Identifying host entry factors underlying respiratory virus tropism for ciliated cells

Respiratory viruses pose a major global health challenge, exhibiting strong tropism for the airway epithelium, with ciliated cells often serving as primary hosts. However, the host factors driving this specificity remain incompletely understood. Human coronavirus HKU1 (HCoV-HKU1) is of particular interest due to its poorly characterised entry mechanisms and difficulty to cultivate in standard immortalised cell culture systems. This study aimed to identify host factors enriched in respiratory ciliated cells that may influence viral entry using computational and experimental approaches. Publicly available single-cell RNA sequencing datasets from healthy adult respiratory tissues were analysed to identify transcripts enriched or depleted in ciliated epithelial cells. Candidates were selected based on expression patterns, subcellular localisation, and known virus-host interactions. CDHR3, CIB1, and EZR emerged as leading candidates and were evaluated using in silico docking and gain-of-function entry assays. To investigate their roles across respiratory viruses, pseudovirus entry assays were performed using glycoproteins from SARS-CoV-1, SARS-CoV-2 wild-type and its variants (Alpha, Beta, Delta, BA.1, BA.2, BA.4, BA.5, XBB, XQ1.1), and influenza A virus. Extensive optimisation was also undertaken to establish an HCoV-HKU1 pseudovirus entry model. Overexpression of CDHR3, CIB1, or EZR did not significantly enhance viral entry in vitro. However, a statistically significant increase in entry was observed for the SARS-CoV-2 Alpha variant in CDHR3 overexpressing cells, suggesting a potential variant-specific interaction. Overall, these findings highlight the complexity of respiratory virus entry and the challenges associated with modelling HCoV-HKU1 infection in vitro. This study provides a rational framework for identifying host factors involved in viral entry and highlights the importance of combining transcriptomic analysis with functional validation.