Characterization of the Mechanisms Underlying Cell Surface ACE2 Receptor Modulation by Seasonal Human Coronavirus NL63

Abstract

Seasonal human coronaviruses, including sHCoV-NL63, sHCoV-229E, sHCoV-OC43, and sHCoV-HKU1, belong to the Coronaviridae family. While typically causing mild respiratory infections, sHCoVs can trigger severe complications in vulnerable populations. Among them, sHCoV-NL63 uses the ACE2 receptor, the same entry receptor used by SARS-CoV-2. Previous work from our laboratory showed that sHCoV-NL63 upregulates the ACE2 receptor after infection, potentially increasing the risk of co-infection and of emerging recombinants. The mechanisms underlying this regulation of ACE2 expression remain unknown. This study investigated whether individual sHCoV-NL63 viral proteins modulate ACE2 surface expression. Expression vectors encoding individual sHCoV-NL63 proteins were transfected into HEK293T+ACE2 and LLC-MK2 permissive cell lines. These were used to analyze protein expression and ACE2 receptor levels by fluorescence microscopy, flow cytometry and western blot. While most viral proteins were successfully expressed, none significantly increased ACE2 surface expression, suggesting that ACE2 receptor regulation may depend on coordinated events associated with productive viral infection and/or the cell-intrinsic antiviral response. Further investigation of viral proteases, host cell signalling pathways, and interferon responses may help elucidate the mechanisms responsible for ACE2 receptor modulation.