Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus

Abstract

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.

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).…”

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines

“…Western blots (data not shown) showed that only SARS vaccine-immunized ferret sera recognized proteins from SARS-CoV-infected cell lysates corresponding to the molecular masses of N and S proteins (Ying et al, 2004) as well as of purified expressed N and S protein (See et al, 2006).…”

“…were only detectable in 3/4 ferrets and all titres were less than 80. At 7 weeks, the titres were 15-fold higher in ferrets vaccinated with WKV plus alum compared with animals vaccinated with Ad-S/N i.m.Western blots (data not shown) showed that only SARS vaccine-immunized ferret sera recognized proteins from SARS-CoV-infected cell lysates corresponding to the molecular masses of N and S proteins (Ying et al, 2004) as well as of purified expressed N and S protein (See et al, 2006). …”

Efficient assembly and release of SARS coronavirus‐like particles by a heterologous expression system

“…SARS-CoV is phylogenetically distinct from other characterized coronaviruses [3]. The genome is composed of a relatively conserved region encoding an RNA-dependent RNA polymerase and a variable region containing open reading frames encoding sequences for viral structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) [4,5]. Like some other coronaviruses, the variable region of SARS-…”