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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
The flavivirus nonstructural protein NS1 (4555 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, precursorproduct 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 065% 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).
|
|
|
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
(23) 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 |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Chambers, T. J., Hahn, C. S., Galler, R. & Rice, C. M. (1990). Flavivirus genome organization, expression and replication. Annual Review of Microbiology 44, 649-688.[Medline]
Crooks, A. J., Lee, J. M., Easterbrook, L. M., Timofeev, A. V. & Stephenson, J. R. (1994). The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell. Journal of General Virology 75, 3453-3460.[Abstract]
Dalgarno, L., Trent, D. W., Strauss, J. H. & Rice, C. M. (1986). Partial nucleotide sequence of the Murray Valley encephalitis virus genome: comparison of the encoded polypeptides with yellow fever virus structural and nonstructural proteins. Journal of Molecular Biology 187, 309-323.[Medline]
Falgout, B. & Markoff, L. (1995). The family Flaviviridae and its diseases. In Exotic Viral Infections , pp. 47-66. Edited by J. S. Porterfield. London:Chapman and Hall Medical.
Flamand, M., Megret, F., Mathieu, M., Lepault, J., Rey, F. A. & Deubel, V. (1999). Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. Journal of Virology 73, 6104-6110.
Gorman, J. J. & Shiell, B. J. (1993). Isolation of carboxyl-termini and blocked amino-termini of viral proteins by high-performance cation-exchange chromatography. Journal of Chromatography 646, 193-205.[Medline]
Hall, R. A., Kay, B. H., Burgess, G. W., Clancy, P. & Fanning, I. D. (1990). Epitope analysis of the envelope and nonstructural glycoproteins of Murray Valley encephalitis virus. Journal of General Virology 71, 2923-2930.[Abstract]
Hall, R. A., Coelen, R. J. & Mackenzie, J. S. (1991). Immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus: selection of the appropriate ligand and optimal conditions for elution. Journal of Virological Methods 32, 11-20.[Medline]
Hall, R. A., Brand, T. N., Lobigs, M., Sangster, M. Y., Howard, M. J. & Mackenzie, J. S. (1996). Protective immune responses to the E and NS1 proteins of Murray Valley encephalitis virus in hybrids of flavivirus-resistant mice. Journal of General Virology 77, 1287-1294.[Abstract]
Khromykh, A. A., Sedlak, P. L., Guyatt, K. J., Hall, R. A. & Westaway, E. G. (1999). Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase. Journal of Virology 73, 10272-10280.
Kornfeld, R. & Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54, 631-664.[Medline]
Lindenbach, B. D. & Rice, C. M. (1997). Trans-complementation of YF virus NS1 reveals a role in early RNA replication. Journal of Virology 71, 9608-9617.[Abstract]
Mackenzie, J. S., Lindsay, M. D., Coelen, R. J., Broom, A. K., Hall, R. A. & Smith, D. W. (1994). Arboviruses causing human disease in the Australasian zoogeographic region. Archives of Virology 136, 447-467.[Medline]
Mackenzie, J. M., Jones, M. K. & Young, P. R. (1996). Immunolocalization of the dengue viral nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 220, 220-240.
Mackow, E., Makino, Y., Zhao, B. T., Zhang, Y. M., Markoff, L., Buckler-White, A., Guiler, M., Chanock, R. & Lai, C. J. (1987). The nucleotide sequence of dengue type 4 virus: analysis of genes coding for nonstructural proteins. Virology 159, 217-228.[Medline]
Mason, P. W. (1989). Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology 169, 354-364.[Medline]
Mason, P. W., McAda, P. C., Dalrymple, J. M., Fournier, M. J. & Mason, T. C. (1987). Expression of Japanese encephalitis virus antigens in Escherichia coli. Virology 158, 361-372.[Medline]
Monath, T. P. & Heinz, F. X. (1996). Flaviviruses. In Fields Virology , pp. 961-1034. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:LippincottRaven.
Muylaert, I. A., Chambers, T. J., Galler, R. & Rice, C. M. (1996). Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse virulence. Virology 222, 159-168.[Medline]
Muylaert, I. A., Galler, R. & Rice, C. M. (1997). Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature sensitive mutation which blocks RNA accumulation. Journal of Virology 71, 291-298.[Abstract]
Nestorowicz, A., Chambers, T. J. & Rice, C. M. (1994). Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication and evidence for alternative processing of the NS2A protein. Virology 199, 114-123.[Medline]
Pryor, M. J. & Wright, P. J. (1993). The effects of site-directed mutagenesis on the dimerization and secretion of the NS1 protein specific by dengue virus. Virology 194, 769-780.[Medline]
Pryor, M. J. & Wright, P. J. (1994). Glycosylation mutants of dengue virus NS1 protein. Journal of General Virology 75, 1183-1187.[Abstract]
Rice, C. M. (1996). Flaviviridae: the viruses and their replication. In Fields Virology , pp. 931-959. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:LippincottRaven.
Schlesinger, J. J., Brandriss, M. W., Cropp, C. B. & Monath, T. P. (1986). Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1. Journal of Virology 60, 1153-1155.[Medline]
Schlesinger, J. J., Brandriss, M. W., Putnak, J. R. & Walsh, E. E. (1990). Cell surface expression of yellow fever virus non-structural glycoprotein NS1: consequences of interaction with antibody. Journal of General Virology 71, 593-599.[Abstract]
Stadler, K., Allison, S. L., Schalich, J. & Heinz, F. X. (1997). Proteolytic activation of tick-borne encephalitis virus by furin. Journal of Virology 71, 8475-8481.[Abstract]
Sumiyoshi, H., Mori, C., Fuke, I., Morita, K., Kuhara, S., Kondou, J., Kikuchi, Y., Nagamatu, H. & Igarashi, A. (1987). Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. Virology 161, 497-510.[Medline]
Timofeev, A. V., Ozherelkov, S. V., Pronin, A. V., Deeva, A. V., Karganova, G. G., Elbert, L. B. & Stephenson, J. R. (1998). Immunological basis for protection in a murine model of tick-borne encephalitis by a recombinant adenovirus carrying the gene encoding the NS1 non-structural protein. Journal of General Virology 79, 689-695.[Abstract]
Winkler, G., Randolph, V. B., Cleaves, G. R., Ryan, T. E. & Stollar, V. (1988). Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 162, 187-196.[Medline]
Winkler, G., Maxwell, S. E., Ruemmler, C. & Stollar, V. (1989). Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 171, 302-305.[Medline]
Young, P. R. & Falconar, A. K. I. (1989). Nonstructural proteins as virus vaccines. Arbovirus Research in Australia 5, 62-67.
Received 19 March 2001;
accepted 17 May 2001.