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. Blitvich1, Denis Scanlonb,2, Brian J. Shiell2, John S. Mackenzie1,3, Kim Pham3 and Roy A. Hall1,3

Department of Microbiology, The University of Western Australia, QE-II Medical Centre, Nedlands 6907, Australia1
Protein Biochemistry, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong 3220, Australia2
Department of Microbiology and Parasitology, The University of Queensland, St Lucia 4072, Australia3

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{at}lamar.colostate.edu


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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.


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Murray Valley encephalitis virus (MVE) is a member of the genus Flavivirus (family Flaviviridae) which consists of approximately 70 members and includes human pathogens of global 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 homologous 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, mutagenesis studies on infectious clones of YF and Kunjin virus have shown that viral RNA accumulation is blocked by specific amino acid substitutions in the NS1 gene and that virus replication can be restored by supplying authentic NS1 in trans (Muylaert et al., 1996 , 1997 ; Lindenbach & Rice, 1997 ; Khromykh et al., 1999 ).

NS1 contains 12 cysteine residues that are strictly conserved among all members of the flaviviruses, with the exception of DEN-4, suggesting a critical role for disulfide linkages in protein stability and/or function (Mackow et al., 1987 ; Chambers et al., 1990 ). Disulfide bridges have been implicated to play an important role in dimerization, as site-directed mutagenesis of any of the final three carboxy-terminal cysteines of DEN-2 NS1 abolishes dimer formation, and mutagenesis of the third amino-terminal cysteine leads to the formation of unstable dimers (Pryor & Wright, 1993 ). Despite this, there are no data available on the disulfide bond arrangement of NS1 for any of the flaviviruses. The positions of the putative N-linked glycosylation sites (Asn-X-Thr/Ser) of NS1 are also remarkably conserved. All mosquito-borne flaviviruses contain potential NS1 glycosylation sites at Asn130 and Asn207 (except YF: Asn130 and Asn208), and a third site is present at Asn175 in all members of the JE serogroup, with the exception of JE (Sumiyoshi et al., 1987 ; Chambers et al., 1990 ). As these post-translational modifications are presumably critical in the maturation of this protein into its functionally active form, we investigated the disulfide and glycosylation arrangements of MVE NS1.

In order to define the disulfide linkages of this protein, first we immunoaffinity-isolated NS1 from the supernatant of MVE-infected Vero cells using an NS1-specific chromatography column, as previously described (Hall et al., 1991 ). The integrity of the isolated protein was determined by Western blot analysis (results not shown) using NS1-specific monoclonal antibodies produced and characterized by Hall et al. (1990) . The protein was freeze-dried, resolubilized in 6 M urea, diluted sixfold in 70 mM NH4HCO3 and digested 1:20 with sequencing grade modified trypsin (Promega). Forty-four NS1 peptides are theoretically generated upon trypsin digestion, as shown in Table 1. Separation of the non-reduced tryptic peptides was performed by reverse-phase high-performance liquid chromatography (RP-HPLC) on a 2·1 mmx250 mm Vydac C18 column (The Separations Group, Hesperia, CA, USA) (Fig. 1a). Peptide separation was achieved using a flow-rate of 200 µl/min, column temperature of 45 °C and gradient of 0–65% buffer B for 78 min (buffer B, 0·05% trifluoroacetic acid, 80% CH3CN; diluted in buffer A, 0·05% trifluoroacetic acid). Eluted peptides were collected and identified by a combination of protein sequencing, amino acid analysis and electrospray mass spectrometry. Protein sequencing was performed using a gas-phase protein sequencer (Applied Biosystems model 470A) with a synchronized on-line PTH analyser (Applied Biosystems model 120A). Amino acid analysis was performed as follows: RP-HPLC fractions were dried, subjected to gas-phase hydrolysis and amino acids derivitized with 6-aminoquinolyl-N-hydroxy-succinimidyl (Waters) for subsequent HPLC analysis. Electrospray mass spectrometry was performed by direct analysis of RP-HPLC fractions on a VG Platform instrument (VG Analytical).


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Table 1. The 44 peptides theoretically generated following trypsin-digestion of NS1

 


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Fig. 1. Analysis of non-reduced tryptic-digested NS1 by (a) reverse-phase and (b) cation-exchange HPLC. The identities of the labelled peaks in the RP-HPLC profile are as follows: Peak#1, peptides 52–58 (Cys55) and 121–147 (Cys143 and Asn130); Peak#2, peptide 190–211 (Asn207); Peak#3, peptides 1–10 (Cys4) and 15–31 (Cys15); Peak#4, peptides 262–293 (Cys280, Cys291), 307–314 (Cys312, Cys313), 315–322 (Cys316) and 323–339 (Cys329); Peak#5, peptides 171–189 (Cys179 and Asn175) and 222–251 (Cys223); Peak#6, partially digested NS1 fragment (residues 171–352). In the cation-exchange HPLC profile, the asterisks denote the tetrapeptide composed of 262–293 (Cys280, Cys291), 307–314 (Cys312, Cys313), 315–322 (Cys316) and 323–339 (Cys329). (c) Possible cysteine-pairing arrangements between the six C-terminal cysteine residues of NS1. Using reverse-phase and cation-exchange HPLC, in conjunction with protein sequencing, amino acid analysis and electrospray mass spectrometry, we identified the three N-terminal cysteine-pairing arrangements, and eliminated three other arrangements, leaving eight possible pairing combinations for the remaining cysteines.

 
Two of the later eluting peaks observed in the RP-HPLC profile showed multiple sequences upon protein sequencing and amino acid analysis, consistent with the pairing of tryptic peptides by disulfide bonds. This allowed us to pair four of the cysteine-containing tryptic peptides, namely: 52–58 to 121–147 (Cys55–Cys143) and 171–189 to 222–251 (Cys179–Cys223) (Fig. 1a, peaks 1 and 5 respectively). A third pairing arrangement was identified between tryptic fragments 1–10 and 15–31 (Cys4–Cys15) (Fig. 1a, peak 3). This pairing arrangement was initially identified by electrospray mass spectrometry, which revealed that this fragment was 2995 Da in size (Fig. 2a), then later confirmed by protein sequencing. Another of the later eluting peaks (Fig. 1a, peak 6) represents a large, partially digested NS1 fragment (residues 171–352), as determined by gel electrophoresis and protein sequencing (results not shown).



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Fig. 2. Electrospray mass spectrometric analysis of tryptic digested NS1. (a) The peptide fraction corresponding to peak#3 on the RP-HPLC profile (as illustrated in Fig. 1a) was subjected to electrospray mass spectrometry and direct protein sequencing and identified as peptides 1–10 (Cys4) and 15–31 (Cys15). (b) The peptide fraction corresponding to peak#4 on the RP-HPLC profile was examined by electrospray mass spectrometry and shown to contain peptides 262–293 (Cys280, Cys291), 307–314 (Cys312, Cys313), 315–322 (Cys316) and 323–339 (Cys329). (c) Molecular mass determination of the tryptic fragment corresponding to peak#2 on the RP-HPLC profile [peptide 190–211 (Asn207)].

 
The tryptic peptides that contained the six remaining cysteines were linked together as a single large fragment. This fragment was initially identified by strong cation-exchange HPLC on a Polysulfoethyl aspartamide silica column (Poly LC, Murietta, CA, USA) (Fig. 1b), as previously described (Gorman & Shiell, 1993 ). Briefly, peptide separation was achieved using a flow-rate of 1 ml/min, column temperature of 22 °C and gradient of 0–40% buffer B for 50 min (buffer B, 10 mM H3PO4, 76 mM Na2SO4, 25% CH3CN; diluted in buffer A, 10 mM H3PO4, 25% CH3CN, pH 3·0). The fragment eluted from the cation-exchange HPLC column in two peaks and subsequent protein sequencing identified amino-terminal residues for four different peptides. The same fragment was then identified in the RP-HPLC profile (Fig. 1a, peak 4) by electrospray mass spectrometry and protein sequencing. This fragment was 7451 Da in size (Fig. 2b) and composed of four tryptic peptides, namely: 262–293, 307–314, 315–322 and 323–339, with the 262–293 peptide generated due to a missed cleavage at the Lys274–Leu275 site. It is likely that the two peaks observed in the cation-exchange HPLC profile are also a consequence of incomplete trypsin digestion. The second peak probably includes a peptide composed of residues 262–294, due to a missed cleavage at the Lys293–Arg294 site. Thus, the additional positive charge resulting from the arginine residue would lead to increased retention of the fragment on the cation-exchange column. Attempts were made to subdigest the tetrapeptide with sequencing grade endoproteinase GluC (Promega) but, presumably due to the complex disulfide arrangement, this was unsuccessful. Therefore, the disulfide bond arrangement between the six carboxy-terminal cysteines was not elucidated, as the corresponding tryptic fragments were linked together by the three undefined disulfide bonds. However, in order for this tetrapeptide disulfide linkage pattern to occur, some disulfide combinations are not possible. For instance, tryptic peptides 315–322 and 323–339 cannot be directly linked which eliminates the Cys316–Cys329 combination. In addition, there cannot be an internal disulfide in either the 262–293 or 307–314 fragments, excluding the Cys280–Cys291 and Cys312–Cys313 combinations respectively. Therefore, this leaves eight possible cysteine-pairing combinations for the six carboxy-terminal cysteine residues (Fig. 1c).

Protein sequencing revealed that all three putative N-linked glycosylation sites of NS1 were indeed modified. This was evident by the loss of the amino acid signal in the protein sequencing RP-HPLC profile at residues 130, 175 and 207, the sites at which asparagine residues of the type Asn-X-Thr/Ser are predicted to occur. The tryptic peptide that contained the Asn207 glycosylation site (Fig. 1a, peak 2) was subjected to electrospray mass spectrometry and shown to have an estimated molecular mass of 4175±162 Da (Fig. 2c). The amino acids of this tryptic peptide contribute an estimated 2472Da to the total molecular mass, suggesting that the carbohydrate moiety is 1703±162 Da in size, consistent to that of a mannose-rich glycan (Kornfeld & Kornfeld, 1985 ). The molecular masses of the other two carbohydrate-containing peptides (Fig. 1a, peaks 1 and 5) could not be determined by electrospray mass spectrometry, as both were disulfide-linked to a second peptide. However, this data, in conjunction with our previous lectin-binding studies that showed secreted NS1 contains a mixture of (i) mannose-rich glycans, (ii) complex glycans that lack terminal sialic acid and (iii) complex glycans with sialic acid linked {alpha}(2–3) to galactose (Blitvich et al., 1999 ), suggest that the Asn130 and Asn175 sites are filled by complex carbohydrate residues. Similarly, analysis of extracellular DEN-2 NS1, by site-directed mutagenesis and endoglycosidase H digestion, revealed that the Asn130 and Asn207 sites are filled by complex and high-mannose glycans respectively (Pryor & Wright, 1994 ).


   Acknowledgments
 
The authors would like to thank Gary Beddome for his expertise in sequencing tryptic peptides from NS1. This study was supported by the National Health and Medical Research Council, Canberra, Australia.


   Footnotes
 
b Present address: Auspep Pty Ltd, Parkville 3052, Australia.


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Received 19 March 2001; accepted 17 May 2001.