The Proton-Activated Chloride Channel Inhibits SARS-CoV-2 Spike Protein-Mediated Viral Entry Through the Endosomal...

Area of Science:

• Virology
• Cell Biology
• Molecular Biology

Background:

• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters cells via endocytosis, a process dependent on endosomal acidification.
• Proton pumps (V-ATPase) drive acidification, while chloride transporters regulate proton gradients.
• The Proton-Activated Chloride (PAC) channel was recently identified as a regulator of endosomal pH.

Purpose of the Study:

• To investigate the role of the PAC channel in SARS-CoV-2 viral entry.
• To determine if PAC can be a therapeutic target against SARS-CoV-2 infection.

Main Methods:

• Overexpression of PAC in ACE2-expressing HEK 293T cells.
• Pseudoviral entry assays.
• Analysis of endosomal acidification and localization.
• Inhibition assays using cathepsin inhibitors (E64-d) and TMPRSS2 expression.
• Plaque assays with live SARS-CoV-2 isolates (B.1 and Omicron XBB.1.16) in Vero E6 cells.

Main Results:

• PAC overexpression significantly inhibited SARS-CoV-2 spike-mediated viral entry.
• PAC's inhibitory effect was dependent on its endosomal localization and channel activity.
• The inhibition mechanism involved suppressing the endosomal entry pathway, similar to cathepsin inhibition.
• PAC overexpression reduced plaque formation by live SARS-CoV-2 isolates.

Conclusions:

• The PAC channel is a crucial inhibitor of SARS-CoV-2 entry.
• PAC functions by negatively regulating endosomal acidification.
• PAC represents a potential novel therapeutic target for SARS-CoV-2 and other viruses utilizing the endosomal pathway.