%0 Journal Article %A Blitvich, Bradley J. %A Scanlon, Denis %A Shiell, Brian J. %A Mackenzie, John S. %A Pham, Kim %A Hall, Roy A. %T Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus %D 2001 %J Journal of General Virology, %V 82 %N 9 %P 2251-2256 %@ 1465-2099 %R https://doi.org/10.1099/0022-1317-82-9-2251 %I Microbiology Society, %X 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: Cys4–Cys15, Cys55–Cys143 and Cys179–Cys223. 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 Asn207 glycosylation site contains a mannose-rich glycan. %U https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-82-9-2251