Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects

Retrovirology

Christopher Aiken³  Angela M Gronenborn¹  Jens Meiler²  Jonathan H Sheehan²  Jinwoo Ahn¹  In-Ja L Byeon¹  Jiong Shi³  Ruifeng Yang³ 

[1] Department of Structural Biology, University of Pittsburgh Medical School, Pittsburgh, PA, USA;Center for Structural Biology, Vanderbilt University, Nashville, TN, USA;Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA

关键词: Inhibitor;    Suppressor mutations;    Uncoating;    Capsid;    HIV-1;   

Others  :  1209315
DOI  :  10.1186/1742-4690-9-30

 received in 2012-01-12, accepted in 2012-04-19,  发布年份 2012

【 摘 要 】

Background

Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection.

Results

To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α.

Conclusions

The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid.

【 授权许可】

   
2012 Yang et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, Sundquist WI, Hill CP: Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell 1996, 87:1285-1294.
• [2]Gamble TR, Yoo S, Vajdos FF, von Schwedler UK, Worthylake DK, Wang H, McCutcheon JP, Sundquist WI, Hill CP: Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science 1997, 278:849-853.
• [3]Gitti RK, Lee BM, Walker J, Summers MF, Yoo S, Sundquist WI: Structure of the amino-terminal core domain of the HIV-1 capsid protein. Science 1996, 273:231-235.
• [4]Li S, Hill CP, Sundquist WI, Finch JT: Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature 2000, 407:409-413.
• [5]Momany C, Kovari LC, Prongay AJ, Keller W, Gitti RK, Lee BM, Gorbalenya AE, Tong L, McClure J, Ehrlich LS, et al.: Crystal structure of dimeric HIV-1 capsid protein. Nat Struct Biol 1996, 3:763-770.
• [6]Bowzard JB, Wills JW, Craven RC: Second-site suppressors of Rous sarcoma virus Ca mutations: evidence for interdomain interactions. J Virol 2001, 75:6850-6856.
• [7]Lanman J, Lam TT, Emmett MR, Marshall AG, Sakalian M, Prevelige PE Jr: Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange. Nat Struct Mol Biol 2004, 11:676-677.
• [8]Lanman J, Lam TT, Barnes S, Sakalian M, Emmett MR, Marshall AG, Prevelige PE Jr: Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry. J Mol Biol 2003, 325:759-772.
• [9]Ganser-Pornillos BK, Cheng A, Yeager M: Structure of full-length HIV-1 CA: a model for the mature capsid lattice. Cell 2007, 131:70-79.
• [10]Pornillos O, Ganser-Pornillos BK, Kelly BN, Hua Y, Whitby FG, Stout CD, Sundquist WI, Hill CP, Yeager M: X-ray structures of the hexameric building block of the HIV capsid. Cell 2009, 137:1282-1292.
• [11]Aiken C: Viral and cellular factors that regulate HIV-1 uncoating. Curr Opin HIV AIDS 2006, 1:194-199.
• [12]Miller MD, Farnet CM, Bushman FD: Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J Virol 1997, 71:5382-5390.
• [13]Farnet CM, Haseltine WA: Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol 1991, 65:1910-1915.
• [14]Fassati A, Goff SP: Characterization of intracellular reverse transcription complexes of human immunodeficiency virus type 1. J Virol 2001, 75:3626-3635.
• [15]Arfi V, Lienard J, Nguyen XN, Berger G, Rigal D, Darlix JL, Cimarelli A: Characterization of the behavior of functional viral genomes during the early steps of human immunodeficiency virus type 1 infection. J Virol 2009, 83:7524-7535.
• [16]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.
• [17]Forshey BM, von Schwedler U, Sundquist WI, Aiken C: Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol 2002, 76:5667-5677.
• [18]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.
• [19]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.
• [20]Casella CR, Raffini LJ, Panganiban AT: Pleiotropic mutations in the HIV-1 matrix protein that affect diverse steps in replication. Virology 1997, 228:294-306.
• [21]Kiernan RE, Ono A, Englund G, Freed EO: Role of matrix in an early postentry step in the human immunodeficiency virus type 1 life cycle. J Virol 1998, 72:4116-4126.
• [22]Davis MR, Jiang J, Zhou J, Freed EO, Aiken C: A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41. J Virol 2006, 80:2405-2417.
• [23]Stremlau M, Perron M, Lee M, Li Y, Song B, Javanbakht H, Diaz-Griffero F, Anderson DJ, Sundquist WI, Sodroski J: Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci USA 2006, 103:5514-5519.
• [24]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.
• [25]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.
• [26]Auewarakul P, Wacharapornin P, Srichatrapimuk S, Chutipongtanate S, Puthavathana P: Uncoating of HIV-1 requires cellular activation. Virology 2005, 337:93-101.
• [27]Campbell S, Vogt VM: Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1. J Virol 1995, 69:6487-6497.
• [28]Ehrlich LS, Agresta BE, Carter CA: Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro. J Virol 1992, 66:4874-4883.
• [29]Gross I, Hohenberg H, Krausslich HG: In vitro assembly properties of purified bacterially expressed capsid proteins of human immunodeficiency virus. Eur J Biochem 1997, 249:592-600.
• [30]von Schwedler UK, Stemmler TL, Klishko VY, Li S, Albertine KH, Davis DR, Sundquist WI: Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly. EMBO J 1998, 17:1555-1568.
• [31]Douglas CC, Thomas D, Lanman J, Prevelige PE Jr: Investigation of N-terminal domain charged residues on the assembly and stability of HIV-1 CA. Biochemistry 2004, 43:10435-10441.
• [32]Yamashita M, Emerman M: Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J Virol 2004, 78:5670-5678.
• [33]Shi J, Zhou J, Shah VB, Aiken C, Whitby K: Small-molecule inhibition of human immunodeficiency virus type 1 infection by virus capsid destabilization. J Virol 2011, 85:542-549.
• [34]Cowan S, Hatziioannou T, Cunningham T, Muesing MA, Gottlinger HG, Bieniasz PD: Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism. Proc Natl Acad Sci USA 2002, 99:11914-11919.
• [35]Hatziioannou T, Perez-Caballero D, Yang A, Cowan S, Bieniasz PD: Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proc Natl Acad Sci USA 2004, 101:10774-10779.
• [36]Keckesova Z, Ylinen LM, Towers GJ: The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc Natl Acad Sci USA 2004, 101:10780-10785.
• [37]Yap MW, Nisole S, Lynch C, Stoye JP: Trim5alpha protein restricts both HIV-1 and murine leukemia virus. Proc Natl Acad Sci USA 2004, 101:10786-10791.
• [38]Nisole S, Lynch C, Stoye JP, Yap MW: A Trim5-cyclophilin A fusion protein found in owl monkey kidney cells can restrict HIV-1. Proc Natl Acad Sci USA 2004, 101:13324-13328.
• [39]Sayah DM, Sokolskaja E, Berthoux L, Luban J: Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1. Nature 2004, 430:569-573.
• [40]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.
• [41]Shi J, Aiken C: Saturation of TRIM5alpha-mediated restriction of HIV-1 infection depends on the stability of the incoming viral capsid. Virology 2006, 350:493-500.
• [42]Noviello CM, Lopez CS, Kukull B, McNett H, Still A, Eccles J, Sloan R, Barklis E: Second-site compensatory mutations of HIV-1 capsid mutations. J Virol 2011, 85:4730-4738.
• [43]Byeon IJ, Meng X, Jung J, Zhao G, Yang R, Ahn J, Shi J, Concel J, Aiken C, Zhang P, Gronenborn AM: Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function. Cell 2009, 139:780-790.
• [44]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.
• [45]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.
• [46]Swingler S, Gallay P, Camaur D, Song J, Abo A, Trono D: The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix. J Virol 1997, 71:4372-4377.
• [47]von Schwedler UK, Stray KM, Garrus JE, Sundquist WI: Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol 2003, 77:5439-5450.
• [48]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.
• [49]Chen C, Okayama H: High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 1987, 7:2745-2752.
• [50]Yee JK, Friedmann T, Burns JC: Generation of high-titer pseudotyped retroviral with very broad host range. Methods Cell Biol 1994, 43:99-112.
• [51]Wehrly K, Chesebro B: p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents. Methods 1997, 12:288-293.
• [52]Aiken C, Trono D: Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J Virol 1995, 69:5048-5056.
• [53]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.
• [54]Charneau P, Alizon M, Clavel F: A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. J Virol 1992, 66:2814-2820.
• [55]Shah VB, Aiken C: In vitro Uncoating of HIV-1 Cores. J Vis Exp 2011, 57:e3384.
• [56]Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI: Assembly and analysis of conical models for the HIV-1 core. Science 1999, 283:80-83.
• [57]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.
• [58]Koradi R, Billeter M, Wuthrich K: MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 1996, 14:51-55. 29-32
• [59]Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE: UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem 2004, 25:1605-1612.