Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped single-stranded positive-sense RNA virus, is a member of the genus Betacoronavirus, family Coronaviridae. The SARS-CoV envelope protein E is a small (∼8.4 kDa) channel-forming membrane protein whose sequence is highly conserved between SARS-CoV and SARS-CoV-2. As a viroporin, it is involved in various aspects of the virus life cycle including assembly, budding, envelope formation, virus release, and inflammasome activation. Here, SARS-CoV E protein was recombinantly expressed in HEK293 cells and channel activity and the effects of viroporin inhibitors studied using patch-clamp electrophysiology and a cell viability assay. We introduced a membrane-directing signal peptide to ensure transfer of recombinant E protein to the plasma membrane. E protein expression induced transmembrane currents that were blocked by various inhibitors. In an ion-reduced buffer system, currents were proton-dependent and blocked by viroporin inhibitors rimantadine and amantadine. I-V relationships of recombinant E protein were not pH-dependent in a classical buffer system with high extracellular Na+ and high intracellular K+. E-protein mediated currents were inhibited by amantadine and rimantadine, as well as 5-(N,N-hexamethylene)amiloride (HMA). We tested a total of 10 flavonoids, finding inhibitory activity of varying potency. Epigallocatechin and quercetin were most effective, with IC50 values of 1.5 ± 0.1 and 3.7 ± 0.2 nM, respectively, similar to the potency of rimantadine (IC50 = 1.7 ± 0.6 nM). Patch-clamp results were independently verified using a modified cell viability assay for viroporin inhibitors. These results contribute to the development of novel antiviral drugs that suppress virus activity and proliferation.

Highlights

  • Coronaviruses (CoVs) cause respiratory diseases in humans ranging from common colds, bronchiolitis to acute respiratory distress syndrome (ARDS) and fatal pneumonias

  • A range of known viroporin inhibitors as well as several flavonoids were tested for their activity against recombinant E protein

  • We confirmed the activity of the classical viroporin inhibitors amantadine, rimantadine, and HMA on severe acute respiratory syndrome (SARS) CoV in a cell viability assay and by patch clamp electrophysiology on recombinant E protein expressed in HEK293 cells

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Summary

Introduction

Coronaviruses (CoVs) cause respiratory diseases in humans ranging from common colds, bronchiolitis to acute respiratory distress syndrome (ARDS) and fatal pneumonias. SARS-CoV-2, SARS-CoV and MERS-CoV (subgenus Merbecovirus) are highly pathogenic zoonotic viruses; the associated diseases are COVID-19 (2019 Wuhan, China), SARS (2002 Guangdong province, China) and MERS (2012 Kingdom of Saudi Arabia) whose mortality severely affected the economies of countries where the viruses spread (Liu et al, 2020). Severe cases result in ARDS with systemic inflammation; lung injury is associated with release of inflammatory cytokines interleukin (IL-6) and IL-1b (Freeman and Swartz, 2020). Release of inflammatory cytokines in hepatitis C (Shrivastava et al, 2013; Farag et al, 2017, 2020; Negash et al, 2019) is associated with ion channel activity of the HCV p7 protein (Farag et al, 2017, 2020). COVID-19 severity is associated with increased proinflammatory cytokines and chemokines and IL-6, is predictive of COVID-19 fatality (Costela-Ruiz et al, 2020)

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