Pseudovirus-mediated proximity labeling identifies candidate host cell membrane proteins involved in viral attachment

Viral infections pose a significant threat to humanity, as exemplified by the COVID-19 pandemic. To mitigate the associated damage, understanding the biological characteristics of causative viruses is essential. In this study, we report a novel method for identifying host cell molecules (including virus receptors) required for viral attachment, referred to as host cell attachment factors (HCAFs). Our approach utilizes proximity labeling (PL) technology; pseudoviruses engineered to express a PL enzyme are applied to host cells, where they bind and initiate PL. Because HCAFs are expected to localize near viral attachment sites, candidate HCAFs were specifically tagged using the PL process. The analysis of influenza HCAFs enabled the identification of multiple candidate molecules. Subsequent viral attachment experiments using Chinese hamster ovary cells suggested that neuropilin-1 may serve as an HCAF for influenza viruses. Given its simplicity and rapid turnaround, this method is adaptable to a wide range of enveloped viruses, including pandemic viruses that require emergency analyses.IMPORTANCEIdentifying cellular receptors for viruses is not only crucial in conventional virology research but also plays a key role in addressing pandemic viruses. The significance of our work lies in establishing a standardized method for viral receptor identification, an area in which no widely accepted protocol has previously existed, by utilizing pseudovirus-mediated proximity labeling technology. Moreover, this approach provides a highly versatile platform that enables rapid and low-cost identification of viral receptors.