Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.A novel coronavirus (CoV), severe acute respiratory syndrome-CoV (SARS-CoV), is the causal agent of severe acute respiratory syndrome, which afflicted thousands of people worldwide in 2002 and 2003 (
10,
39). SARS-CoV is an enveloped, single- and positive-strand RNA virus that encodes four major structural proteins: S, spike glycoprotein (GP); E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein (
46,
55). Similar to other coronaviruses, the S glycoprotein of the virus mediates the initial attachment of the virus to host cell receptors, angiotensin-converting enzyme 2 (ACE2) (
44) and/or DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-related molecule; also CD209L or L-SIGN[liver/lymph node-SIGN]) (
32) and subsequent fusion of the viral and cellular membranes to allow viral entry into susceptible target cells. The S glycoprotein of SARS-CoV (SARS-S) is a 1,255-amino-acid (aa) type I membrane glycoprotein (
46) with 23 potential N-linked glycosylation sites (
55). The S glycoproteins of some coronaviruses are translated as a large polypeptide that is subsequently proteolytically cleaved into two functional subunits, S1 (harboring the receptor-binding domain [RBD]) and S2 (containing the membrane fusion domains) (
1,
31,
51), during biogenesis, but others are not. The S glycoprotein on mature SARS-CoV virions does not appear to be cleaved (
50,
61), but sequence alignments with other coronavirus S glycoproteins allow definition of S1 and S2 regions (
46,
55). More recently, studies showed the proteolysis of the S glycoprotein of SARS-CoV on mature virions by cathepsin L (CTSL) (
28,
59), as well as trypsin (
43,
61) and factor Xa (
11), suggesting that a critical cleavage event may occur during cell entry rather than during virion biogenesis.Mannose-binding lectin (MBL; also known as mannose-binding or mannan-binding protein [MBP]) is a Ca
2+-dependent (C-type) serum lectin that plays an important role in innate immunity by binding to carbohydrates on the surface of a wide range of pathogens (including bacteria, viruses, fungi, and protozoa) (
8,
14,
18), where it activates the complement system or acts directly as an opsonin (
30,
40,
52). In order to activate the complement system, MBL must be in complex with a group of MBL-associated serine proteases (MASPs), MASP-1, -2, and -3. Currently, only the role of MASP-2 in complement activation has been clearly defined (
65). The MBL-MASP-2 complex cleaves C4 and C2 to form C3 convertase (C4bC2a), which, in turn, activates the downstream complement cascade. MBL is a pattern recognition molecule (
9), and surface recognition is mediated through its C-terminal carbohydrate recognition domains (CRDs), which are linked to collagenous stems by a short coiled-coil of alpha-helices. MBL is a mixture of oligomers assembled from subunits that are formed from three identical polypeptide chains (
9) and usually has two to six clusters of CRDs. Within each of the clusters, the carbohydrate-binding sites have a fixed orientation, which allows selective recognition of patterns of carbohydrate residues on the surfaces of a wide range of microorganisms (
8,
14,
18). The concentration of MBL in the serum varies greatly and is affected by mutations of the promoter and coding regions of the human MBL gene (
45). MBL deficiency is associated with susceptibility to various infections, as well as autoimmune, metabolic, and cardiovascular diseases, although MBL-deficient individuals are generally healthy (
13,
37,
67).There are conflicting results with regard to the role of MBL in SARS-CoV infection (
29,
42,
72,
73). While the association of MBL gene polymorphisms with susceptibility to SARS-CoV infection was reported in some studies (
29,
73), Yuan et al. demonstrated that there were no significant differences in MBL genotypes and allele frequencies among SARS patients and controls (
72). Ip et al. observed binding to, and inhibition of, SARS-CoV by MBL (
29). However, in other studies, no binding of MBL to purified SARS-CoV S glycoprotein was detected (
42).In this study, retroviral particles pseudotyped with SARS-S and
in vitro assays were used to characterize the role of MBL in SARS-CoV infection. The data indicated that MBL selectively bound to SARS-S and mediated inhibition of viral infection in susceptible cell lines. Moreover, we identified a single N-linked glycosylation site, N330, on SARS-S that is critical for the specific interactions with MBL.
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