Evidence for Vpr-dependent HIV-1 Replication in Human CD4+ CEM.NKR T-Cells

Retrovirology

Yong-Hui Zheng¹  Jiajun Zhou¹  Jacob J Baker¹  Ying Dang¹  Tao Zhou¹ 

[1] Department of Microbiology and Molecular Genetics, Michigan State University, 567 Wilson Road, 2215, Biomedical and Physical Sciences Building, East Lansing, MI, 48824-4320, USA

关键词: DCAF1;    G2 cell cycle arrest;    SAMHD1;    HIV-1;    Host Restriction;    Vpx;    Vpr;   

Others  :  1209241
DOI  :  10.1186/1742-4690-9-93

 received in 2012-08-12, accepted in 2012-10-11,  发布年份 2012

【 摘 要 】

Background

Vpr is exclusively expressed in primate lentiviruses and contributes to viral replication and disease progression in vivo. HIV-1 Vpr has two major activities in vitro: arrest of cell cycle in the G2 phase (G2 arrest), and enhancement of viral replication in macrophages. Previously, we reported a potent HIV-1 restriction in the human CD4⁺ CEM.NKR (NKR) T cells, where wild-type (WT) HIV-1 replication was inhibited by almost 1,000-fold. From the parental NKR cells, we isolated eight clones by limiting dilution. These clones showed three levels of resistance to the WT HIV-1 infection: non-permissive (NP), semi-permissive (SP), and permissive (P). Here, we compared the replication of WT, Vif-defective, Vpr-defective, and Vpu-defective viruses in these cells.

Results

Although both WT and Vpu-defective viruses could replicate in the permissive and semi-permissive clones, the replication of Vif-defective and Vpr-defective viruses was completely restricted. The expression of APOBEC3G (A3G) cytidine deaminase in NKR cells explains why Vif, but not Vpr, was required for HIV-1 replication. When the Vpr-defective virus life cycle was compared with the WT virus life cycle in the semi-permissive cells, it was found that the Vpr-defective virus could enter the cell and produce virions containing properly processed Gag and Env proteins, but these virions showed much less efficiency for reverse transcription during the next-round of infection. In addition, although viral replication was restricted in the non-permissive cells, treatment with arsenic trioxide (As₂O₃) could completely restore WT, but not Vpr-defective virus replication. Moreover, disruption of Vpr binding to its cofactor DCAF1 and/or induction of G2 arrest activity did not disrupt the Vpr activity in enhancing HIV-1 replication in NKR cells.

Conclusions

These results demonstrate that HIV-1 replication in NKR cells is Vpr-dependent. Vpr promotes HIV-1 replication from the 2nd cycle likely by overcoming a block at early stage of viral replication; and this activity does not require DCAF1 and G2 arrest. Further studies of this mechanism should provide new understanding of Vpr function in the HIV-1 life cycle.

【 授权许可】

   
2012 Zhou et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Ayinde D, Maudet C, Transy C, Margottin-Goguet F: Limelight on two HIV/SIV accessory proteins in macrophage infection: is Vpx overshadowing Vpr? Retrovirology 2010, 7:35. BioMed Central Full Text
• [2]Sharp PM, Bailes E, Stevenson M, Emerman M, Hahn BH: Gene acquisition in HIV and SIV. Nature 1996, 383(6601):586-587.
• [3]Tristem M, Marshall C, Karpas A, Hill F: Evolution of the primate lentiviruses: evidence from vpx and vpr. EMBO J 1992, 11(9):3405-3412.
• [4]Paxton W, Connor RI, Landau NR: Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol 1993, 67(12):7229-7237.
• [5]Yu XF, Matsuda M, Essex M, Lee TH: Open reading frame vpr of simian immunodeficiency virus encodes a virion-associated protein. J Virol 1990, 64(11):5688-5693.
• [6]Cohen EA, Dehni G, Sodroski JG, Haseltine WA: Human immunodeficiency virus vpr product is a virion-associated regulatory protein. J Virol 1990, 64(6):3097-3099.
• [7]Accola MA, Bukovsky AA, Jones MS, Gottlinger HG: A conserved dileucine-containing motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeficiency viruses SIV(mac) and SIV(agm). J Virol 1999, 73(12):9992-9999.
• [8]Wu X, Conway JA, Kim J, Kappes JC: Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein. J Virol 1994, 68(10):6161-6169.
• [9]Lang SM, Weeger M, Stahl-Hennig C, Coulibaly C, Hunsmann G, Muller J, Muller-Hermelink H, Fuchs D, Wachter H, Daniel MM, et al.: Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus. J Virol 1993, 67(2):902-912.
• [10]Goh WC, Rogel ME, Kinsey CM, Michael SF, Fultz PN, Nowak MA, Hahn BH, Emerman M: HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo. Nat Med 1998, 4(1):65-71.
• [11]Caly L, Saksena NK, Piller SC, Jans DA: Impaired nuclear import and viral incorporation of Vpr derived from a HIV long-term non-progressor. Retrovirology 2008, 5:67. BioMed Central Full Text
• [12]Jacquot G, Le Rouzic E, Maidou-Peindara P, Maizy M, Lefrere JJ, Daneluzzi V, Monteiro-Filho CM, Hong D, Planelles V, Morand-Joubert L, et al.: Characterization of the molecular determinants of primary HIV-1 Vpr proteins: impact of the Q65R and R77Q substitutions on Vpr functions. PLoS One 2009, 4(10):e7514.
• [13]Somasundaran M, Sharkey M, Brichacek B, Luzuriaga K, Emerman M, Sullivan JL, Stevenson M: Evidence for a cytopathogenicity determinant in HIV-1 Vpr. Proc Natl Acad Sci U S A 2002, 99(14):9503-9508.
• [14]Zhao Y, Chen M, Wang B, Yang J, Elder RT, Song XQ, Yu M, Saksena NK: Functional conservation of HIV-1 Vpr and variability in a mother-child pair of long-term non-progressors. Virus Res 2002, 89(1):103-121.
• [15]Gibbs JS, Lackner AA, Lang SM, Simon MA, Sehgal PK, Daniel MD, Desrosiers RC: Progression to AIDS in the absence of a gene for vpr or vpx. J Virol 1995, 69(4):2378-2383.
• [16]Hoch J, Lang SM, Weeger M, Stahl-Hennig C, Coulibaly C, Dittmer U, Hunsmann G, Fuchs D, Muller J, Sopper S, et al.: vpr deletion mutant of simian immunodeficiency virus induces AIDS in rhesus monkeys. J Virol 1995, 69(8):4807-4813.
• [17]Belshan M, Kimata JT, Brown C, Cheng X, McCulley A, Larsen A, Thippeshappa R, Hodara V, Giavedoni L, Hirsch V, et al.: Vpx is critical for SIVmne infection of pigtail macaques. Retrovirology 2012, 9:32. BioMed Central Full Text
• [18]Hirsch VM, Sharkey ME, Brown CR, Brichacek B, Goldstein S, Wakefield J, Byrum R, Elkins WR, Hahn BH, Lifson JD, et al.: Vpx is required for dissemination and pathogenesis of SIV(SM) PBj: evidence of macrophage-dependent viral amplification. Nat Med 1998, 4(12):1401-1408.
• [19]Kogan M, Rappaport J: HIV-1 accessory protein Vpr: relevance in the pathogenesis of HIV and potential for therapeutic intervention. Retrovirology 2011, 8:25. BioMed Central Full Text
• [20]Zhao RY, Li G, Bukrinsky MI: Vpr-host interactions during HIV-1 viral life cycle. J Neuroimmune Pharmacol 2011, 6(2):216-229.
• [21]Planelles V: Restricted access to myeloid cells explained. Viruses 2011, 3(9):1624-1633.
• [22]Goujon C, Jarrosson-Wuilleme L, Bernaud J, Rigal D, Darlix JL, Cimarelli A: With a little help from a friend: increasing HIV transduction of monocyte-derived dendritic cells with virion-like particles of SIV(MAC). Gene Ther 2006, 13(12):991-994.
• [23]Negri DR, Rossi A, Blasi M, Michelini Z, Leone P, Chiantore MV, Baroncelli S, Perretta G, Cimarelli A, Klotman ME, et al.: Simian immunodeficiency virus-Vpx for improving integrase defective lentiviral vector-based vaccines. Retrovirology 2012, 9(1):69. BioMed Central Full Text
• [24]Andersen JL, DeHart JL, Zimmerman ES, Ardon O, Kim B, Jacquot G, Benichou S, Planelles V: HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT. PLoS Pathog 2006, 2(12):e127.
• [25]Planelles V, Benichou S: Vpr and its interactions with cellular proteins. Curr Top Microbiol Immunol 2009, 339:177-200.
• [26]Ward J, Davis Z, DeHart J, Zimmerman E, Bosque A, Brunetta E, Mavilio D, Planelles V, Barker E: HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response. PLoS Pathog 2009, 5(10):e1000613.
• [27]Casey L, Wen X, de Noronha CM: The functions of the HIV1 protein Vpr and its action through the DCAF1.DDB1.Cullin4 ubiquitin ligase. Cytokine 2010, 51(1):1-9.
• [28]Gramberg T, Sunseri N, Landau NR: Evidence for an activation domain at the amino terminus of simian immunodeficiency virus Vpx. J Virol 2010, 84(3):1387-1396.
• [29]Sharova N, Wu Y, Zhu X, Stranska R, Kaushik R, Sharkey M, Stevenson M: Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction. PLoS Pathog 2008, 4(5):e1000057.
• [30]Srivastava S, Swanson SK, Manel N, Florens L, Washburn MP, Skowronski J: Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog 2008, 4(5):e1000059.
• [31]Zhao RY, Elder RT, Bukrinsky M: Interactions of HIV-1 viral protein R with host cell proteins. Adv Pharmacol 2007, 55:233-260.
• [32]Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik-Brodacka M, Srivastava S, Florens L, Washburn MP, Skowronski J: Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 2011, 474(7353):658-661.
• [33]Laguette N, Sobhian B, Casartelli N, Ringeard M, Chable-Bessia C, Segeral E, Yatim A, Emiliani S, Schwartz O, Benkirane M: SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 2011, 474(7353):654-657.
• [34]Gummuluru S, Emerman M: Cell cycle- and Vpr-mediated regulation of human immunodeficiency virus type 1 expression in primary and transformed T-cell lines. J Virol 1999, 73(7):5422-5430.
• [35]Iijima S, Nitahara-Kasahara Y, Kimata K, Zhong Zhuang W, Kamata M, Isogai M, Miwa M, Tsunetsugu-Yokota Y, Aida Y: Nuclear localization of Vpr is crucial for the efficient replication of HIV-1 in primary CD4+ T cells. Virology 2004, 327(2):249-261.
• [36]de Silva S, Planelles V, Wu L: Differential effects of Vpr on single-cycle and spreading HIV-1 infections in CD4+ T-cells and dendritic cells. PLoS One 2012, 7(5):e35385.
• [37]Campbell BJ, Hirsch VM: Vpr of simian immunodeficiency virus of African green monkeys is required for replication in macaque macrophages and lymphocytes. J Virol 1997, 71(7):5593-5602.
• [38]Ueno F, Shiota H, Miyaura M, Yoshida A, Sakurai A, Tatsuki J, Koyama AH, Akari H, Adachi A, Fujita M: Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells. Microbes Infect 2003, 5(5):387-395.
• [39]Gibbs JS, Regier DA, Desrosiers RC: Construction and in vitro properties of SIVmac mutants with deletions in "nonessential" genes. AIDS Res Hum Retroviruses 1994, 10(5):607-616.
• [40]Guyader M, Emerman M, Montagnier L, Peden K: VPX mutants of HIV-2 are infectious in established cell lines but display a severe defect in peripheral blood lymphocytes. EMBO J 1989, 8(4):1169-1175.
• [41]Yu XF, Yu QC, Essex M, Lee TH: The vpx gene of simian immunodeficiency virus facilitates efficient viral replication in fresh lymphocytes and macrophage. J Virol 1991, 65(9):5088-5091.
• [42]Han Y, Wang X, Dang Y, Zheng YH: Demonstration of a novel HIV-1 restriction phenotype from a human T cell line. PLoS One 2008, 3(7):e2796.
• [43]Zhou T, Han Y, Dang Y, Wang X, Zheng YH: A novel HIV-1 restriction factor that is biologically distinct from APOBEC3 cytidine deaminases in a human T cell line CEM.NKR. Retrovirology 2009, 6:31. BioMed Central Full Text
• [44]Stopak K, de Noronha C, Yonemoto W, Greene WC: HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability. Mol Cell 2003, 12(3):591-601.
• [45]Berthoux L, Towers GJ, Gurer C, Salomoni P, Pandolfi PP, Luban J: As(2)O(3) enhances retroviral reverse transcription and counteracts Ref1 antiviral activity. J Virol 2003, 77(5):3167-3180.
• [46]Turelli P, Doucas V, Craig E, Mangeat B, Klages N, Evans R, Kalpana G, Trono D: Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegration complexes: interference with early steps of viral replication. Mol Cell 2001, 7(6):1245-1254.
• [47]Belzacq AS, El Hamel C, Vieira HL, Cohen I, Haouzi D, Metivier D, Marchetti P, Brenner C, Kroemer G: Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437. Oncogene 2001, 20(52):7579-7587.
• [48]Larochette N, Decaudin D, Jacotot E, Brenner C, Marzo I, Susin SA, Zamzami N, Xie Z, Reed J, Kroemer G: Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Exp Cell Res 1999, 249(2):413-421.
• [49]Belzile JP, Duisit G, Rougeau N, Mercier J, Finzi A, Cohen EA: HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase. PLoS Pathog 2007, 3(7):e85.
• [50]Richard J, Sindhu S, Pham TN, Belzile JP, Cohen EA: HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell-mediated killing. Blood 2010, 115(7):1354-1363.
• [51]Sakai K, Dimas J, Lenardo MJ: The Vif and Vpr accessory proteins independently cause HIV-1-induced T cell cytopathicity and cell cycle arrest. Proc Natl Acad Sci U S A 2006, 103(9):3369-3374.
• [52]Liu L, Oliveira NM, Cheney KM, Pade C, Dreja H, Bergin AM, Borgdorff V, Beach DH, Bishop CL, Dittmar MT, et al.: A whole genome screen for HIV restriction factors. Retrovirology 2011, 8:94. BioMed Central Full Text
• [53]Pertel T, Reinhard C, Luban J: Vpx rescues HIV-1 transduction of dendritic cells from the antiviral state established by type 1 interferon. Retrovirology 2011, 8:49. BioMed Central Full Text
• [54]Bolton DL, Lenardo MJ: Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells. J Virol 2007, 81(17):8878-8890.
• [55]Wang X, Han Y, Dang Y, Fu W, Zhou T, Ptak RG, Zheng YH: Moloney leukemia virus 10 (MOV10) protein inhibits retrovirus replication. J Biol Chem 2010, 285(19):14346-14355.
• [56]Butler SL, Hansen MS, Bushman FD: A quantitative assay for HIV DNA integration in vivo. Nat Med 2001, 7(5):631-634.