Specific asparagine-linked glycosylation sites are critical for DC-SIGN- and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry

Abstract

Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus (CoV) designated SARS-CoV. The virus utilizes angiotensin-converting enzyme 2 (ACE2) as the primary receptor. Although the idea is 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 independently 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 residues of the ACE2-binding domain (amino acids 318 to 510). Amino acid sequence analyses of S proteins encoded by viruses isolated from animals and humans suggest that glycosylation sites N227 and N699 have facilitated zoonotic transmission.

FIG. 1.

DC-SIGN and L-SIGN serve as alternative receptors. (A) HeLa cells transfected with plasmids expressing ACE2, DC-SIGN, or L-SIGN were infected with SARS pseudoviruses. An empty vector (pcDNA) was used as a negative control. (B) ACE2- and L-SIGN-mediated infections were examined in the presence of various concentrations of an inhibitory peptide (P6) derived from ACE2 (13). (C) L-SIGN-mediated, but not ACE2-mediated, infections are inhibited by mannan in a dose-dependent manner. (D) Specific inhibition of ACE2-mediated infections by MAbs. A polyclonal anti-S-protein antiserum (poly Ab) from mice is able to inhibit infections mediated by both ACE2 and L-SIGN.

FIG. 2.

Minimal synergy between ACE2 and DC/L-SIGN in SARS infection. SARS pseudovirus infections were carried out in HeLa cells transfected with various amounts of plasmids expressing DC-SIGN (A) or L-SIGN (B) with or without ACE2 (0.25 μg). A total of 1.25 μg of DNA was used, with pcDNA plasmid as a filler DNA. Dashed lines represent additive levels of infection.

FIG. 3.

Effects of deglycosylation on SARS-CoV infectivity. SARS pseudoviruses were treated with Endo H, buffer, or culture medium for various times, as indicated, at 37°C. Pseudoviruses subsequently were added to HeLa cells expressing DC-SIGN (A), L-SIGN (B), or ACE2 (C).

FIG. 4.

Importance of specific glycosylation sites within cluster I for infections mediated by L-SIGN. (A) A schematic diagram of S glycoprotein and three clusters of potential N-linked glycosylation sites. Functional S1 and S2 domains, RBD and RBM, heptad repeat regions HR1 and HR2, and the transmembrane domain (TM) are indicated. Thirteen glycosylation sites, utilization of which was verified by mass spectrometry or biochemical analyses, are indicated by inverted triangles. (B) Effects of individual glycosylation site mutations on ACE2- or DC-SIGN-mediated SARS pseudovirus infectivity. (C) Western blot analyses of S-protein expression in TELCeB6 cells.

FIG. 5.

Infection kinetic analyses of glycosylation site mutants. (A) HeLa cells expressing L-SIGN were infected with either wild-type (WT) or mutant SARS pseudoviruses. Pseudoviruses were adsorbed to cells for either 20 or 60 min before removing the inoculum. (B) Detailed infection kinetics for mutant pseudoviruses N109Q and N118Q in HeLa cells expressing either ACE2 or L-SIGN compared to those of the wild type. Pseudoviruses were adsorbed to cells for 0, 5, 10, 20, 40, 60, 90, or 120 min before removing inoculum. Data were normalized to the wild-type virus titer from the 60-min adsorption period.

FIG. 6.

Infectivity of multiple glycosylation site mutants. (A) HeLa cells expressing ACE2 or L-SIGN were infected with wild-type (WT) or mutant pseudoviruses. (B) Western blot analyses of mutant μ5 S-protein expression in TELCeB6 cells compared to that of the wild type or mutant μ4, which exhibited normal infectivity using ACE2.

FIG. 7.

Importance of specific glycosylation sites within cluster II for infections mediated by L-SIGN. HeLa cells expressing ACE2 or L-SIGN were infected with wild-type (WT) or mutant SARS pseudoviruses.

FIG. 8.

N-linked glycosylation site mutants of SARS-CoV S protein affect both DC-SIGN and L-SIGN usage similarly. HeLa cells expressing DC-SIGN or L-SIGN were infected with wild-type (WT) or mutant SARS pseudoviruses. Mutant pseudovirus infectivity was normalized to that of the wild-type virus.

FIG. 9.

Role of N-linked glycosylation sites in S-protein function. (A) A schematic diagram of S glycoprotein. Locations of seven glycosylation sites important for DC/L-SIGN-mediated infections are shown. Yellow lines indicate positions of 17 amino acids that are different between SZ3 and Urbani. Amino acid changes critical for efficient usage of human ACE2 (K479N and S487T) are identified by inverted triangles. Amino acid changes that introduced two novel N-linked glycosylation sites are shown with red dots (K227N and L701S). (B) Amino acid sequences of S proteins of viruses isolated from civets and humans. Viruses isolated from humans at different phases of the epidemic are indicated (S, sporadic; E, early; M, middle; and L, late). Amino acid sequences are compared to that of the SZ3 isolate, and they are color coded as follows: tan, changes found predominantly in civets; yellow, changes found mostly in human isolates; green, changes found both in human and civet isolates; light blue, changes critical for human ACE2 usage; orange, changes that introduce two novel glycosylation sites; and purple, sporadic changes. Numbers indicate amino acid positions. (C) A crystal structure of an RBD of S protein complexed with neutralizing antibody 80R (15) (Protein Data Bank file 2GHW). The RBM and locations of glycosylation sites N330 and N357 are shown. The first residue of the RBD (I319) shows the likely position of glycosylation site N318. The 2GHW file was used because it included residues closest to N318. (D) Likely ability of different SARS-CoV isolates to replicate in humans. Isolate names and critical amino acid changes that could have facilitated zoonotic transmission of the virus are indicated.