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|Title:||HIV-1 Env-mediated cell-cell fusion assay that utilizes a green fluorescent protein from the planktonic copepod Pontellina plumata|
|Abstract:||The successful entry of human immunodeficiency virus type 1 (HIV-1) relies on active functions of the viral envelope glycoproteins (Env) and human CD4 together with proper chemokine co-receptors. To specifically search for novel HIV-1 entry inhibitors, we established a cell-cell fusion assay that mimicked the entry step of the virus. The assay utilized two cell populations; effector cells that expressed HIV-1 Env on the cell surface as well as a trans-activator Tat protein, and target cells that expressed human CD4 and CXCR4 as viral receptor and co-receptor, respectively. To assess fusion, we exploited a copepod Pontellina plumata green fluorescent protein (copGFP) to be expressed under the control of a truncated HIV-1 LTR promoter in the target cells. By co-culturing the effector and the target cells, multinucleated cells or syncytia were formed by 24 hours as a result of cell-cell fusion,allowing Tat from the effector cells to activate the truncated LTR promoter in the target cells. Therefore, the amounts of syncytial formations could be directly quantified by measuring fluorescent intensity, which was demonstrated to be optimal on day 4 after co-cultivation. The assay was validated by showing that cell-cell fusion mediated by HIV-1 Env was inhibited by a known fusion inhibitor (enfuvirtide, T20) in a dose-dependent manner. The 50% inhibitory concentration (IC50) of T20 in our assay was 0.0242 µg/ml. These results indicate that our system of HIV-1 Env-mediated cell-cell fusion assay is suitable for identification and evaluation of potential HIV-1 entry inhibitors.|
|Appears in Collections:||Microbiology: International Proceedings|
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