Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus

Retrovirology

Jeremy Luban¹  Alberto De Iaco¹ 

[1] Department of Microbiology and Molecular Medicine, University of Geneva, 1 Rue Michel Servet, CH-1211 Geneva, Switzerland

关键词: TNPO3;    integrase;    capsid;    HIV-1;   

Others  :  1209380
DOI  :  10.1186/1742-4690-8-98

 received in 2011-10-17, accepted in 2011-12-06,  发布年份 2011

【 摘 要 】

Background

HIV-1 infects non-dividing cells. This implies that the virus traverses the nuclear pore before it integrates into chromosomal DNA. Recent studies demonstrated that TNPO3 is required for full infectivity of HIV-1. The fact that TNPO3 is a karyopherin suggests that it acts by directly promoting nuclear entry of HIV-1. Some studies support this hypothesis, while others have failed to do so. Additionally, some studies suggest that TNPO3 acts via HIV-1 Integrase (IN), and others indicate that it acts via capsid (CA).

Results

To shed light on the mechanism by which TNPO3 contributes to HIV-1 infection we engineered a panel of twenty-seven single-cycle HIV-1 vectors each bearing a different CA mutation and characterized them for the ability to transduce cells in which TNPO3 had been knocked down (KD). Fourteen CA mutants were relatively TNPO3-independent, as compared to wild-type (WT) HIV-1. Two mutants were more TNPO3-dependent than the WT, and eleven mutants were actually inhibited by TNPO3. The efficiency of the synthesis of viral cDNA, 2-LTR circles, and proviral DNA was then assessed for WT HIV-1 and three select CA mutants. Controls included rescue of TNPO3 KD with non-targetable coding sequence, RT- and IN- mutant viruses, and pharmacologic inhibitors of RT and IN. TNPO3 KD blocked transduction and establishment of proviral DNA by wild-type HIV-1 with no significant effect on the level of 2-LTR circles. PCR results were confirmed by achieving TNPO3 KD using two different methodologies (lentiviral vector and siRNA oligonucleotide transfection); by challenging three different cell types; by using two different challenge viruses, each necessitating different sets of PCR primers; and by pseudotyping virus with VSV G or using HIV-1 Env.

Conclusion

TNPO3 promotes HIV-1 infectivity at a step in the virus life cycle that is detectable after the preintegration complex arrives in the nucleus and CA is the viral determinant for TNPO3 dependence.

【 授权许可】

   
2011 De Iaco and Luban; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Hulme AE, Perez O, Hope TJ: Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription. Proc Natl Acad Sci USA 2011, 108:9975-9980.
• [2]McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, Emerman M, Hope TJ: Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 2002, 159:441-452.
• [3]Arhel NJ, Souquere-Besse S, Munier S, Souque P, Guadagnini S, Rutherford S, Prevost MC, Allen TD, Charneau P: HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore. EMBO J 2007, 26:3025-3037.
• [4]Yamashita M, Perez O, Hope TJ, Emerman M: Evidence for direct involvement of the capsid protein in HIV infection of nondividing cells. PLoS Pathog 2007, 3:1502-1510.
• [5]Santoni FA, Hartley O, Luban J: Deciphering the code for retroviral integration target site selection. PLoS Comput Biol 2010, 6:e1001008.
• [6]Lee K, Ambrose Z, Martin TD, Oztop I, Mulky A, Julias JG, Vandegraaff N, Baumann JG, Wang R, Yuen W, et al.: Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe 2010, 7:221-233.
• [7]Ocwieja KE, Brady TL, Ronen K, Huegel A, Roth SL, Schaller T, James LC, Towers GJ, Young JA, Chanda SK, et al.: HIV integration targeting: a pathway involving Transportin-3 and the nuclear pore protein RanBP2. PLoS Pathog 2011, 7:e1001313.
• [8]Brown PO, Bowerman B, Varmus HE, Bishop JM: Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci USA 1989, 86:2525-2529.
• [9]Bukrinsky M, Sharova N, Stevenson M: Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex. J Virol 1993, 67:6863-6865.
• [10]Brussel A, Sonigo P: Evidence for gene expression by unintegrated human immunodeficiency virus type 1 DNA species. J Virol 2004, 78:11263-11271.
• [11]Wu Y, Marsh JW: Early transcription from nonintegrated DNA in human immunodeficiency virus infection. J Virol 2003, 77:10376-10382.
• [12]Cimarelli A, Sandin S, Hoglund S, Luban J: Rescue of multiple viral functions by a second-site suppressor of a human immunodeficiency virus type 1 nucleocapsid mutation. J Virol 2000, 74:4273-4283.
• [13]Lewis PF, Emerman M: Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994, 68:510-516.
• [14]Bukrinsky MI, Sharova N, Dempsey MP, Stanwick TL, Bukrinskaya AG, Haggerty S, Stevenson M: Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc Natl Acad Sci USA 1992, 89:6580-6584.
• [15]Lewis P, Hensel M, Emerman M: Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J 1992, 11:3053-3058.
• [16]Weinberg JB, Matthews TJ, Cullen BR, Malim MH: Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. J Exp Med 1991, 174:1477-1482.
• [17]Roe T, Reynolds TC, Yu G, Brown PO: Integration of murine leukemia virus DNA depends on mitosis. EMBO J 1993, 12:2099-2108.
• [18]Miller MD, Farnet CM, Bushman FD: Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J Virol 1997, 71:5382-5390.
• [19]Mattaj IW, Englmeier L: Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem 1998, 67:265-306.
• [20]von Schwedler U, Kornbluth RS, 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 USA 1994, 91:6992-6996.
• [21]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, 70:1027-1032.
• [22]Gallay P, Hope T, Chin D, Trono D: HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. Proc Natl Acad Sci USA 1997, 94:9825-9830.
• [23]Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P: HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 2000, 101:173-185.
• [24]Yamashita M, Emerman M: Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J Virol 2004, 78:5670-5678.
• [25]Hearps AC, Jans DA: HIV-1 integrase is capable of targeting DNA to the nucleus via an importin alpha/beta-dependent mechanism. Biochem J 2006, 398:475-484.
• [26]Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, Lieberman J, Elledge SJ: Identification of host proteins required for HIV infection through a functional genomic screen. Science 2008, 319:921-926.
• [27]Fassati A, Gorlich D, Harrison I, Zaytseva L, Mingot JM: Nuclear import of HIV-1 intracellular reverse transcription complexes is mediated by importin 7. EMBO J 2003, 22:3675-3685.
• [28]Christ F, Thys W, De Rijck J, Gijsbers R, Albanese A, Arosio D, Emiliani S, Rain JC, Benarous R, Cereseto A, Debyser Z: Transportin-SR2 imports HIV into the nucleus. Curr Biol 2008, 18:1192-1202.
• [29]Konig R, Zhou Y, Elleder D, Diamond TL, Bonamy GM, Irelan JT, Chiang CY, Tu BP, De Jesus PD, Lilley CE, et al.: Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 2008, 135:49-60.
• [30]Matreyek KA, Engelman A: The requirement for nucleoporin NUP153 during human immunodeficiency virus type 1 infection is determined by the viral capsid. J Virol 2011, 85:7818-7827.
• [31]Bushman FD, Malani N, Fernandes J, D'Orso I, Cagney G, Diamond TL, Zhou H, Hazuda DJ, Espeseth AS, Konig R, et al.: Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog 2009, 5:e1000437.
• [32]Kataoka N, Bachorik JL, Dreyfuss G: Transportin-SR, a nuclear import receptor for SR proteins. J Cell Biol 1999, 145:1145-1152.
• [33]Krishnan L, Matreyek KA, Oztop I, Lee K, Tipper CH, Li X, Dar MJ, Kewalramani VN, Engelman A: The requirement for cellular transportin 3 (TNPO3 or TRN-SR2) during infection maps to human immunodeficiency virus type 1 capsid and not integrase. J Virol 2010, 84:397-406.
• [34]Zhou L, Sokolskaja E, Jolly C, James W, Cowley SA, Fassati A: Transportin 3 Promotes a Nuclear Maturation Step Required for Efficient HIV-1 Integration. PLoS Pathog 2011, 7:e1002194.
• [35]Zhang R, Mehla R, Chauhan A: Perturbation of host nuclear membrane component RanBP2 impairs the nuclear import of human immunodeficiency virus -1 preintegration complex (DNA). PLoS One 2010, 5:e15620.
• [36]Pertel T, Hausmann S, Morger D, Zuger S, Guerra J, Lascano J, Reinhard C, Santoni FA, Uchil PD, Chatel L, et al.: TRIM5 is an innate immune sensor for the retrovirus capsid lattice. Nature 2011, 472:361-365.
• [37]Braaten D, Franke EK, Luban J: Cyclophilin A is required for an early step in the life cycle of human immunodeficiency virus type 1 before the initiation of reverse transcription. J Virol 1996, 70:3551-3560.
• [38]Chatterji U, Bobardt MD, Stanfield R, Ptak RG, Pallansch LA, Ward PA, Jones MJ, Stoddart CA, Scalfaro P, Dumont JM, et al.: Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl monkey cells. J Biol Chem 2005, 280:40293-40300.
• [39]Yoo S, Myszka DG, Yeh C, McMurray M, Hill CP, Sundquist WI: Molecular recognition in the HIV-1 capsid/cyclophilin A complex. J Mol Biol 1997, 269:780-795.
• [40]Yamashita M, Emerman M: Cellular restriction targeting viral capsids perturbs human immunodeficiency virus type 1 infection of nondividing cells. J Virol 2009, 83:9835-9843.
• [41]Qi M, Yang R, Aiken C: Cyclophilin A-dependent restriction of human immunodeficiency virus type 1 capsid mutants for infection of nondividing cells. J Virol 2008, 82:12001-12008.
• [42]Yin L, Braaten D, Luban J: Human immunodeficiency virus type 1 replication is modulated by host cyclophilin A expression levels. J Virol 1998, 72:6430-6436.
• [43]Braaten D, Aberham C, Franke EK, Yin L, Phares W, Luban J: Cyclosporine A-resistant human immunodeficiency virus type 1 mutants demonstrate that Gag encodes the functional target of cyclophilin A. J Virol 1996, 70:5170-5176.
• [44]Forshey BM, Shi J, Aiken C: Structural requirements for recognition of the human immunodeficiency virus type 1 core during host restriction in owl monkey cells. J Virol 2005, 79:869-875.
• [45]Yang R, Aiken C: A mutation in alpha helix 3 of CA renders human immunodeficiency virus type 1 cyclosporin A resistant and dependent: rescue by a second-site substitution in a distal region of CA. J Virol 2007, 81:3749-3756.
• [46]Dismuke DJ, Aiken C: Evidence for a functional link between uncoating of the human immunodeficiency virus type 1 core and nuclear import of the viral preintegration complex. J Virol 2006, 80:3712-3720.
• [47]Blair WS, Pickford C, Irving SL, Brown DG, Anderson M, Bazin R, Cao J, Ciaramella G, Isaacson J, Jackson L, et al.: HIV capsid is a tractable target for small molecule therapeutic intervention. PLoS Pathog 2010, 6:e1001220.
• [48]Buckman JS, Bosche WJ, Gorelick RJ: Human immunodeficiency virus type 1 nucleocapsid zn(2+) fingers are required for efficient reverse transcription, initial integration processes, and protection of newly synthesized viral DNA. J Virol 2003, 77:1469-1480.
• [49]Berthoux L, Sebastian S, Muesing MA, Luban J: The role of lysine 186 in HIV-1 integrase multimerization. Virology 2007, 364:227-236.
• [50]Butler SL, Hansen MS, Bushman FD: A quantitative assay for HIV DNA integration in vivo. Nat Med 2001, 7:631-634.
• [51]Ruegsegger U, Blank D, Keller W: Human pre-mRNA cleavage factor Im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits. Mol Cell 1998, 1:243-253.
• [52]Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP: Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell 1993, 73:1067-1078.
• [53]Li Y, Kar AK, Sodroski J: Target cell type-dependent modulation of human immunodeficiency virus type 1 capsid disassembly by cyclophilin A. J Virol 2009, 83:10951-10962.
• [54]Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D: Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998, 72:9873-9880.
• [55]Berthoux L, Sebastian S, Sokolskaja E, Luban J: Lv1 inhibition of human immunodeficiency virus type 1 is counteracted by factors that stimulate synthesis or nuclear translocation of viral cDNA. J Virol 2004, 78:11739-11750.
• [56]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, 77:3167-3180.
• [57]He J, Chen Y, Farzan M, Choe H, Ohagen A, Gartner S, Busciglio J, Yang X, Hofmann W, Newman W, et al.: CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia. Nature 1997, 385:645-649.
• [58]Neagu MR, Ziegler P, Pertel T, Strambio-De-Castillia C, Grutter C, Martinetti G, Mazzucchelli L, Grutter M, Manz MG, Luban J: Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components. J Clin Invest 2009, 119:3035-3047.
• [59]Pizzato M, Erlwein O, Bonsall D, Kaye S, Muir D, McClure MO: A one-step SYBR Green I-based product-enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture supernatants. J Virol Methods 2009, 156:1-7.
• [60]Maudru T, Peden KW: Adaptation of the fluorogenic 5'-nuclease chemistry to a PCR-based reverse transcriptase assay. Biotechniques 1998, 25:972-975.