The establishment of the screening analysis for viral entry inhibitor using flow cytometry.

Abstract

The combination anti-retroviral therapy (cART) was introduced into the treatment of AIDS, the mortality due to the AIDS markedly decreased, and the convalescence of the patient was improved drastically. Although, the membrane fusion of host's T cells and HIV-1 is an essential step of early infection of HIV-1, there is low number of drugs targeting this step and it will be hope to develop new HIV-1 entry inhibitors. Moreover, it was reported that decreasing of cell membrane fluidity suppresses HIV-1 entry and cell-cell fusion. Here, we focused on the viral entry as initial step of HIV-1 infection and evaluated to establish the novel screening analysis using flow cytometry. Firstly, we screened the reagents modified cell membrane fluidity using fluorescent depolarization methods. Next, we performed cell-cell fusion inhibition assay. In this model, Tetinducible HIV-1 envelope protein (Env) expressing T cell and Target T cell were labeled with green and red fluorescent dyes, respectively and cells were co-cultured. Then, fused cells are taken on yellow color due to merge green and red color with fluorescent microscopy and can detect as double positive cells by flow cytometric analysis. We identified Cepharanthine and GUT-70 as novel viral entry/fusion inhibitors targeting host cell membrane and the availability of our screening analysis was confirmed. Our study suggests the possibility of rapidly screening for viral entry inhibitors using flow cytometry. The rapidly screening of anti-viral reagents is possible to reduce the time to apply for clinical use.