Abstract
The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587–10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587–10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Briggs JA, Simon MN, Gross I, Krausslich HG, Fuller SD, Vogt VM, Johnson MC (2004) The stoichiometry of Gag protein in HIV-1. Nat Struct Mol Biol 11:672–675
Briggs JA, Wilk T, Welker R, Krausslich HG, Fuller SD (2003) Structural organization of authentic, mature HIV-1 virions and cores. EMBO J 22:1707–1715
Lanman J, Lam TT, Emmett MR, Marshall AG, Sakalian M, Prevelige PE Jr (2004) Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange. Nat Struct Mol Biol 11:676–677
McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, Emerman M, Hope TJ (2002) Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159:441–452
Fassati A, Goff SP (2001) Characterization of intracellular reverse transcription complexes of human immunodeficiency virus type 1. J Virol 75:3626–3635
Stremlau M, Perron M, Lee M, Li Y, Song B, Javanbakht H, Diaz-Griffero F, Anderson DJ, Sundquist WI, Sodroski J (2006) Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci U S A 103:5514–5519
Diaz-Griffero F, Kar A, Lee M, Stremlau M, Poeschla E, Sodroski J (2007) Comparative requirements for the restriction of retrovirus infection by TRIM5alpha and TRIMCyp. Virology 369:400–410, Epub 2007 Oct 24
Roa A, Hayashi F, Yang Y, Lienlaf M, Zhou J, Shi J, Watanabe S, Kigawa T, Yokoyama S, Aiken C, Diaz-Griffero F (2012) RING domain mutations uncouple TRIM5alpha restriction of HIV-1 from inhibition of reverse transcription and acceleration of uncoating. J Virol 86:1717–1727
Arfi V, Lienard J, Nguyen XN, Berger G, Rigal D, Darlix JL, Cimarelli A (2009) Characterization of the behavior of functional viral genomes during the early steps of human immunodeficiency virus type 1 infection. J Virol 83:7524–7535
Yamashita M, Emerman M (2004) Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J Virol 78:5670–5678
Yamashita M, Perez O, Hope TJ, Emerman M (2007) Evidence for direct involvement of the capsid protein in HIV infection of nondividing cells. PLoS Pathog 3:1502–1510
Yamashita M, Emerman M (2006) Retroviral infection of non-dividing cells: old and new perspectives. Virology 344:88–93
Diaz-Griffero F (2012) The role of TNPO3 in HIV-1 replication. Mol Biol Int 2012:868597
Yang Y, Fricke T, Diaz-Griffero F (2013) Inhibition of reverse transcriptase activity increases stability of the HIV-1 core. J Virol 87:683–687
Forshey BM, von Schwedler U, Sundquist WI, Aiken C (2002) Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol 76:5667–5677
Ohagen A, Gabuzda D (2000) Role of Vif in stability of the human immunodeficiency virus type 1 core. J Virol 74:11055–11066
Diaz-Griffero F, Kar A, Perron M, Xiang SH, Javanbakht H, Li X, Sodroski J (2007) Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain. J Virol 81:10362–10378
Shi J, Zhou J, Shah VB, Aiken C, Whitby K (2011) Small-molecule inhibition of human immunodeficiency virus type 1 infection by virus capsid destabilization. J Virol 85:542–549
De Iaco A, Santoni F, Vannier A, Guipponi M, Antonarakis S, Luban J (2013) TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm. Retrovirology 10:20
Perron MJ, Stremlau M, Lee M, Javanbakht H, Song B, Sodroski J (2007) The human TRIM5alpha restriction factor mediates accelerated uncoating of the N-tropic murine leukemia virus capsid. J Virol 81:2138–2148
Diaz-Griffero F, Perron M, McGee-Estrada K, Hanna R, Maillard PV, Trono D, Sodroski J (2008) A human TRIM5alpha B30.2/SPRY domain mutant gains the ability to restrict and prematurely uncoat B-tropic murine leukemia virus. Virology 378:233–242
Ohkura S, Goldstone DC, Yap MW, Holden-Dye K, Taylor IA, Stoye JP (2011) Novel escape mutants suggest an extensive TRIM5alpha binding site spanning the entire outer surface of the murine leukemia virus capsid protein. PLoS Pathog 7, e1002011
Berube J, Bouchard A, Berthoux L (2007) Both TRIM5alpha and TRIMCyp have only weak antiviral activity in canine D17 cells. Retrovirology 4:68
Fricke T, Brandariz-Nunez A, Wang X, Smith AB 3rd, Diaz-Griffero F (2013) Human cytosolic extracts stabilize the HIV-1 core. J Virol 87:10587–10597
Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI (1999) Assembly and analysis of conical models for the HIV-1 core. Science 283:80–83
Sticht J, Humbert M, Findlow S, Bodem J, Muller B, Dietrich U, Werner J, Krausslich HG (2005) A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol 12:671–677
Blair WS, Pickford C, Irving SL, Brown DG, Anderson M, Bazin R, Cao J, Ciaramella G, Isaacson J, Jackson L, Hunt R, Kjerrstrom A, Nieman JA, Patick AK, Perros M, Scott AD, Whitby K, Wu H, Butler SL (2010) HIV capsid is a tractable target for small molecule therapeutic intervention. PLoS Pathog 6, e1001220
Goudreau N, Lemke CT, Faucher AM, Grand-Maitre C, Goulet S, Lacoste JE, Rancourt J, Malenfant E, Mercier JF, Titolo S, Mason SW (2013) Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography. ACS Chem Biol 8:1074–1082
Lemke CT, Titolo S, Goudreau N, Faucher AM, Mason SW, Bonneau P (2013) A novel inhibitor-binding site on the HIV-1 capsid N-terminal domain leads to improved crystallization via compound-mediated dimerization. Acta Crystallogr D Biol Crystallogr 69:1115–1123
Fricke T, White TE, Schulte B, de Souza Aranha Vieira DA, Dharan A, Campbell EM, Brandariz-Nunez A, Diaz-Griffero F (2014) MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1. Retrovirology 11:68
Fricke T, Valle-Casuso JC, White TE, Brandariz-Nunez A, Bosche WJ, Reszka N, Gorelick R, Diaz-Griffero F (2013) The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6. Retrovirology 10:46
Acknowledgments
Projects AI087390, AI10282401, R56AI108432, and AI104476 to F.D.-G supported this work. We are grateful to the NIH HIV-1/AIDS repository for providing reagents such as antibodies and small-molecule inhibitors that were crucial for this work.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Fricke, T., Diaz-Griffero, F. (2016). HIV-1 Capsid Stabilization Assay. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3046-3_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3045-6
Online ISBN: 978-1-4939-3046-3
eBook Packages: Springer Protocols