Caroline Silveira Falleiros Silva, University of Ottawa

Caroline Silveira Falleiros Silva, Negar Joharinia, Marc-André Langlois

Fighting & Responding

Contributions of Human Coronavirus NL63 Viral Proteins to ACE2 Cell-Surface Dynamics Caroline Silveira Falleiros Silva, Negar Joharinia, Marc-André Langlois Department of Biochemistry, Microbiology & Immunology, Faculty of Medicine, University of Ottawa Introduction Seasonal human coronaviruses (sHCoVs), including HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1, belong to the Coronaviridae family. While typically associated with mild respiratory infections, sHCoVs can cause severe complications in vulnerable populations. Given the limited antiviral treatments available, a better understanding of sHCoV–host interactions is essential for developing preventative strategies against severe disease and emerging variants. NL63 utilizes ACE2, the same receptor required for SARS-CoV-2 entry. Previous work from our laboratory demonstrated that HCoV-NL63 infection induces ACE2 upregulation over infection, potentially increasing co-infection risks. However, the mechanisms underlying host receptor modulation remain unclear. Results We aimed to identify which HCoV-NL63 viral proteins contribute to ACE2 upregulation by transfecting individual viral expression vectors into cell lines and analyzing receptor expression by flow cytometry and Western blot. Flow cytometry demonstrated considerable viral protein expression, followed by modest modulation of ACE2 receptor levels. However, we encountered challenges in detecting protein expression by Western blot, limiting protein characterization. Additionally, due to the large size of ORF1, the expression wasn't successful. Conclusion To overcome these limitations, we're developing a lentiviral expression system to enhance protein expression and enable more robust analysis of receptor modulation. Furthermore, given the difficulty in expressing the ORF1 protein, the corresponding viral proteases will be cloned and evaluated as alternative candidates to assess their potential role in ACE2 receptor regulation.