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Measuring T Cell-to-T Cell HIV-1 Transfer, Viral Fusion, and Infection Using Flow Cytometry

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1354)

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.

Key words

HIV entry Cell-to-cell transfer Cell-to-cell infection Virological synapse Neutralization assay Fluorescent reporter virus Gag-iGFP β-lactamase (BlaM) fusion assay 
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Notes

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.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Infectious Diseases, Department of Medicine, Immunology InstituteIcahn School of Medicine at Mt. SinaiNew YorkUSA

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