Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural proteinNS1 of Murray Valley encephalitis virus

The 12 cysteine residues in the flavivirus NS1 protein are strictly conserv ed, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the in tramolecular disulfide bond arrangement of NS1 of Murray Valley encephaliti s virus and elucidated three of the six cysteine-pairing arrangements. Disu lfide linkages were identified by separating tryptic-digested NS1 by revers e-phase high pressure liquid chromatography and analysing the resulting pep tide peaks by protein sequencing, amino acid analysis and/or electrospray m ass spectrometry. The pairing arrangements between the six amino-terminal c ysteines were identified as follows: Cys(4)-Cys(15), Cys(55)-Cys(143) and C ys(179)-Cys(223). Although the pairing arrangements between the six carboxy terminal cysteines were not determined, we were able to eliminate several c ysteine-pairing combinations. Furthermore, we demonstrated that all three p utative N-linked glycosylation sites of NS1 are utilized and that the Asn(2 07) glycosylation site contains a mannose-rich glycan.