Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus.
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.
Blitvich
Bradley J
BJ
Department of Microbiology, The University of Western Australia, QE-II Medical Centre, Nedlands 6907, Australia1.
Scanlon
Denis
D
Protein Biochemistry, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong 3220, Australia2.
Shiell
Brian J
BJ
Protein Biochemistry, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong 3220, Australia2.
Mackenzie
John S
JS
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.
Pham
Kim
K
Department of Microbiology and Parasitology, The University of Queensland, St Lucia 4072, Australia3.
Hall
Roy A
RA
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.
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*
D004220:Disulfides; C100315:NS1 protein, Flavivirus; D017361:Viral Nonstructural Proteins