A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

Abstract

Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.

Keywords: Cre recombinase; Drug screening; Env; Gag; HIV-1; Viral membrane fusion; Virus entry.

Fig. 1

HIV Gag-iCre carries the Cre gene inserted into HIV gag. (A) Insertion of Cre between the MA and CA domains allow high levels of Cre to be packaged into virus particles, with the Cre enzyme cleaved out of this precursor by the viral protease. (B) ELISA quantitation of p24 produced by Gag-iCre particles compared to NL4-3. (C) Western analysis of producer cell lysates using anti-cre antibody. (D) Western analysis of virus particles from supernatants using anti-cre antibody.

Fig. 2

HIV Gag-iCre reporter signal is specific to fusion. (A) Jurkat floxRG target cells are transduced with the MSCV floxR-G cassette that expresses dsRed (upper) and switches to GFP expression upon fusion by recombination by Cre. (B) Fluorescent microscopy of infection of RG Jurkats incubated with Gag-iCre in the presence of fusion inhibitor (AMD3100), reverse transcription inhibitor (AZT) or untreated. (C) Red channel fluorescence of samples shown in C. (D) FACS analysis of RG Jurkats exposed to HIV Gag-iCre in the presence and absence of inhibitors.

Fig. 3

Viral membrane fusion after cell-to-cell infection. (A) FACS analysis of RG Jurkat target cells co-cultured with indicated donor cells for 48 h with or without inhibitors. (B) Titrations of AMD3100, Indinavir, AZT and integrase inhibitors in Gag-iCre infected cells co-cultured with RG Jurkat for 48 h. Values normalized to uninfected controls (8% GFP positive).

Fig. 4

HIV Gag-iCre reporter signal is comparable to Vpr Blam. (A) FACS analysis of Jurkat floxRG cells spinoculated with Gag-iCre virus and Jurkat cells spinoculated with NL43 Vpr Blam 16 h after spinnoculation. (B) Dose response plot of viral input versus percent fusion evident in target cells following Gag-iCre of Vpr–Blam assays. Bars represent error from 2 replicates. (C) Z factor plotted as a function of increasing inhibitor concentration for Gag-iCre and Vpr Blam assays.

Fig. 5

Small molecule library screening overview. (A) Nucleofected Gag-iCre donor cells were co-cultured with Jurkat floxRG target cell line in the presence of each of the 1998 library compounds, performed in duplicate. Plates were incubated for 48 h followed by fixation and quantitation of fusion by flow cytometry. (B) Percent inhibition was calculated by normalizing to DMSO treated and uninfected controls. All compounds that inhibited fusion by 30% or more were considered first round hits.

Fig. 6

TZM assay follow up for select compounds. TZM-bl cells were infected with NL4-3 virus particles and treated with indicated compound for 48 h. % inhibition is relative to DMSO-treated control samples. Plots on the left represent data from GagiCre titration follow up, while plots on the right represent TZM-bl assay. For both assays viability is measured by Cell-titer Glo.

Fig. 7

Modifications of Gag-iCre assay to study viral tropism and heterologous viral fusion glycoproteins. (A) Donor Jurkat cells were nucleofected with HIV Gag-iCre JRFL and co-cultured with the CCR5 expressing T cell Cre-responsive cell line, A3R5-RG for 48 h untreated or in presence of fusion antagonists, TAK779 or AMD3100. Entry efficiency was measured by flow cytometry. (B) Pseudotypted Gag-iCreΔEnv was produced by co transfection of HIV Gag-iCre with Ebola GP, Ebola mutant GP or VSV envelope. Cell free virus co-cultured with Hela RG target cell line for 48 hours. Entry efficiency was measured by flow cytometry.