Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection

Qing, Enya; Hantak, Michael; Perlman, Stanley; Gallagher, Tom

Virus binding to sialic acids facilitates protein receptor-mediated entry. We next aimed to identify the biological importance of the JHM spike-sialate interaction. We reasoned that this interaction could be sufficient for viral entry or may instead be an early step preceding viral engagement with protein (mCEACAM) receptors. To assess virus entry, we utilized VLPs containing N proteins fused to only one of the DSP fragments (DSP [8] [9] [10] [11] ), such that VLP entry into target cells containing DSP 1-7 fragments could be measured by reporter complementation into active Rluc or GFP moieties ( Fig. 2A) . We compared VLP entries in HeLa and HeLa-mCEACAM cells and found that Rluc entry signals required mCEACAM presence (Fig. 2B) . The entry signals were elevated by the presence of TMPRSS2 (Fig. 2B) , a known viral fusion-triggering protease (11, 12, (43) (44) (45) (46) (47) (48) . This facilitating effect of TMPRSS2 supported the contention that the VLP-based entry assay faithfully reflected authentic CoV entry processes. In conjunction with VLP binding data, these findings suggest that JHM-CoV cell entry is a multistep process, with initial virus-sialate binding and subsequent mCEACAM receptor-driven entry.