Min Jin, Victoria A Jefferson, Zhe Zhao, Nicholas J Catanzaro, Luca De Sabato, Emma L Keeler, Bridget L Menasche, Cameron Hoffman, Boyd L Yount, Anna Castelli, Ilaria Di Bartolo, Gabriele Vaccari, Tyler N Starr, Stephanie N Seifert, Adrian A Castellanos, Barbara A Han, Leonid Serebryannyy, Alicen B Spaulding, Daniel C Douek, Ana Moreno, Ralph S Baric, Craig B Wilen, James M Rini, Michael Letko
Merbecoviruses, closely related to the Middle East respiratory syndrome coronavirus, circulate in hedgehogs throughout Europe and Asia, raising concerns about zoonotic transmission to humans and domestic animals. However, it is not clear how these viruses enter host cells. Here we tested three coronavirus receptors from European hedgehogs (Erinaceus europaeus) in cell-based assays and identified aminopeptidase N (APN) as a receptor for hedgehog merbecoviruses. We verified it by pseudotype experiments using an in vitro reporter system based on replication competent vesicular stomatitis virus and protein binding assays. Hedgehog coronavirus spike proteins showed enhanced infectivity when produced at 33 °C, approximating the physiological hedgehog body temperature, as compared with 37 °C. A screen of 30 mammalian APN orthologues showed restricted cross-species receptor use, including the inability to use human APN. Electron cryomicroscopy revealed a distinct glycoprotein-receptor interface unlike known coronavirus spike-APN interactions, clarifying species barriers. These findings broaden our understanding of receptor use across merbecoviruses and betacoronaviruses and inform risk assessments for viral emergence.