A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

Abstract

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

Figure 1. Cellular electrical impedance as a measure of viral induced cell fusion.

(A) Assay schematic. Cells are grown in microtitre plates (96- or 384-well) with embedded gold microelectrodes. Viral fusion proteins expressed on the surface of infected or transfected cells are triggered to undergo a conformational change following receptor binding or exposure to low pH depending on viral fusion class. This conformational change initially involves the formation of a short-lived, elongated intermediate that collapses into a trimeric hairpin, which drives the merger of neighboring cell membranes (dengue virus E protein mediated membrane fusion is schematically shown in the lower inset). (B) SEM of cells growing on microelectrodes. Cells were fixed and imaged when infected cells showed maximum cell impedance. The large syncytia that accompany cell fusion are clearly visible in both DENV and RSV infected cells (C6/36 insect cells and BEAS2B human bronchial airway epithelial cells, respectively). (C) Maximum cell index, CI (bars represent SE; N = 3), following infection of cells by RSV and DENV or transient expression of recombinant VSV G. show significant membrane fusion for all three fusion classes. (D–F) Raw, real-time output of impedance measurements over the time course of the fusion assay reveals quantitative fusion detection that correlates with live cell microscopy (lower panels) for all three classes of viral fusion proteins. Microscopy based assays were performed with C6/36 cells for DENV and imaged in bright field, while RSV and VSV fusion were visualized using fluorescent microcopy of a mixed population of stable COS-7 cells expressing GFP and mCherry, fusion is visualised as an overlap of the GFP and mCherry signals. Measurements were performed on infected (RSV and DENV) cells and cells transiently expressing recombinant VSV-G. Maximal cell impedance for DENV and VSV G was only observed following treatment of cells with low pH.

Figure 2. Label-free quantification of dengue fusion inhibition by a monoclonal antibody.

(A) Schematic of the mode of action for the DENV neutralizing monoclonal antibody 4G2, which binds to the fusion peptide, FP. After acidification, molecular rearrangement of the E protein exposes the hydrophobic FP (green), which associates with the host cell membrane. 4G2 binding to the exposed FP prevents membrane insertion and therefore blocks membrane fusion. (B) Measurement of the anti-fusion effects of 4G2 using cellular impedance. Cells were infected at an MOI of 2 or mock infected. Twenty-four hour post infection the culture media was replaced with fresh media at pH 6 and the CI recorded. The cell impedance measurement at 1.5 h post media change is shown. The presence of 4G2 (500 μg/ml) within the low pH media was sufficient to completely abrogate the fusion signal. An isotype matched antibody control, 9C4 (specific for the RSV F protein) had no significant effect. (C) Parallel fusion assay setup to (B) was observed using live cell bright field microscopy. Similar anti-fusion effects of 4G2 were observed, with fusion extent inversely proportional to cell number. (D) A dose response analysis of the inhibitory effect of 4G2 using both cell impedance and live cell microscopy revealed near identical activity profiles. The CEI measurement is indicated on the left axis and BFM measurements are plotted on an inverted right axis to allow direct comparison of the dose response curves observed. (E) 4G2 inhibition of DENV cell infection analyzed using PRNT analysis. Control antibody 9C4 was observed to have no antiviral activity in this assay format.

Figure 3. Label-free quantification of RSV F mediated fusion by TMC-353121.

(A) Schematic of the mode of action of the small molecule RSV fusion inhibitor TMC-353121. Upon activation of the F protein through receptor engagement, an elongated form exposing a trimeric helical bundle is formed. TMC-353121 (yellow) binds to the trimeric helical bundle, thus preventing the collapse of F into the hairpin post-fusion conformation, which drives membrane fusion. (B) TMC-353121 is a potent inhibitor of virus infection as demonstrated by PRNT (IC₅₀ of 5.8 nM). (C) Anti-fusion activity of TMC-353121 against transiently expressed recombinant F measured using cell impedance (blue). No effect on cell impedance of the compound was observed for cells transfected with a noncoding empty vector (eV, black). (D) A luciferase reporter based fusion assay demonstrates the anti-fusion activity of TMC-353121 against both live virus (red) and transiently expressed recombinant F protein (blue). (E) Antifusion activity of TMC-353121 against RSV can be measured using cell impedance (red). No change in cell impedance following treatment of control cells with the compound was observed (black).

Figure 4. Demonstration of QSAR using a small panel of RSV fusion inhibitors.

(A) Chemical structures of the inhibitors (BTA-1 to 7 and TMC-353121) used in the assay validation. (B–D) Tabular IC₅₀ and corresponding scatter plots demonstrating a high correlation between the impedance based assays, whether in 96- or 384-well format with other standard assays over a wide range of inhibitor potencies. Tabulated summary of compound IC₅₀ or EC₅₀ values (log₁₀ M, SE shown in parentheses) measured either by cellular electrical impedance (in 96- and 384-well formats), luciferase reporter cell content mixing assay or a cytopathic effect (CPE) assay. A spread of IC₅₀ values over three orders of magnitude was observed using both fusion assays against live virus infection in a 96-well format and a spread of two orders of magnitude is observed for transiently expressed recombinant F (recF) based fusion assays in a 384-well format. The SE reported for the impedance fusion assay is generally smaller than the corresponding luciferase reporter, cell content mixing assay. R² values shown were obtained via linear regression analysis.