Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection

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

® JHM HEϪ -CoV entry by 38% (DBT), 38% (HeLa-mCEACAM), and 54% (BHK) (Fig. 2C) . After neuraminidase pretreatments, the cells were similarly resistant to IAV pp transduction, but not VSV G protein (VSV G)-containing pseudoparticle (VSVG pp) transduction (Fig. 2C) . Partial resistance to IAV pp, a known sialic acid-requiring virus (49) (50) (51) , is best explained by incomplete removal of sialic acids by neuraminidase. We infer that the partial neuraminidase resistance of CoV VLPs is similarly explained by incomplete sialic acid removal. These results support a model in which initial virus binding to sialic acids facilitates protein receptor-mediated entry. Sialic acid receptors promote JHM viral spread without requiring mCEACAM protein receptors. CoV infections spread through the canonical process of progeny virus release and reinfection, and also through fusion, i.e., syncytial spread, with uninfected cells. JHM S proteins can uniquely mediate syncytia in several conditions, even when mCEACAM protein receptors are absent (33, 35) . To determine whether sialic acids operate independently of mCEACAM receptors in JHM cell-cell membrane fusion and syncytial development, we cultured JHM CoV-infected mouse (DBT) cells with mCEACAM-negative human (HeLa) cells that separately harbored either DSP 1-7 or DSP [8] [9] [10] [11] , such that HeLa cell-cell fusions would permit DSP complementation into active Rluc (Fig. 3A) . Using live-cell Rluc substrate, the development of active Rluc was measured over time, and in the presence of increasing neuraminidase doses. The results (Fig. 3B ) revealed neuraminidase dose-dependent reductions in JHM CoVinduced syncytia, up to 7.4-fold at the highest neuraminidase dose (2 U/ml). Similar, but less pronounced reductions were observed in parallel cultures of CEACAM-positive HeLa cells (Fig. 3C ). Of note, the neuraminidase treatments significantly reduced cell-surface sialic acids, as revealed by adherence of fluorescent wheat germ agglutinin (WGA) to treated cells (Fig. S2 ). From these findings, we conclude that sialic acids can enable a CoV S-mediated cell-cell fusion process without requiring a prototypic proteinaceous receptor.