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.
Highlights
Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses
To demonstrate practical advantages and broad potential of the assay we assessed cellular fusion induced by all three viral fusion classes, represented by respiratory syncytial virus (RSV, class I), dengue virus (DENV, class II) and vesicular stomatitis virus (VSV, class III)
Fusion is triggered by the addition of acidified media, which mimics the low pH environment of the endosome where viral entry occurs for some viruses (DENV and VSV), or once viral fusion glycoproteins are transported to the cell surface where they can interact with receptor molecules on a neighboring cell (RSV)
Summary
Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. 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. Despite significant differences in structure, a common fusion mechanism has been proposed, where either receptor binding or low pH within the cellular endosome, triggers the formation of an elongated intermediate fusion protein that inserts a hydrophobic fusion peptide (FP) into the target host membrane Subsequent collapse of these intermediates into a low energy hairpin-like structure provides the driving force required for membrane fusion. Electrical impedance assays are one such approach suitable for plate-based screening of live cells[14,15]
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