title: 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.
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, China.
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
authors.forename: Yunwei
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authors.lastname: Hao
authors.forename: Yangjun
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authors.lastname: Zhang
authors.forename: Wenming
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authors.lastname: Peng
authors.forename: Ede
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authors.lastname: Qin
authors.forename: Yun
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authors.lastname: Cai
authors.forename: Kaihua
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authors.forename: Jie
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authors.forename: Guohui
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authors.lastname: Chang
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authors.forename: Shujia
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authors.lastname: Dai
authors.forename: Xiaohai
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authors.forename: Yunping
authors.initials: Y
authors.lastname: Zhu
authors.forename: Jianqi
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authors.forename: Songfeng
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authors.lastname: Wu
authors.forename: Lihai
authors.initials: L
authors.lastname: Guo
authors.forename: Jingquan
authors.initials: J
authors.lastname: Dai
authors.forename: Jinglan
authors.initials: J
authors.lastname: Wang
authors.forename: Ping
authors.initials: P
authors.lastname: Wan
authors.forename: Tinggui
authors.initials: T
authors.lastname: Chen
authors.forename: Chunjuan
authors.initials: C
authors.lastname: Du
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.forename: Jia
authors.initials: J
authors.lastname: Wan
authors.forename: Xuezhang
authors.initials: X
authors.lastname: Kuai
authors.forename: Weihua
authors.initials: W
authors.lastname: Li
authors.forename: Rong
authors.initials: R
authors.lastname: Shi
authors.forename: Handong
authors.initials: H
authors.lastname: Wei
authors.forename: Cheng
authors.initials: C
authors.lastname: Cao
authors.forename: Man
authors.initials: M
authors.lastname: Yu
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Fangting
authors.initials: F
authors.lastname: Dong
authors.forename: Donggen
authors.initials: D
authors.lastname: Wang
authors.forename: Xuemin
authors.initials: X
authors.lastname: Zhang
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Zhu
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism; D002522:Chlorocebus aethiops; D017934:Coronavirus / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008168:Lung / Q000821:virology*; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D045169:Severe Acute Respiratory Syndrome / Q000821:virology*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D019590:Nucleocapsid Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C505407:CASP3 protein, human; C505514:CASP6 protein, human; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases