Abstract
Direct T cell-to-T cell HIV-1 infection is a distinct mode of HIV-1 infection that requires physical contact between an HIV-1-infected “donor” cell and an uninfected, CD4-expressing “target” cell. In vitro studies indicate that HIV-1 cell-to-cell infection is much more efficient than infection by cell-free viral particles; however, the exact mechanisms of the enhanced efficiency of this infection pathway are still unclear. Several assays have been developed to study the mechanism of direct cell-to-cell HIV-1 transmission and to assess sensitivity to neutralizing antibodies and pharmacologic inhibitors. These assays are based on the coculture of donor and target cells. Here, we describe methods that utilize flow cytometry, which can discriminate donor and target cells and can assess different stages of entry and infection following cell-to-cell contact. HIV Gag-iGFP, a clone that makes fluorescent virus particles, can be used to measure cell-to-cell transfer of virus particles. HIV NL-GI, a clone that expresses GFP as an early gene, facilitates the measure of productive infection after cell-to-cell contact. Lastly, a variation of the β-lactamase (BlaM)-Vpr fusion assay can be used to measure the viral membrane fusion process after coculture of donor and target cells in a manner that is independent of cell-cell fusion. These assays can be performed in the presence of neutralizing antibodies/inhibitors to determine the 50 % inhibitory concentration (IC50) required to block infection specifically in the target cells.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
McDonald D, Wu L, Bohks SM, KewalRamani VN, Unutmaz D, Hope TJ (2003) Recruitment of HIV and its receptors to dendritic cell-T cell junctions. Science 300:1295–1297. doi:10.1126/science.1084238
Cameron PU, Freudenthal PS, Barker JM, Gezelter S, Inaba K, Steinman RM (1992) Dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to CD4+ T cells. Science 257:383–387
Arrighi JF, Pion M, Garcia E, Escola JM, van Kooyk Y, Geijtenbeek TB, Piguet V (2004) DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells. J Exp Med 200:1279–1288. doi:10.1084/jem.20041356
Jolly C, Kashefi K, Hollinshead M, Sattentau QJ (2004) HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J Exp Med 199:283–293. doi:10.1084/jem.20030648
Piguet V, Sattentau Q (2004) Dangerous liaisons at the virological synapse. J Clin Invest 114:605–610. doi:10.1172/jci22812
Dale BM, Alvarez RA, Chen BK (2013) Mechanisms of enhanced HIV spread through T-cell virological synapses. Immunol Rev 251:113–124. doi:10.1111/imr.12022
Puigdomenech I, Massanella M, Cabrera C, Clotet B, Blanco J (2009) On the steps of cell-to-cell HIV transmission between CD4 T cells. Retrovirology 6:89. doi:10.1186/1742-4690-6-89
Igakura T, Stinchcombe JC, Goon PK, Taylor GP, Weber JN, Griffiths GM, Tanaka Y, Osame M, Bangham CR (2003) Spread of HTLV-I between lymphocytes by virus-induced polarization of the cytoskeleton. Science 299:1713–1716. doi:10.1126/science.1080115
Chen P, Hubner W, Spinelli MA, Chen BK (2007) Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses. J Virol 81:12582–12595. doi:10.1128/jvi.00381-07
Sourisseau M, Sol-Foulon N, Porrot F, Blanchet F, Schwartz O (2007) Inefficient human immunodeficiency virus replication in mobile lymphocytes. J Virol 81:1000–1012. doi:10.1128/jvi.01629-06
Arthos J, Cicala C, Martinelli E, Macleod K, Van Ryk D, Wei D, Xiao Z, Veenstra TD, Conrad TP, Lempicki RA, McLaughlin S, Pascuccio M, Gopaul R, McNally J, Cruz CC, Censoplano N, Chung E, Reitano KN, Kottilil S, Goode DJ, Fauci AS (2008) HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol 9:301–309. doi:10.1038/ni1566
Rudnicka D, Feldmann J, Porrot F, Wietgrefe S, Guadagnini S, Prevost MC, Estaquier J, Haase AT, Sol-Foulon N, Schwartz O (2009) Simultaneous cell-to-cell transmission of human immunodeficiency virus to multiple targets through polysynapses. J Virol 83:6234–6246. doi:10.1128/jvi.00282-09
Jolly C, Mitar I, Sattentau QJ (2007) Adhesion molecule interactions facilitate human immunodeficiency virus type 1-induced virological synapse formation between T cells. J Virol 81:13916–13921. doi:10.1128/jvi.01585-07
Haller C, Fackler OT (2008) HIV-1 at the immunological and T-lymphocytic virological synapse. Biol Chem 389:1253–1260. doi:10.1515/bc.2008.143
Vasiliver-Shamis G, Dustin ML, Hioe CE (2010) HIV-1 virological synapse is not simply a copycat of the immunological synapse. Viruses 2(5):1239–1260. doi:10.3390/v2051239
Sattentau Q (2010) Cell-to-cell spread of retroviruses. Viruses 2:1306–1321
Martin N, Welsch S, Jolly C, Briggs JA, Vaux D, Sattentau QJ (2010) Virological synapse-mediated spread of human immunodeficiency virus type 1 between T cells is sensitive to entry inhibition. J Virol 84:3516–3527. doi:10.1128/jvi.02651-09
Gupta P, Balachandran R, Ho M, Enrico A, Rinaldo C (1989) Cell-to-cell transmission of human immunodeficiency virus type 1 in the presence of azidothymidine and neutralizing antibody. J Virol 63:2361–2365
Dale BM, McNerney GP, Thompson DL, Hubner W, de Los RK, Chuang FY, Huser T, Chen BK (2011) Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion. Cell Host Microbe 10:551–562. doi:10.1016/j.chom.2011.10.015
Hubner W, McNerney GP, Chen P, Dale BM, Gordon RE, Chuang FY, Li XD, Asmuth DM, Huser T, Chen BK (2009) Quantitative 3D video microscopy of HIV transfer across T cell virological synapses. Science 323:1743–1747. doi:10.1126/science.1167525
Sloan RD, Kuhl BD, Mesplede T, Munch J, Donahue DA, Wainberg MA (2013) Productive entry of HIV-1 during cell-to-cell transmission via dynamin-dependent endocytosis. J Virol 87:8110–8123. doi:10.1128/jvi.00815-13
Sigal A, Kim JT, Balazs AB, Dekel E, Mayo A, Milo R, Baltimore D (2011) Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature 477:95–98. doi:10.1038/nature10347
Abela IA, Berlinger L, Schanz M, Reynell L, Gunthard HF, Rusert P, Trkola A (2012) Cell-cell transmission enables HIV-1 to evade inhibition by potent CD4bs directed antibodies. PLoS Pathog 8:e1002634. doi:10.1371/journal.ppat.1002634
Durham ND, Yewdall AW, Chen P, Lee R, Zony C, Robinson JE, Chen BK (2012) Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail. J Virol 86:7484–7495. doi:10.1128/jvi.00230-12
Swartz TH, Esposito AM, Durham ND, Hartmann B, Chen BK (2014) P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection. J Virol 88:11504. doi:10.1128/jvi.01158-14
Massanella M, Puigdomenech I, Cabrera C, Fernandez-Figueras MT, Aucher A, Gaibelet G, Hudrisier D, Garcia E, Bofill M, Clotet B, Blanco J (2009) Antigp41 antibodies fail to block early events of virological synapses but inhibit HIV spread between T cells. AIDS 23:183–188. doi:10.1097/QAD.0b013e32831ef1a3
Sanchez-Palomino S, Massanella M, Carrillo J, Garcia A, Garcia F, Gonzalez N, Merino A, Alcami J, Bofill M, Yuste E, Gatell JM, Clotet B, Blanco J (2011) A cell-to-cell HIV transfer assay identifies humoral responses with broad neutralization activity. Vaccine 29:5250–5259. doi:10.1016/j.vaccine.2011.05.016
Malbec M, Porrot F, Rua R, Horwitz J, Klein F, Halper-Stromberg A, Scheid JF, Eden C, Mouquet H, Nussenzweig MC, Schwartz O (2013) Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission. J Exp Med 210:2813–2821. doi:10.1084/jem.20131244
Agosto LM, Zhong P, Munro J, Mothes W (2014) Highly active antiretroviral therapies are effective against HIV-1 cell-to-cell transmission. PLoS Pathog 10:e1003982. doi:10.1371/journal.ppat.1003982
Titanji BK, Aasa-Chapman M, Pillay D, Jolly C (2013) Protease inhibitors effectively block cell-to-cell spread of HIV-1 between T cells. Retrovirology 10:161. doi:10.1186/1742-4690-10-161
Hubner W, Chen P, Del Portillo A, Liu Y, Gordon RE, Chen BK (2007) Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. J Virol 81:12596–12607. doi:10.1128/jvi.01088-07
Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chen BK, Strominger JL, Baltimore D (1999) The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10:661–671
Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59:284–291
Cavrois M, De Noronha C, Greene WC (2002) A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20:1151–1154. doi:10.1038/nbt745
Tobiume M, Lineberger JE, Lundquist CA, Miller MD, Aiken C (2003) Nef does not affect the efficiency of human immunodeficiency virus type 1 fusion with target cells. J Virol 77:10645–10650
Cavrois M, Neidleman J, Bigos M, Greene WC (2004) Fluorescence resonance energy transfer-based HIV-1 virion fusion assay. Methods Mol Biol 263:333–344. doi:10.1385/1-59259-773-4:333
Acknowledgments
We thank members of the B. K. Chen Lab for helpful comments and the Flow Cytometry Shared Resource Facility, Icahn School of Medicine at Mount Sinai, for assistance. This work was supported by NIH/NIDA DA028866 and NIH/NIAID A1074420.
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
Durham, N.D., Chen, B.K. (2016). Measuring T Cell-to-T Cell HIV-1 Transfer, Viral Fusion, and Infection Using Flow Cytometry. 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_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3046-3_2
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