A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Abstract

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.

Purinergic Receptors: Elucidating the Role of these Immune Mediators in HIV-1 Fusion

“…NF449 and NF279 were further reported as fusion inhibitors in primary monocyte-derived macrophage (MDM), CEM-A, Jurkat, and NP2 cells as measured by the BlaM-Vpr assay [93,106]. Our laboratory developed a novel HIV-1 Cre-lox assay to measure cell-to-cell mediated virus-membrane fusion [96,116,117]. In this assay, transduced Jurkat cells express a reporter cassette containing a lox-P flanked dsRed reporter followed by a Cre-inducible GFP site.…”

“…In this assay, transduced Jurkat cells express a reporter cassette containing a lox-P flanked dsRed reporter followed by a Cre-inducible GFP site. Transfected donor Jurkat cells produce HIV-1 that is derived from NL4-3 and packages the Cre Recombinase enzyme (HIV-1 Gag-iCre); HIV-1 Gag-iCre viral fusion with target Jurkat floxRG donor cells releases Cre into the target cell cytoplasm, thereby inducing a red-to-green switch in fluorescence [116,117]. Using this assay, we identified the P2RX1-selective compounds NF279, PPNDS, and NF023 as potent inhibitors of cell-to-cell mediated virus-membrane fusion.…”

P2X1 Selective Antagonists Block HIV-1 Infection through Inhibition of Envelope Conformation-Dependent Fusion

“…Our earlier studies demonstrated that nonselective P2X antagonists could inhibit HIV-1 productive infection at the level of viral membrane fusion (32,37). We began by probing the role of more selective inhibitors of P2X receptor subtypes.…”

“…However, the manner and site by which these antagonists are able to block HIV-1 infection have not been fully determined. Inhibition of these receptors through various antagonists has been demonstrated to impact the HIV-1 life cycle at the stage of viral membrane fusion (31,32,(36)(37)(38). Giroud et al demonstrated that P2X1 antagonists blocked HIV-1 fusion by blocking virus interactions with coreceptors C-C chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) (31,38).…”

Purinergic Receptors: Elucidating the Role of These Immune Mediators in HIV-1 Fusion

“…In this assay, transduced Jurkat cells express a reporter cassette containing a lox-P flanked dsRed reporter followed by a Cre-inducible GFP site. Transfected donor Jurkat cells produce HIV-1 that is derived from NL4-3 and packages the Cre Recombinase enzyme (HIV-1 Gag-iCre); HIV-1 Gag-iCre viral fusion with target Jurkat floxRG donor cells releases Cre into the target cell cytoplasm thereby inducing a red-togreen switch in fluorescence(115,116). Using this assay, we identified the P2X1R-selective compounds NF279, PPNDS, and NF023 as potent inhibitors of cell-to-cell mediated virus-membrane fusion.…”