Biol 2010, 21:712-718.
• [66]Geleziunas R, Xu W, Takeda K, Ichijo H, Greene WC: HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell. Nature 2001, 410:834-838.
• [67]Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, Wu L, Kominami E, Ueno T, Yamamoto A, et al.: Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. J Cell Biol 2009, 186:255-268.
• [68]Lenassi M, Cagney G, Liao M, Vaupotic T, Bartholomeeusen K, Cheng Y, Krogan NJ, Plemenitas A, Peterlin BM: HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells. Traffic 2010, 11:110-122.
• [69]Shelton MN, Huang MB, Ali SA, Powell MD, Bond VC: Secretion modification region-derived peptide disrupts HIV-1 Nef’s interaction with mortalin and blocks virus and Nef exosome release. J Virol 2012, 86:406-419.
• [70]Baur AS: HIV-Nef and AIDS pathogenesis: are we barking up the wrong tree? Trends Microbiol 2011, 19:435-440.
• [71]Denton PW, Krisko JF, Powell DA, Mathias M, Kwak YT, Martinez-Torres F, Zou W, Payne DA, Estes JD, Garcia JV: Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice. PLoS One 2010, 5:e8829.
• [72]Denton PW, Olesen R, Choudhary SK, Archin NM, Wahl A, Swanson MD, Chateau M, Nochi T, Krisko JF, Spagnuolo RA, et al.: Generation of HIV latency in humanized BLT mice. J Virol 2012, 86:630-634.
• [73]Denton PW, Othieno F, Martinez-Torres F, Zou W, Krisko JF, Fleming E, Zein S, Powell DA, Wahl A, Kwak YT, et al.: One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates. J Virol 2011, 85:7582-7593.
• [74]Kim SS, Peer D, Kumar P, Subramanya S, Wu H, Asthana D, Habiro K, Yang YG, Manjunath N, Shimaoka M, Shankar P: RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther 2010, 18:370-376.
• [75]Lan P, Tonomura N, Shimizu A, Wang S, Yang YG: Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood 2006, 108:487-492.
• [76]Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno FA, Wege AK, Haase AT, Garcia JV: Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med 2006, 12:1316-1322.
• [77]Rajesh D, Zhou Y, Jankowska-Gan E, Roenneburg DA, Dart ML, Torrealba J, Burlingham WJ: Th1 and Th17 immunocompetence in humanized NOD/SCID/IL2rgammanull mice. Hum Immunol 2010, 71:551-559.
• [78]Peden K, Emerman M, Montagnier L: Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology 1991, 185:661-672.
• [79]Wei BL, Denton PW, O’Neill E, Luo T, Foster JL, Garcia JV: Inhibition of lysosome and proteasome function enhances human immunodeficiency virus type 1 infection. J Virol 2005, 79:5705-5712.
• [80]Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, Polis M, Dewar RL, Planta A, Liu S, Metcalf JA, et al.: New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 2003, 41:4531-4536.
• [81]Denton PW, Garcia JV: Mucosal HIV-1 transmission and prevention strategies in BLT humanized mice. Trends Microbiol 2012, 20:268-274.
• [82]Kimpton J, Emerman M: Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol 1992, 66:2232-2239.
• [83]Chang AH, Hoxie JA, Cassol S, O’Shaughnessy M, Jirik F: Construction of single-chain antibodies that bind an overlapping epitope of HIV-1 Nef. FEBS Lett 1998, 441:307-312.