Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

Abstract

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.T he M2 gene of influenza virus encodes a 97-amino-acid, homotetrameric, proton-selective ion channel necessary for influenza virus infectivity (1, 2). Activation of M2 is triggered by low pH in host cell endosomes, resulting in an influx of protons into the virus and acidification that releases the viral RNA into the host cell (2-4). M2 also helps to package viral RNA (5, 6) and regulate pH in the Golgi apparatus of infected cells during viral assembly (7). Two FDA-approved adamantane-based drugs, amantadine and rimantadine (2,8), are potent M2 inhibitors that can neutralize influenza virus in humans and other animals. However, the emergence of widespread highly virulent adamantane-resistant strains such as avian and swine influenza virus strains (9, 10) has prompted the need for newer antivirals. Drug discovery efforts targeting M2 have been difficult due to the toxicity of M2 in cells (11), the difficulty of reconstituting M2 in liposomes for largescale application (12, 13), and the labor-intensive, low-throughput electrophysiological approaches typically used to study ion channels.To address these challenges, we developed a rapid, cell-free assay to measure the movement of proton ions directly across the lipid bilayer of virus-like particles (VLPs). High-throughput screening (HTS) of M2-VLPs using Ͼ100,000 compounds identified 19 M2-specific inhibitors, including two novel chemical scaffolds, that inhibited both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) protonophore activity also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane but that is not t...

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“…In addition, there was no difference between DMSO-treated and AMT-treated FCCP VLPs with respect to proton migration kinetics. Meanwhile, consistent with a previous report (29), VLPs spiked with PR8M2-S and PR8M2-R proteins allowed proton influx under acidic conditions ( Fig. 5B, lower left and right graphs, respectively).…”

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure‐guided surface engineering

“…Virus‐like particles (VLPs) were produced by transfection of HEK‐293T cells with the retroviral (murine leukemia virus [MLV]) Gag protein, as previously described . Three different Gag proteins were utilized for this study: wild‐type Gag (Gag‐only), fusion proteins containing either eGFP (Gag‐GFP), or TGP (Gag‐TGP).…”

Tauroursodeoxycholic acid (TUDCA) inhibits influenza A viral infection by disrupting viral proton channel M2

“…IVLPs were obtained by transfection of HEK293T cells with plasmids encoding IAV elements. IVLP-based M2 proton channel activity was measured by modifying previously published protocols [25]. Details are given in Supplemental Experimental Procedures.…”

“…Challenging iVLPm1-2 (Fig. 3e) or iVLP incorporated with Gag and M2 (iVLPgm2) [25] (Fig. S3k online) with acidic buffer resulted in membrane depolarization, an indication of M2 activation.…”