12 Altered mechanisms of cell entry in clinical isolates of the seasonal coronavirus OC43

Objectives/Goals: The human coronavirus OC43 is a Betacoronavirus, like SARS-CoV-2, but causes limited disease. Current clinical OC43 strains replicate in airway epithelial cultures while the lab-adapted strains only replicate in immortalized cells. We will identify the host and viral factors causing this difference. Methods/Study Population: To observe the effects of specific mutations present in clinical isolates of OC43, we will construct infectious clones of clinical and lab-adapted strains. Through targeted mutagenesis, we will characterize structural differences present in the Spike protein of clinical strains and identify the tropism-determining genome regions. By manipulating expression levels of enzymes involved in sialic acid production including SIAE and CasD1 and using a carbohydrate microarray to further evaluate carbohydrate binding, we will evaluate whether clinical OC43 isolates use the same 9-O-acetylated sialic acid identified as a receptor for the lab-adapted OC43 strain (VR-1558). Results/Anticipated Results: In primary human lung epithelial cell cultures, clinical strains of OC43 persistently replicate through 30 days post-infection. In contrast, the lab-adapted strain did not replicate. Growth on standard immortalized cells demonstrated the reverse phenotype with extensive replication of VR-1558 but no replication by the clinical isolates. Using AlphaFold to predict the 3D structure of Spike from a clinical isolate of OC43, we identified differences at a region previously identified as the binding site for its 9-O-acetylated sialic acid receptor. Further testing will determine whether these structural differences in Spike are responsible for the observed differences in viral tropism. Discussion/Significance of Impact: This project will deepen knowledge of coronavirus biology and the drivers of respiratory cell tropism. It will also develop novel tools for the study of OC43 and particularly of clinically relevant circulating strains which are better models for developing broad-spectrum therapies.