title: An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors.
abstract: HIV-1 envelope glycoproteins (Envs) mediate virus entry by fusing the viral and target cell membranes, a multi-step process that represents an attractive target for inhibition. Entry inhibitors with broad-range activity against diverse isolates of HIV-1 may be extremely useful as lead compounds for the development of therapies or prophylactic microbicides. To facilitate the identification of such inhibitors, we have constructed a cell-cell fusion system capable of simultaneously monitoring inhibition efficiency and specificity. In this system, effector cells stably express a tetracycline-controlled transactivator (tTA) that enables tightly inducible expression of both HIV-1 Env and the Renilla luciferase (R-Luc) reporter protein. Target cells express the HIV-1 receptors, CD4 and CCR5, and carry the firefly luciferase (F-Luc) reporter gene under the control of a tTA-responsive promoter. Thus, Env-mediated fusion of these two cell types allows the tTA to diffuse to the target cell and activate the expression of the F-Luc protein. The efficiency with which an inhibitor blocks cell-cell fusion is measured by a decrease in the F-Luc activity, while the specificity of the inhibitor is evaluated by its effect on the R-Luc activity. The system exhibited a high dynamic range and high Z'-factor values. The assay was validated with a reference panel of inhibitors that target different steps in HIV-1 entry, yielding inhibitory concentrations comparable to published virus inhibition data. Our system is suitable for large-scale screening of chemical libraries and can also be used for detailed characterization of inhibitory and cytotoxic properties of known entry inhibitors.
authors: Department of Immunology Cancer and AIDS, Dana-Farber Cancer Institute and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Alon
authors: A
authors: Herschhorn
authors: Andres
authors: A
authors: Finzi
authors: David M
authors: DM
authors: Jones
authors: Joel R
authors: JR
authors: Courter
authors: Akihiro
authors: A
authors: Sugawara
authors: Amos B
authors: AB
authors: Smith
authors: Joseph G
authors: JG
authors: Sodroski
mesh_terms: D065100:CCR5 Receptor Antagonists; D015704:CD4 Antigens / Q000378:metabolism; D002459:Cell Fusion; D005434:Flow Cytometry; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D023581:HIV Fusion Inhibitors / Q000494:pharmacology*; D015658:HIV Infections / Q000188:drug therapy / Q000235:genetics / Q000821:virology; D015497:HIV-1 / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D049410:Luciferases, Renilla / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects*; D019713:Receptors, CCR5 / Q000378:metabolism*; D015728:Receptors, HIV / Q000378:metabolism*; D053586:Virus Internalization / Q000187:drug effects*
chemical_list: D065100:CCR5 Receptor Antagonists; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D023581:HIV Fusion Inhibitors; D019713:Receptors, CCR5; D015728:Receptors, HIV; D049410:Luciferases, Renilla