Mass Spectrometric Characterization of Proteins from the SARS Virus

Abstract

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ϳ46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ϳ139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS. Molecular & Cellular Proteomics 2: 346 -356, 2003.

Identification of Two Neutralizing Regions on the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Produced from the Mammalian Expression System

“…The molecular weight shifts after deglycosylation for every S segment in the analysis (Fig. 1B) supported the notion that the SARS-CoV S protein was glycosylated throughout its entire length (7,26).…”

“…It is a large type-I transmembrane glycoprotein containing 23 potential N glycosylation sites (Fig. 1A) (7,26) and is responsible for receptor binding and membrane fusion during virus entry (4,10,11,21,28). The angiotensin-converting enzyme 2 (ACE2) receptor was identified as a receptor for SARS-CoV (10), and a small 193-amino-acid fragment of the S protein (Asn318-Val510) was shown to bind efficiently to ACE2 (24).…”

“…There has been no direct experimental evidence to date suggesting that SARS-CoV S protein is cleaved into S1 and S2 subunits. The designation of S1 (Ser12-Ser798) and S2 (Arg797-Thr1255) was based on the alignment of the SARS-CoV S protein sequence with those of other coronaviruses with known cleavage between their S1 and S2 domains (1,3,5,7,9,13,16). Molecular modeling has proposed a hypothetic division of these two subunits between Leu681-Asp727 ( Fig.…”

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

“…Although there is only 20-27% amino acid identity between SARS-CoV encoded S protein and those of the other coronavirus family members, a number of features are conserved, particularly in S2 (6). Additionally, SARS-CoV S contains 23 predicted N-linked glycosylation sites (6), of which at least 12 appear to be used (8).…”

Use of the COOH Portion of the Nucleocapsid Protein in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay for Specific and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus

“…For serodiagnosis of the SARS-CoV infection in clinical microbiology laboratories, the recombinant antigen-based enzymelinked immunosorbent assay (ELISA) for detecting specific antibodies is well known to offer higher reproducibility and to be more easily standardized and less laborious than either an indirect immunofluorescence assay based on virus-infected cells or an ELISA based on whole-virus lysate and does not require cultivation of the SARS-CoV (12). Since the specific antibodies to nucleocapsid (N) protein are most abundant in the sera from SARS patients (5), several groups have developed some recombinant N protein-based ELISAs. In general, these new assays are more specific and sensitive than the ELISA based on whole-virus lysate, but some false-positive results still occurred with sera from non-SARS patients or healthy people, even with sera collected several years before the SARS outbreak (3,7,8,(10)(11)(12).…”