Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Tue, 15 Mar 2016 02:08:08 Journal of General Virology (2001), 82, 2251–2256. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus Bradley J. Blitvich, 1 Denis Scanlon, 2 † Brian J. Shiell, 2 John S. Mackenzie, 1, 3 Kim Pham 3 and Roy A. Hall 1, 3 1 Department of Microbiology, The University of Western Australia, QE-II Medical Centre, Nedlands 6907, Australia 2 Protein Biochemistry, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong 3220, Australia 3 Department of Microbiology and Parasitology, The University of Queensland, St Lucia 4072, Australia 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 chromato- graphy 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 com- binations. 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. Murray Valley encephalitis virus (MVE) is a member of the genus Flavivirus (family Flaviviridae) which consists of approxi- mately 70 members and includes human pathogens of global Author for correspondence : Bradley Blitvich. Present address : Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA. Fax 1 970 491 8323. e-mail blitvichlamar.colostate.edu † Present address : Auspep Pty Ltd, Parkville 3052, Australia. importance such as yellow fever (YF), Japanese encephalitis virus (JE) and dengue (DEN). MVE belongs to the JE serogroup, is active in Australia, Papua New Guinea and possibly Indonesia, and is the major aetiological agent of the severe and potentially fatal neurological disease, Australian encephalitis (Mackenzie et al., 1994). The genomic RNA of MVE, like that of the other flaviviruses, is a single-stranded, positive-sense molecule of approximately 11 kb, with a 5 cap structure and a nonpolyadenylated 3 terminus. The viral RNA contains a single open reading frame that encodes a single polyprotein that is co- and post-translationally cleaved to generate three mature structural proteins and seven nonstructural proteins in the gene order: 5-C-prM(M)-E-NS1-NS2A-NS2B-NS3- NS4A-NS4B-NS5-3. Cleavage events are mediated by a combination of endoplasmic reticulum enzyme signalases, furin and the viral trypsin-like NS2B–NS3 serine protease (Chambers et al., 1990; Falgout & Markoff, 1995; Monath & Heinz, 1996 ; Rice, 1996 ; Stadler et al., 1997). The flavivirus nonstructural protein NS1 (45–55 kDa) exists predominantly as a heat-labile homodimer that can be detected within infected cells, expressed on the cell surface and secreted in the extracellular medium (Mason, 1989 ; Winkler et al., 1988, 1989). Hexameric forms of NS1, formed from dimeric subunits, have also been detected in the extracellular medium (Crooks et al., 1994 ; Flamand et al., 1999). Multiple species of this protein exist due to glycosylation variations, precursor– product relationships andor alternative cleavage sites in the viral polyprotein (Young & Falconar, 1989 ; Mason et al., 1987 ; Nestorowicz et al., 1994 ; Blitvich et al., 1999). Active immunization with NS1 or passive transfer of NS1-specific antibodies can protect laboratory animals against the hom- ologous flavivirus (Schlesinger et al., 1986, 1990; Hall et al., 1996 ; Timofeev et al., 1998). NS1 has been implicated to play a role in RNA replication, as demonstrated by the colocalization of NS1 with the viral double-stranded RNA replicative form (Mackenzie et al., 1996). Furthermore, muta- 0001-7740  2001 SGM CCFB