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.
Highlights
Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS) in humans
Design of cell-cell fusion system We have designed and built a unique cell-cell fusion system that incorporates several features optimized for the study of HIV-1 envelope glycoproteins (Envs)-mediated membrane fusion and its inhibition
Cell-cell fusion occurs when effector cells expressing HIV-1 Envs are cocultivated with target cells that express the appropriate receptors
Summary
Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS) in humans. Antiretroviral therapy for HIV-1 infection combines inhibitors against several functional proteins of HIV-1, including the viral reverse transcriptase, protease, gp and integrase, and includes a ligand of the CCR5 co-receptor that blocks viral entry [3]. The use of a combination of drugs efficiently decreases virus loads and extends the lifespan of HIV-1-infected individuals. It is essential to identify additional new inhibitors with low cytotoxicity and broadrange activity against diverse HIV-1 strains for future success in treating HIV-1 infection. In addition to their use as therapeutics, such inhibitors may be used to prevent HIV-1 transmission. This strategy has been validated in the recent partial success of tenofovir, a reverse transcriptase inhibitor, in preventing sexual transmission of HIV-1 when it was administrated either orally or as a topical microbicide [9,10]
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