title: Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus.
abstract: The 12 cysteine residues in the flavivirus NS1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the intramolecular disulfide bond arrangement of NS1 of Murray Valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. Disulfide linkages were identified by separating tryptic-digested NS1 by reverse-phase high pressure liquid chromatography and analysing the resulting peptide peaks by protein sequencing, amino acid analysis and/or electrospray mass spectrometry. The pairing arrangements between the six amino-terminal cysteines were identified as follows: Cys(4)-Cys(15), Cys(55)-Cys(143) and Cys(179)-Cys(223). Although the pairing arrangements between the six carboxy-terminal cysteines were not determined, we were able to eliminate several cysteine-pairing combinations. Furthermore, we demonstrated that all three putative N-linked glycosylation sites of NS1 are utilized and that the Asn(207) glycosylation site contains a mannose-rich glycan.
authors.affiliation: Department of Microbiology, The University of Western Australia, QE-II Medical Centre, Nedlands 6907, Australia1.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Blitvich
authors.affiliation: Protein Biochemistry, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong 3220, Australia2.
authors.forename: Denis
authors.initials: D
authors.lastname: Scanlon
authors.affiliation: Protein Biochemistry, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong 3220, Australia2.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Shiell
authors.affiliation: Department of Microbiology and Parasitology, The University of Queensland, St Lucia 4072, Australia3.|Department of Microbiology, The University of Western Australia, QE-II Medical Centre, Nedlands 6907, Australia1.
authors.forename: John S
authors.initials: JS
authors.lastname: Mackenzie
authors.affiliation: Department of Microbiology and Parasitology, The University of Queensland, St Lucia 4072, Australia3.
authors.forename: Kim
authors.initials: K
authors.lastname: Pham
authors.affiliation: Department of Microbiology and Parasitology, The University of Queensland, St Lucia 4072, Australia3.|Department of Microbiology, The University of Western Australia, QE-II Medical Centre, Nedlands 6907, Australia1.
authors.forename: Roy A
authors.initials: RA
authors.lastname: Hall
mesh_terms: D000595:Amino Acid Sequence; D004220:Disulfides; D018068:Encephalitis Virus, Murray Valley / Q000737:chemistry*; D006031:Glycosylation; D008969:Molecular Sequence Data; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
chemical_list: D004220:Disulfides; C100315:NS1 protein, Flavivirus; D017361:Viral Nonstructural Proteins