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bioRxiv · 10.1101/2024.04.12.589332

Characterization of spike processing and entry mechanisms of seasonal human coronaviruses NL63, 229E and HKU1

Neerukonda, S. N.·Vassell, R.·Lusvarghi, S.·Liu, S.·Akue, A.·Kukuruga, M.·Wang, T. T.·Weiss, C. D.·Wang, W.
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Abstract

Although much has been learned about the entry mechanism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the details of entry mechanisms of seasonal human coronaviruses (HCoVs) remain less well understood. In the present study, we established that 293T cell lines that stably express angiotensin converting enzyme (ACE2), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) support high level transduction of lentiviral pseudoviruses bearing spike proteins of seasonal HCoVs, HCoV-NL63, -229E, or -HKU1, respectively. Our results showed that entry of HCoV-NL63, -229E and -HKU1 pseudoviruses is sensitive to endosomal acidification inhibitors (chloroquine and NH4Cl), indicating virus entry via the endocytosis route. Although HCoV-HKU1 pseudovirus infection requires TMPRSS2 expression on cell surface, endocytosis-mediated HCoV-HKU1 entry requires the serine protease domain but not the serine protease activity of TMPRSS2. We also show that amino acids in the predicted S1/S2 junctions of spike proteins of HCoV-NL63, and - 229E are essential for optimal entry but non-essential for spike-mediated entry of HCoV-HKU1. Our findings provide insights into entry mechanism of seasonal HCoVs that may support the development of novel treatment strategies. ImportanceDetails of the entry mechanisms of seasonal human coronaviruses (HCoVs) remain to be fully explored. To investigate the entry of HCoV-NL63, -229E and -HKU1 CoVs, we employed 293T cells that stably express angiotensin converting enzyme (ACE2) aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) to study entry mechanisms of pseudoviruses bearing spike proteins of HCoV-NL63, -229E and - HKU1 respectively. Our results provide new insights into the predicted S1/S2 subunit junctions, cellular receptor, and protease requirements for seasonal HCoV pseudovirus entry via endocytic route and may support the development of novel treatment strategies.

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Neerukonda, S. N., Vassell, R., Lusvarghi, S., Liu, S., Akue, A., Kukuruga, M., Wang, T. T., Weiss, C. D., Wang, W.. 2024-04-15. Characterization of spike processing and entry mechanisms of seasonal human coronaviruses NL63, 229E and HKU1. https://doi.org/10.1101/2024.04.12.589332

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