title: Measuring T Cell-to-T Cell HIV-1 Transfer, Viral Fusion, and Infection Using Flow Cytometry.
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.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Immunology Institute, Icahn School of Medicine at Mt. Sinai, One Gustave Levy Place, Box 1090, New York, NY, 10029, USA.
authors.forename: Natasha D
authors.initials: ND
authors.lastname: Durham
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Immunology Institute, Icahn School of Medicine at Mt. Sinai, One Gustave Levy Place, Box 1090, New York, NY, 10029, USA. benjamin.chen@mssm.edu.
authors.forename: Benjamin K
authors.initials: BK
authors.lastname: Chen
mesh_terms: D000818:Animals; D002450:Cell Communication; D018929:Cell Culture Techniques / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D015658:HIV Infections / Q000473:pathology* / Q000821:virology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D008561:Membrane Fusion; D013601:T-Lymphocytes / Q000166:cytology / Q000473:pathology* / Q000821:virology*; D053586:Virus Internalization
keywords: Cell-to-cell infection; Cell-to-cell transfer; Fluorescent reporter virus; Gag-iGFP; HIV entry; Neutralization assay; Virological synapse; β-lactamase (BlaM) fusion assay