Esposito Anthony M, Soare Alexandra Y, Patel Foramben, Satija Namita, Chen Benjamin K, Swartz Talia H
Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute; Department of Biology, New Jersey City University.
Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute.
J Vis Exp. 2018 Aug 14(138):58074. doi: 10.3791/58074.
This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.
该检测方法旨在通过流式细胞术或荧光显微镜检测绿色荧光蛋白（GFP）的表达，专门报告HIV-1融合情况。通过将Cre重组酶插入HIV-1基因组中Gag多聚蛋白的基质蛋白和衣壳蛋白之间，产生一种HIV-1报告病毒（HIV-1 Gag-iCre）。这导致Cre重组酶被包装到病毒颗粒中，然后释放到稳定表达Cre重组酶激活的红色荧光蛋白（RFP）到GFP转换盒的靶细胞系中。在基础状态下，该盒仅表达RFP。将Cre重组酶递送至靶细胞后，两侧带有loxP位点的RFP切除，导致GFP表达。该检测方法可用于测试无细胞和细胞间感染系统中任何病毒进入抑制剂（特别是在融合步骤），并已用于鉴定一类嘌呤能受体拮抗剂作为HIV-1病毒膜融合的新型抑制剂。
