Specific asparagine-linked glycosylation sites are critical for DC-SIGN- and L-SIGN-mediated SARS-CoV entry

Departments of Medicine, Biochemistry, Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

^* To whom correspondence should be addressed. Email: mcho{at}case.edu.

	Abstract

Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus designated SARS-CoV. The virus utilizes angiotensin-converting enzyme 2 (ACE2) as the primary receptor. Although less clear and somewhat controversial, SARS-CoV is thought to use C-type lectins DC-SIGN and/or L-SIGN (collectively referred to as DC/L-SIGN) as alternative receptors or as enhancer factors that facilitate ACE2-mediated virus infection. In this study, the function of DC/L-SIGN in SARS-CoV infection was examined in detail. The results of our study clearly demonstrate that both proteins serve as receptors independent of ACE2, and that there is a minimal level of synergy between DC/L-SIGN and ACE2. As expected, glycans on spike (S) glycoprotein are important for DC/L-SIGN-mediated virus infection. Site-directed mutagenesis analyses have identified seven glycosylation sites on the S protein critical for DC/L-SIGN-mediated virus entry. They include asparagine residues at amino acid positions 109, 118, 119, 158, 227, 589 and 699, which are distinct from the ACE-2 binding domain (a.a. 318-510). Amino acid sequence analyses of S proteins encoded by viruses isolated from animals and humans suggest that glycosylation sites N227 and N699 may have facilitated zoonotic transmission.