The role of ANO6/TMEM16F phospholipid scramblase in SARS-CoV-2 infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is enveloped virus and has the Spike (S) glycoprotein on the virion envelope. The proteolytic cleavage of S glycoprotein facilitates membrane fusion of the viral and cell membrane for host cell entry. Externalization of phosphatidylserine (PS) on the cell surface, mediated by the activation of phospholipid scramblase, is associated with membrane fusion between the viral envelope and host cell membrane. However, the molecular mechanism of SARS-CoV-2 and host cell membrane fusion remains largely unknown. One of the Ca2+-activated ion channel, Anoctamin 6 (ANO6) has critical roles in PS scrambling and various membrane fusion events including blood coagulation. In this study, we demonstrate that ANO6/TMEM16F-mediated cell surface exposure of PS is required for SARS-CoV-2 viral entry into the host cells and ANO6 inhibition is effective against SARS-CoV-2 infection. Application of the SARS-CoV-2 Spike pseudotyped virus (SARS2-PsV) evoked Ca2+ and ANO6-dependent PS scrambling and membrane fusion. Treatment with potent ANO6 inhibitor, A6-001 downregulated the SARS2-PsV-induced membrane fusion events. Furthermore, A6-001 inhibited the single-round infection of SARS2-PsV. Lastly, we also found that A6-001 strongly inhibits authentic SARS-CoV-2-induced PS scrambling and SARS-CoV-2 viral replications in Vero, Calu-3, and in vivo hACE2-transgenic mouse model. These results provide mechanistic insights into the viral entry process as well as a potential target for pharmacological intervention to treat SARS-CoV-2 infection.