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Mass spectrometric identification and characterisation of the nucleocapsid protein of Menangle virus
Journal of Virological Methods 102 (2002) 27 – 35 www.elsevier.com/locate/jviromet
Mass spectrometric identification and characterisation of the nucleocapsid protein of Menangle virus Brian J. Shiell, Gary Beddome, Wojtek P. Michalski * Australian Animal Health Laboratory, CSIRO Li6estock Industries, Pri6ate Bag 24, Geelong, Vic. 3220, Australia Received 23 July 2001; received in revised form 26 November 2001; accepted 27 November 2001
Abstract The recent emergence of novel viruses requires reliable methodology for their identification and confirmation both on a cellular and molecular level. Mass spectrometry offers a suitable approach for the identification and characterisation of viral proteins and its application is demonstrated in this study. Menangle virus is a previously unclassified member of the family Paramyxoviridae isolated in Australia in 1997. Menangle virus caused disease in pregnant pigs, and like the other newly emergent Hendra, Nipah and Tioman viruses, appears to be a virus of fruit bats (flying foxes) in the genus Pteropus. The 61 kDa gel-purified protein isolated from cell-associated Menangle virus ribonucleoprotein (RNP) was identified as the nucleocapsid protein (NP) by peptide mapping, mass spectrometry and amino acid sequencing. Over 69% of the amino acid sequence was obtained and found to be identical with that derived from gene analysis (Virology, 283 (2001), 358). The first residue of the mature NP was found to be serine (second residue in the gene derived amino acid sequence). The NP was found to be acetylated at the N-terminus (at Ser-2) and appears to be not modified by phosphorylation. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Nucleocapsid protein; Mass spectrometry; Menangle virus; Rubula6irus; Paramyxoviridae
1. Introduction Advances in gene analysis technology have led to the ability to sequence genes with relative ease and deduce protein sequences as a consequence. However, there are still a variety of reasons why it is desirable to perform a secondary check of these deduced sequences. In some instances mistakes have been made during nucleic acid sequencing. * Corresponding author. Tel.: + 61-3-5227-5772; fax: + 613-5227-5555. E-mail address: [email protected] (W.P. Michalski).
Messenger RNA may also be subject to base insertions or contain multiple initiation codons. Furthermore, gene sequence data alone may be misleading due to the potential for protein splicing events to produce final products with primary structure that could not be predicted from the original gene coding sequences. Protein trans-peptidation has been documented with the product of a single gene and between products of separate genes. Acquisition of information regarding other post-translational modifications also requires protein structure analysis. A strategy for comprehensive comparison of viral protein sequences
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with the products predicted from the corresponding gene sequences is described in this paper. Mass spectrometry plays a principal role in this strategy (Michalski and Shiell, 1999), which also includes other protein analysis techniques such as high performance liquid chromatography (HPLC), amino acid analysis and N-terminal sequencing. In this paper, we demonstrate an application for this strategy in the identification and characterisation of a protein from a newly emergent virus. Menangle virus is a recent addition to the family Paramyxoviridae, subfamily Paramyxovirinae and is the causative agent of a single outbreak of disease in a commercial piggery characterised by a decrease in the farrowing rate and the number of live piglet births. Stillborn piglets had severe degeneration of the brain and spinal cord (Philbey et al., 1998) but infection of adult, non-pregnant pigs was not apparent. Menangle virus also appeared to cause an influenza-like illness of two humans in close contact with infected pigs (Chant et al., 1998). The presence of neutralising antiMenangle virus antibodies in fruit bats (Pteropus genus) suggests they may be the natural host of the virus (Philbey et al., 1998). Serological surveys have implicated flying foxes as the natural host of Hendra virus in Australia (Young et al., 1996). Hendra virus and the closely related Nipah virus (Chua et al., 2000; Harcourt et al., 2000) are also members of the family Paramyxoviridae but cannot be readily classified in any of the current genera within the family (Wang et al., 1998; Yu et al., 1998; Wang et al., 2000, 2001). Menangle virus ultrastructure is consistent with its membership in the family Paramyxoviridae (Philbey et al., 1998) but lacks antigenic cross reactivity with antiserum to viruses in the Rubula6irus, Morbilli6irus and Respiro6irus genera. Comparison of nucleotide and deduced amino acid sequences for genes of Menangle virus with those of members of the family Paramyxoviridae and phylogenetic analyses confirmed that Menangle virus is a member of the genus Rubula6irus (Bowden et al., 2001). Menangle virus is antigenetically related to Tioman virus, another member of the genus Rubula6irus, which was also identified in fruit bats (Chua et al., 2001).
2. Materials and methods
2.1. Purification of Menangle 6irus RNP and NP Monolayers of Vero cells were infected with Menangle virus at a multiplicity of 0.01 TCID50 per ml. At 48 h post infection cells were washed with phosphate buffered saline (PBS) and resuspended in TNM (10 mM Tris, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2) at 4 °C and lysed by addition of NP-40 to 1% (v/v). Nuclei were pelleted and the associated cytoskeleton removed by shearing in a Dounce homogeniser. The nuclei were pelleted, cytoplasmic and cytoskeletal fractions pooled and ethylenediaminetetraacetic acid disodium salt (EDTA) and sodium deoxycholate added to 10 mM and 1% (w/v), respectively. After clarification at 10 000 rpm in a SW55 rotor for 10 min at 4 °C the lysate was layered over 20–40% CsCl gradients in TNE (10 mM Tris, 100 mM NaCl, 1 mM EDTA) with 0.2% sodium lauroyl sarcosinate. After centrifugation in a SW41 rotor for 24 h at 25 000 rpm the visible ribonucleoprotein (RNP) band at 1.31 g cm − 3 was collected and pelleted at 35 000 rpm for 1.5 h in a SW41 rotor (Bowden et al., 2001). Virus purification was performed at Bio-Security Level 4 (BSL 4) at the Australian Animal Health Laboratory by Gary Crameri and Bryan Eaton. Pelleted virus was inactivated in 2% sodium dodecyl sulphate (SDS) prior to its transfer to the non-secure environment and viral proteins were analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS (SDSPAGE).
2.2. Electrophoresis Proteins of SDS-treated virus were separated under reducing conditions on 10% Tris–glycine gels (Novex, San Diego, CA). Proteins in the gels were then either, (i) visualised by Coomasie Brilliant Blue R-250 staining; or (ii) by reverse-staining using a Zinc-imidazole stain kit (Bio-Rad Laboratories, Hercules, CA) and excised for subsequent ‘in gel’ tryptic digestion and mass spectrometry; or (iii) electro-transferred to polyvinylidene fluoride membrane and stained
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(see Section 2.4) prior to direct sequencing and/or chemical cleavage.
2.3. ‘In gel’ digestion Gel bands were cut into 1× 2 mm pieces, transferred to 0.5 ml Eppendorf tubes and washed twice in 0.2 M NH4HCO3/50% acetonitrile. Protein in the gel pieces was then reduced with 10 mM dithiothreitol for 1 h in 0.2 M NH4HCO3, washed twice with 0.2 M NH4HCO3, and then alkylated with 50 mM iodoacetamide in 0.2 M NH4HCO3 for 20 min in the dark. Following two more washes in 0.2 M NH4HCO3 and a final wash in 0.2 M NH4HCO3/50% acetonitrile, the gel pieces were dried for 30 min in a SpeedVac concentrator. Gel pieces were then rehydrated twice with 10 ml of trypsin solution (modified sequencing grade, Roche) of 1 mg/20 ml in 50 mM NH4HCO3 for 10 min at 37 °C, 150 ml 50 mM NH4HCO3 added and digestion continued overnight at 37 °C. After overnight incubation the sample was centrifuged at 12 000×g and supernatant containing peptide fragments removed and kept aside. Residual peptides in the gel pieces were then extracted twice with 200 ml of 60% acetonitrile/1% trifluoroacetic acid (TFA) at 37 °C for 30 min, centrifuged and supernatants combined. Finally, the pooled supernatants were concentrated in a SpeedVac concentrator to approximately 50 ml for subsequent analysis by liquid chromatography – mass spectrometry or isolation by reverse-phase chromatography.
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rated solution of CNBr in 70% formic acid for 24 h. CNBr-generated peptides were eluted from the membrane with 40% acetonitrile containing 0.05% TFA for subsequent isolation by reverse-phase chromatography.
2.5. Re6erse-phase chromatography Chromatography of trypsin and CNBr digested protein samples was performed on a Beckman System Gold HPLC. Peptides from the trypsin digest were separated on a Vydac C18 column (2.1× 250 mm, Separations Group, Hysperia, CA) using a binary gradient with 0.1% TFA as buffer A and 0.09% TFA/80% acetonitrile as buffer B. The gradient was developed as follows; 2–37.5% B over 0–50 min, 37.5–75% B over 50–70 min and finally 75–100% B over 75–85 min at a flow rate of 200 ml min − 1. Peptides from CNBr cleavage were separated on a Vydac C4 column (2.1× 250 mm, Separations Group) using the same buffer system as above but with a gradient of 5–100% B over 45 min. In both cases, purified peptides were hand collected and stored at − 20 °C prior to N-terminal sequencing.
2.6. N-terminal amino acid sequencing Hand collected peptides and polyvinylidene fluoride immobilised protein were subjected to amino acid sequence analysis with vapour-phase delivery of critical reagents in an automated sequenator (Model 470A, Applied Biosystems, Foster City, CA).
2.4. Chemical clea6age 2.7. Liquid chromatography–mass spectrometry Polyvinylidene fluoride membranes were stained for approximately 30 s in a freshly made and filtered 0.1% solution of Coomassie Brilliant Blue R-250, and destained in freshly made 1% aldehyde-free acetic acid/40% methanol until proteins bands were visible. A band identified as the NP protein was excised from the membrane and either subjected to direct amino acid sequencing or to cyanogen bromide (CNBr) cleavage. Protein immobilised on polyvinylidene fluoride was visualised with Ponceau S and protein bands excised from membranes were exposed to satu-
The trypsin digest was separated by reversephase chromatography on a Vydac C18 column (2.1× 250 mm, 5 mm, 300 A, , The Separations Group) using a Thermo Separations Products HPLC system (Finnigan, San Jose, CA) comprising P4000 gradient pump, SCM1000 degasser and AS3000 autosampler. Mobile phase buffers were 0.1% v/v TFA (A) and 0.1% TFA/80% acetonitrile (B), run at 200 ml min − 1 with a linear gradient of 5–100% B over 40 min, held at 100% B for 5 min, then returned to 5% B over 5 min.
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The effluent from the HPLC column was connected directly to the electrospray ion source of a Finnigan LCQ Classic (San Jose, CA) quadrupole ion-trap mass spectrometer. Liquid chromatography tandem mass spectrometry (LC–MS – MS) analysis was carried out in a data dependent mode known as the ‘triple play’ where the most intense ion in each full scan (m/z 400–2000) was automatically selected and subjected to a high resolution zoom scan followed by a tandem mass spectrometry product ion scan. The zoom scan allowed determination of the precise mass/charge state of the selected ion while tandem mass spectrometry determination of the product ion patterns derived from collision induced dissociation of the selected ion. These provided information on the sequence of the peptides and their possible modifications. Tandem mass spectrometry scans were performed with collision energy of 32%. The electrospray ion source settings were as follows: ion spray voltage 5 kV, capillary temperature 200 °C, capillary voltage 42 V and tube lens offset 20 V. Sequence information for Menangle virus NP protein was obtained from the GeneBank database with accession number AF326114.
thereof, than that predicted for tryptic peptides from a theoretical trypsin digest of NP.
3. Results Amino acid sequence analysis requires protein samples to be free from other proteins and peptides. As Menangle virus preparations were routinely inactivated with 2% SDS (a procedure required for the safe transfer of a zoonotic agent from BSL 4 laboratory to non-secure environment), electrophoresis (SDS-PAGE) was employed for the separation of individual proteins and their isolation for sequence analysis. Menangle virus nucleocapsid protein (NP) separates on SDS-PAGE gels as a well-defined protein band (Fig. 1) that can easily be excised from gels, or membranes following electro-transfer. Gel purified protein was analysed after ‘in gel’ trypsin digestion by mass spectrometry and N-terminal sequencing, whereas protein transferred onto PVDF membrane was subjected to chemical di-
2.8. Prediction and detection of phosphorylation Prediction of possible phosphorylation sites was performed using NETPHOS 2.0 (Blom et al., 1999) and PHOSPHOBASE 2.0 (Kreegipuu et al., 1999) prediction databases. Immunodetection of phospho-amino acids was performed on Menangle virus NP protein transferred onto polyvinylidene fluoride membrane and probed with rabbit antibody (antigen affinity purified) raised against phospho-tyrosine, phospho-threonine and phospho-serine (Zymed Laboratories Inc., CA). Antiphosphoamino acid antibodies were probed with goat anti-rabbit IgG conjugated to horse-radish peroxidase (HRP). Visualisation of reactive protein bands was performed using an enhanced chemiluminescence HRP substrate kit (Amersham Pharmacia). Mass spectrometry analysis of phosphorylation was performed by manual searching of the data for masses 80 Da higher (corresponding to addition of phosphate), or multiples
Fig. 1. SDS-PAGE separation of proteins from three different preparations (A – C) of RNP isolated from Menangle virus-infected cells (lanes 2, 4 and 6, 15 mg protein; lanes 3, 5 and 7, 5 mg protein). Lane 1 contains Novex Mark 12 and lane 8 Novex SeeBlue™ Plus 2 standard marker proteins (10 ml of supplied sample). The marked (arrow) 61 kDa protein is the putative Menangle virus NP.
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Fig. 2. LC – MS of Menangle virus NP protein digested with trypsin. Conditions for MS analysis are given in Section 2. T1 – T3 denote peptides identified as autolytic products of trypsin. U1 – U3 denote unidentified peptides. Peptide masses and sequence assignment is given in Table 1.
gestion by cyanogen bromide prior to analysis by N-terminal sequencing.
3.1. Peptide mapping and sequence analysis Trypsin was used to generate internal peptides from the NP protein samples excised from polyacrylamide gels. The digests were analysed by liquid chromatography tandem mass spectrometry (a LC –MS of tryptic digest is shown in Fig. 2) and N-terminal amino acid sequencing. No sequence coverage was obtained for a number of small peptides 312–348 and 432– 474, or the large peptide from residues 120 to 180. Both peptides 120– 180 and 312–348 with respective monoiso-
topic masses of 6995.24 and 4313.86 are outside the mass range of the LCQ mass spectrometer. It is possible that the third peptide 432– 474 is too negatively charged and thus suppressed in the MS analysis. No amino acid sequence data was obtained by direct sequencing of gel purified NP suggesting that the protein was blocked at the N-terminus (see above). Amino acid sequencing and mass spectrometry analyses of HPLC-purified peptides derived from ‘in gel’ trypsin digestion generated a large number of peptides of which 40 provided maximum coverage of the NP protein and are included in Table 1. Chemical cleavage with CNBr generated eight peptides and provided confirmatory sequence information for regions
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Table 1 NP peptides identified and confirmed by MS and sequencing analyses Peak
Sequence assignment
Mass found
Mass predictedf
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 T1 17 18 19 20 21 T2 22 23 24 25 26 27 U1 28 U2 T3 29 30 U3 31 32 33 34 35 36 37 38 39 40
Leu-205–Arg-209 Asn-482–Arg-488 Leu-401–Arg-405 Leu-114–Arg-119 Leu-196–Arg-204 Cys-181–Arg-195 Leu-58–Arg-65 Asp-393–Arg-400 Val-37–Arg-48e Leu-475–Arg-481e Ile-90–Arg-101e Ser225–Arg230 Ser-2–Arg-6a Asp-489–Arg-507e Thr-33–Arg-48c His-49–Arg-57 Ser-126–Lys-136 Tyr-209–Arg-217e Trp-281–Lys-295 Gly-406–Arg-420b His-49–Arg-57b Gln-231–Arg-241e Leu-50–Lys-69 Gln-372–Arg-392 Gln-231–Arg-241d Asp-489–Met-519c Gly-406–Arg-420e Gln-372–Arg-392d Leu-401–Arg-420 Unknown Asp-489–Met-519b Unknown Ser-70–Lys-89 Ile-90–Arg-113 Trp-281–Arg-304 Unknown Asp-489–Met-519 Ala-7–Arg-32 Asn-349–Lys-378 Asn-349–Arg-371 Thr-70–Arg-89c Ala-66–Arg-89b Thr-70–Arg-89b Ser-2–Arg-32a Ala-66–Arg-89 Thr-70–Arg-89
531.21 818.42 547.35 700.46 1091.41 1760.63 831.49 850.42 1429.47 886.49 1357.69 676.42 637.31 2075.19 1885.93 1301.47 1153.27 1145.69 1612.99 1573.71 1285.27 1343.75 2162.99 2276.13 1326.55 3524.90 1557.95 2259.27 2086.07 3444.53 3508.91 3462.56 2273.21 2719.55 2745.11 2733.38 3492.53 3069.61 3431.9 2689.61 2213.13 2604.23 2197.05 3688.10 2588.18 2181.21
531.29 818.39 547.32 700.42 1091.60 1760.74 831.47 850.41 1429.69 886.51 1357.67 676.40 637.33 2075.03 1885.96 1301.49 1153.57 1145.64 1612.91 1573.78 1285.50 1343.71 2163.06 2276.12 1326.71 3524.62 1557.78 2259.12 2086.08 – 3508.63 – 2273.16 2719.33 2745.55 – 3492.64 3069.48 3431.71 2689.35 2213.18 2604.41 2197.18 3687.79 2588.42 2181.19
a, N-terminal acetylation; b, one oxidised methionine residue; c, two oxidised methionine residues; d, N-terminal pyroglutamylation; e, confirmed by sequencing; f, predicted masses were determined using the software program MS-DIGEST from the UCSF Mass Spectrometry Facility (http:// prospector.ucsf.edu/ucsfhtml3.4/msdigest.htm).
122–132, 182–185, 253–278, 347–350, 394–416 and 513–518. The sequence coverage obtained from all these peptides was identical with over 69% of that deduced from the nucleotide sequence of the Menangle virus NP gene (Fig. 3).
3.2. Phosphorylation of NP protein NP protein has 18 serine and 25 threonine residues (Bowden et al., 2001), with many of these residues located in predicted phosphorylation-site consensus sequences (16 predicted sites). Detection of phosphoproteins was performed using immunodetection of phosphorylated amino acids with antibodies specifically reacting with phosphotyrosine, phosphothreonine and phosphoserine (P-Tyr, P-Ser, P-Thr). None of the viral proteins reacted with anti-P-Tyr, anti P-Thr and P-Ser antibodies indicating the absence of phosphorylated residues in the NP protein (data not shown). Phospho-proteins from Hendra and Nipah viruses were used as positive controls. Also, liquid chromatography tandem mass spectrometry analysis of the trypsin ‘in gel’ digest of the NP protein did not reveal the presence of any phosphorylated peptide fragments. No unexplained masses corresponding to peptides with the addition of phosphate (+80 Da) were found.
3.3. N-terminal modification Direct N-terminal amino acid sequence analysis of the NP protein immobilised on PVDF membrane yielded no data, thereby indicating that the N-terminus of the protein was chemically blocked. It should be noted that similar results were obtained in experiments when aldehyde-free acetic acid was used in staining procedures suggesting that the NP protein may have its N terminus modified ‘in vivo’. Liquid chromatography tandem mass spectrometry analysis of the N-terminal peptide from the ‘in gel’ trypsin digest revealed that the NP protein has the N-terminal Met cleaved and is indeed acetylated at Ser-2 (Table 1, peptides/peaks c13 and c 38). This indicates that serine is actually the first residue of the mature Menangle virus NP protein. An identical modification has been found in Hendra virus
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Fig. 3. Correlation of amino acid sequence determined for peptides derived from purified NP with deduced amino acid sequence from mRNA sequence obtained by PCR-Select cDNA subtraction cloning (Bowden et al., 2001). The amino acid sequences of peptides derived from the 61 kDa protein in purified RNP that were confirmed by mass spectrometry are underlined while those confirmed by N-terminal sequencing are in bold.
NP protein (B. Shiell and W. Michalski, personal communication).
4. Conclusions In recent years, with improvements in instrumentation and sample handling, mass spectrometry has become the analytical method of choice not only for the identification of proteins, but also for the structural investigation of post-translational modifications. In this paper, we report on identification and comprehensive characterisation of Menangle virus NP protein using peptide mapping (69% coverage), mass spectrometry (61% coverage) and N-terminal sequencing (31% coverage). The application of automated LC– MS – MS methodology using the Finnigan LCQ ESI– MS instrument for analysis of NP protein from the newly emerged Menangle virus provided a comprehensive analysis, in a single chromatographic run on sample derived from a protein band obtained from a single electrophoretic separation. The genome of Menangle virus has recently been characterised and the comparison of nucleotide and deduced amino acid sequences for genes
of Menangle virus with those of members of the family Paramyxoviridae and phylogenetic analyses confirmed that Menangle virus is a member of the genus Rubula6irus. The deduced protein sequence of the NP protein gene exhibited significant homology with the NP proteins of a number of viruses. Database alignment of the partial Menangle virus NP sequence suggested that the virus bore the closest relationship to human parainfluenza virus type 4a and 4b, mumps virus and La Piedad–Michoacan virus, members of the Rubula6irus genus (Bowden et al., 2001). A number of new members of the family Paramyxoviridae that appear to have fruit bats as their native host, have emerged from obscurity and caused significant diseases in man and animals. Hendra and Nipah viruses, which have emerged in Australia and Malaysia, cause acute disease in man and animals and constitute what appears to be a new genus in the subfamily Paramyxovirinae (Wang et al., 2000). Menangle virus caused reproductive failure in pigs and an influenza-like illness in man and is antigenetically related to Tioman virus, also a member of the Rubula6irus genus (Chua et al., 2001). Bats constitute a most abundant vertebrate species with al-
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most 1000 species (Wilson, 1997). They appear to be a rich source of novel viruses, some of which clearly have the potential to cause significant disease in man and domestic animals. Hendra, Nipah, Menangle and Tioman viruses are members of the family Paramyxoviridae. Other uncharacterised members of the same family, Mossman and J viruses have also been isolated from Australian native and introduced rodent species (Campbell et al., 1977; Jun et al., 1977), but their relationship to other members of the Paramyxoviridae is unclear. Detailed study of Mossman and J viruses is warranted to determine their relationship to new and existing members of the Paramyxoviridae and to explore their potential for emergence as new disease agents of man and animals. As zoonotic agents these viruses are rather difficult and costly to purify (requirements of BSL4 facilities) and as a consequence are available to detailed analyses in relatively small quantities. The application of mass spectrometric analysis to such viral proteins offers a powerful and effective approach to rapid identification and characterisation of structural proteins of newly emergent viruses, and its usefulness has been demonstrated in studies of Hendra and Nipah viruses (Wang et al., 1998, 2001; Michalski and Shiell, 1999; Michalski et al., 2000).
Acknowledgements We wish to thank Gary Crameri and Bryan Eaton for the preparation of viral material and stimulating discussions.
References Blom, N., Gammeltoft, S., Brunak, S., 1999. Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294, 1351 –1362. Bowden, T.R., Westenberg, M., Wang, L.-F., Eaton, B.T., Boyle, D.B., 2001. Molecular characterization of Menangle virus, a novel Paramyxo6irus which infects pigs, fruit bats and humans. Virology 283, 358 –373. Campbell, R.W., Carley, J.G., Doherty, R.L., Domrow, R., Filippich, C.G.B., Karabatsos, N., 1977. Mossman virus, a
Paramyxo6irus of rodents isolated in Queensland. Search 8, 435 – 436. Chant, K., Chan, R., Smith, M., Dwyer, D.E., Kirkland, P., 1998. Probable human infection with a newly described virus in the family Paramyxoviridae. Emerg. Infect. Dis. 4, 273 – 275. Chua, K.B., Bellini, W.J., Rota, P.A., Harcourt, B.H., Tamin, A., Lam, S.K., Ksiazek, T.G., Rollin, P.E., Zaki, S.R., Shieh, W.-J., Goldsmith, C.S., Gubler, D.J., Roehrig, J.T., Eaton, B., Gould, A.R., Olson, J., Field, H., Daniels, P., Ling, A.E., Peters, C.J., Anderson, L.J., Mahy, B.W.J., 2000. Nipah virus: a recently emergent deadly Paramyxo6irus. Science 288, 1432 – 1435. Chua, K.B., Wang, L.F., Lam, S.K., Crameri, G., Yu, M., Wise, T., Boyle, D., Hyatt, A.D., Eaton, B.T., 2001. Tioman virus, a novel Paramyxo6irus isolated from fruit bats in Malaysia. Virology 283, 215 – 229. Harcourt, B.H., Tamin, A., Ksiazek, T.G., Rollin, P.E., Anderson, L.J., Bellini, W.J., Rota, P.A., 2000. Molecular characterisation of Nipah virus, a newly emergent Paramyxo6irus. Virology 271, 334 – 349. Jun, M.H., Karabatsos, N., Johnson, R.H., 1977. A new mouse Paramyxo6irus (J virus). Aust. J. Exp. Biol. 55, 646 – 647. Kreegipuu, A., Blom, N., Brunak, S., 1999. PhosphoBase, a database of phosphorylation sites: release 2.0. Nucleic Acids Res. 27, 237 – 239. Michalski, W.P., Shiell, B.J., 1999. Strategies for analysis of electrophoretically separated proteins and peptides. Anal. Chim. Acta 383, 27 – 46. Michalski, W.P., Shiell, B.J., Beddome, G., Crameri, G., Eaton, B.T., 2000. Phosphorylation sites pf P protein from Hendra and Nipah viruses: new members of Paramyxoviridae. The 4th European Symposium of the Protein Society (Proceedings), Paris, France. Protein science 10, Suppl. 1, p. 61. Philbey, A.W., Kirkland, P.D., Ross, A.D., Davis, R.J., Gleeson, A.B., Love, R.J., Daniels, P.W., Gould, A.R., Hyatt, A.D., 1998. An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4, 269 – 271. Wang, L.F., Michalski, W.P., Yu, M., Pritchard, L.I., Crameri, G., Shiell, B., Eaton, B., 1998. A novel P/V/C gene in a new member of the Paramyxoviridae family, which causes lethal infection in humans, horses, and other animals. J. Virol. 72, 1482 – 1490. Wang, L.F., Yu, M., Hannson, E., Pritchard, L.I., Shiell, B., Michalski, W.P., Eaton, B.T., 2000. The exceptionally large genome of Hendra virus: support for creation of a new genus within the family Paramyxoviridae. J. Virol. 74, 9972 – 9979. Wang, L.F., Harcourt, B.H., Yu, M., Tamin, A., Rota, P.A., Bellini, W.J., Eaton, B., 2001. Molecular biology of Hendra and Nipah viruses. Microbes Infect. 3, 279 – 287. Wilson, D.E., 1997. Bats in Question. The Smithsonian Answer Book. CSIRO Publishing, Melbourne.
B.J. Shiell et al. / Journal of Virological Methods 102 (2002) 27–35 Young, P.L., Halpin, K., Selleck, P.W., Field, H., Gravel, J.L., Kelly, M.A., MacKenzie, J.S., 1996. Serologic evidence for the presence in Pteropus bats of a paramyxovirus related to Equine Morbillivirus. Emerg. Infect. Dis. 2, 239 –240.
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Yu, M., Hansson, E., Shiell, B.J., Michalski, W.P., Eaton, B.T., Wang, L.F., 1998. Sequence analysis of the Hendra virus nucleoprotein gene: comparison with other members of the subfamily Paramyxovirinae. J. Virol. 79, 1775 – 1780.
Mass spectrometric identification and characterisation of the nucleocapsid protein of Menangle virus Brian J. Shiell, Gary Beddome, Wojtek P. Michalski * Australian Animal Health Laboratory, CSIRO Li6estock Industries, Pri6ate Bag 24, Geelong, Vic. 3220, Australia Received 23 July 2001; received in revised form 26 November 2001; accepted 27 November 2001
Abstract The recent emergence of novel viruses requires reliable methodology for their identification and confirmation both on a cellular and molecular level. Mass spectrometry offers a suitable approach for the identification and characterisation of viral proteins and its application is demonstrated in this study. Menangle virus is a previously unclassified member of the family Paramyxoviridae isolated in Australia in 1997. Menangle virus caused disease in pregnant pigs, and like the other newly emergent Hendra, Nipah and Tioman viruses, appears to be a virus of fruit bats (flying foxes) in the genus Pteropus. The 61 kDa gel-purified protein isolated from cell-associated Menangle virus ribonucleoprotein (RNP) was identified as the nucleocapsid protein (NP) by peptide mapping, mass spectrometry and amino acid sequencing. Over 69% of the amino acid sequence was obtained and found to be identical with that derived from gene analysis (Virology, 283 (2001), 358). The first residue of the mature NP was found to be serine (second residue in the gene derived amino acid sequence). The NP was found to be acetylated at the N-terminus (at Ser-2) and appears to be not modified by phosphorylation. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Nucleocapsid protein; Mass spectrometry; Menangle virus; Rubula6irus; Paramyxoviridae
1. Introduction Advances in gene analysis technology have led to the ability to sequence genes with relative ease and deduce protein sequences as a consequence. However, there are still a variety of reasons why it is desirable to perform a secondary check of these deduced sequences. In some instances mistakes have been made during nucleic acid sequencing. * Corresponding author. Tel.: + 61-3-5227-5772; fax: + 613-5227-5555. E-mail address: [email protected] (W.P. Michalski).
Messenger RNA may also be subject to base insertions or contain multiple initiation codons. Furthermore, gene sequence data alone may be misleading due to the potential for protein splicing events to produce final products with primary structure that could not be predicted from the original gene coding sequences. Protein trans-peptidation has been documented with the product of a single gene and between products of separate genes. Acquisition of information regarding other post-translational modifications also requires protein structure analysis. A strategy for comprehensive comparison of viral protein sequences
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with the products predicted from the corresponding gene sequences is described in this paper. Mass spectrometry plays a principal role in this strategy (Michalski and Shiell, 1999), which also includes other protein analysis techniques such as high performance liquid chromatography (HPLC), amino acid analysis and N-terminal sequencing. In this paper, we demonstrate an application for this strategy in the identification and characterisation of a protein from a newly emergent virus. Menangle virus is a recent addition to the family Paramyxoviridae, subfamily Paramyxovirinae and is the causative agent of a single outbreak of disease in a commercial piggery characterised by a decrease in the farrowing rate and the number of live piglet births. Stillborn piglets had severe degeneration of the brain and spinal cord (Philbey et al., 1998) but infection of adult, non-pregnant pigs was not apparent. Menangle virus also appeared to cause an influenza-like illness of two humans in close contact with infected pigs (Chant et al., 1998). The presence of neutralising antiMenangle virus antibodies in fruit bats (Pteropus genus) suggests they may be the natural host of the virus (Philbey et al., 1998). Serological surveys have implicated flying foxes as the natural host of Hendra virus in Australia (Young et al., 1996). Hendra virus and the closely related Nipah virus (Chua et al., 2000; Harcourt et al., 2000) are also members of the family Paramyxoviridae but cannot be readily classified in any of the current genera within the family (Wang et al., 1998; Yu et al., 1998; Wang et al., 2000, 2001). Menangle virus ultrastructure is consistent with its membership in the family Paramyxoviridae (Philbey et al., 1998) but lacks antigenic cross reactivity with antiserum to viruses in the Rubula6irus, Morbilli6irus and Respiro6irus genera. Comparison of nucleotide and deduced amino acid sequences for genes of Menangle virus with those of members of the family Paramyxoviridae and phylogenetic analyses confirmed that Menangle virus is a member of the genus Rubula6irus (Bowden et al., 2001). Menangle virus is antigenetically related to Tioman virus, another member of the genus Rubula6irus, which was also identified in fruit bats (Chua et al., 2001).
2. Materials and methods
2.1. Purification of Menangle 6irus RNP and NP Monolayers of Vero cells were infected with Menangle virus at a multiplicity of 0.01 TCID50 per ml. At 48 h post infection cells were washed with phosphate buffered saline (PBS) and resuspended in TNM (10 mM Tris, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2) at 4 °C and lysed by addition of NP-40 to 1% (v/v). Nuclei were pelleted and the associated cytoskeleton removed by shearing in a Dounce homogeniser. The nuclei were pelleted, cytoplasmic and cytoskeletal fractions pooled and ethylenediaminetetraacetic acid disodium salt (EDTA) and sodium deoxycholate added to 10 mM and 1% (w/v), respectively. After clarification at 10 000 rpm in a SW55 rotor for 10 min at 4 °C the lysate was layered over 20–40% CsCl gradients in TNE (10 mM Tris, 100 mM NaCl, 1 mM EDTA) with 0.2% sodium lauroyl sarcosinate. After centrifugation in a SW41 rotor for 24 h at 25 000 rpm the visible ribonucleoprotein (RNP) band at 1.31 g cm − 3 was collected and pelleted at 35 000 rpm for 1.5 h in a SW41 rotor (Bowden et al., 2001). Virus purification was performed at Bio-Security Level 4 (BSL 4) at the Australian Animal Health Laboratory by Gary Crameri and Bryan Eaton. Pelleted virus was inactivated in 2% sodium dodecyl sulphate (SDS) prior to its transfer to the non-secure environment and viral proteins were analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS (SDSPAGE).
2.2. Electrophoresis Proteins of SDS-treated virus were separated under reducing conditions on 10% Tris–glycine gels (Novex, San Diego, CA). Proteins in the gels were then either, (i) visualised by Coomasie Brilliant Blue R-250 staining; or (ii) by reverse-staining using a Zinc-imidazole stain kit (Bio-Rad Laboratories, Hercules, CA) and excised for subsequent ‘in gel’ tryptic digestion and mass spectrometry; or (iii) electro-transferred to polyvinylidene fluoride membrane and stained
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(see Section 2.4) prior to direct sequencing and/or chemical cleavage.
2.3. ‘In gel’ digestion Gel bands were cut into 1× 2 mm pieces, transferred to 0.5 ml Eppendorf tubes and washed twice in 0.2 M NH4HCO3/50% acetonitrile. Protein in the gel pieces was then reduced with 10 mM dithiothreitol for 1 h in 0.2 M NH4HCO3, washed twice with 0.2 M NH4HCO3, and then alkylated with 50 mM iodoacetamide in 0.2 M NH4HCO3 for 20 min in the dark. Following two more washes in 0.2 M NH4HCO3 and a final wash in 0.2 M NH4HCO3/50% acetonitrile, the gel pieces were dried for 30 min in a SpeedVac concentrator. Gel pieces were then rehydrated twice with 10 ml of trypsin solution (modified sequencing grade, Roche) of 1 mg/20 ml in 50 mM NH4HCO3 for 10 min at 37 °C, 150 ml 50 mM NH4HCO3 added and digestion continued overnight at 37 °C. After overnight incubation the sample was centrifuged at 12 000×g and supernatant containing peptide fragments removed and kept aside. Residual peptides in the gel pieces were then extracted twice with 200 ml of 60% acetonitrile/1% trifluoroacetic acid (TFA) at 37 °C for 30 min, centrifuged and supernatants combined. Finally, the pooled supernatants were concentrated in a SpeedVac concentrator to approximately 50 ml for subsequent analysis by liquid chromatography – mass spectrometry or isolation by reverse-phase chromatography.
29
rated solution of CNBr in 70% formic acid for 24 h. CNBr-generated peptides were eluted from the membrane with 40% acetonitrile containing 0.05% TFA for subsequent isolation by reverse-phase chromatography.
2.5. Re6erse-phase chromatography Chromatography of trypsin and CNBr digested protein samples was performed on a Beckman System Gold HPLC. Peptides from the trypsin digest were separated on a Vydac C18 column (2.1× 250 mm, Separations Group, Hysperia, CA) using a binary gradient with 0.1% TFA as buffer A and 0.09% TFA/80% acetonitrile as buffer B. The gradient was developed as follows; 2–37.5% B over 0–50 min, 37.5–75% B over 50–70 min and finally 75–100% B over 75–85 min at a flow rate of 200 ml min − 1. Peptides from CNBr cleavage were separated on a Vydac C4 column (2.1× 250 mm, Separations Group) using the same buffer system as above but with a gradient of 5–100% B over 45 min. In both cases, purified peptides were hand collected and stored at − 20 °C prior to N-terminal sequencing.
2.6. N-terminal amino acid sequencing Hand collected peptides and polyvinylidene fluoride immobilised protein were subjected to amino acid sequence analysis with vapour-phase delivery of critical reagents in an automated sequenator (Model 470A, Applied Biosystems, Foster City, CA).
2.4. Chemical clea6age 2.7. Liquid chromatography–mass spectrometry Polyvinylidene fluoride membranes were stained for approximately 30 s in a freshly made and filtered 0.1% solution of Coomassie Brilliant Blue R-250, and destained in freshly made 1% aldehyde-free acetic acid/40% methanol until proteins bands were visible. A band identified as the NP protein was excised from the membrane and either subjected to direct amino acid sequencing or to cyanogen bromide (CNBr) cleavage. Protein immobilised on polyvinylidene fluoride was visualised with Ponceau S and protein bands excised from membranes were exposed to satu-
The trypsin digest was separated by reversephase chromatography on a Vydac C18 column (2.1× 250 mm, 5 mm, 300 A, , The Separations Group) using a Thermo Separations Products HPLC system (Finnigan, San Jose, CA) comprising P4000 gradient pump, SCM1000 degasser and AS3000 autosampler. Mobile phase buffers were 0.1% v/v TFA (A) and 0.1% TFA/80% acetonitrile (B), run at 200 ml min − 1 with a linear gradient of 5–100% B over 40 min, held at 100% B for 5 min, then returned to 5% B over 5 min.
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The effluent from the HPLC column was connected directly to the electrospray ion source of a Finnigan LCQ Classic (San Jose, CA) quadrupole ion-trap mass spectrometer. Liquid chromatography tandem mass spectrometry (LC–MS – MS) analysis was carried out in a data dependent mode known as the ‘triple play’ where the most intense ion in each full scan (m/z 400–2000) was automatically selected and subjected to a high resolution zoom scan followed by a tandem mass spectrometry product ion scan. The zoom scan allowed determination of the precise mass/charge state of the selected ion while tandem mass spectrometry determination of the product ion patterns derived from collision induced dissociation of the selected ion. These provided information on the sequence of the peptides and their possible modifications. Tandem mass spectrometry scans were performed with collision energy of 32%. The electrospray ion source settings were as follows: ion spray voltage 5 kV, capillary temperature 200 °C, capillary voltage 42 V and tube lens offset 20 V. Sequence information for Menangle virus NP protein was obtained from the GeneBank database with accession number AF326114.
thereof, than that predicted for tryptic peptides from a theoretical trypsin digest of NP.
3. Results Amino acid sequence analysis requires protein samples to be free from other proteins and peptides. As Menangle virus preparations were routinely inactivated with 2% SDS (a procedure required for the safe transfer of a zoonotic agent from BSL 4 laboratory to non-secure environment), electrophoresis (SDS-PAGE) was employed for the separation of individual proteins and their isolation for sequence analysis. Menangle virus nucleocapsid protein (NP) separates on SDS-PAGE gels as a well-defined protein band (Fig. 1) that can easily be excised from gels, or membranes following electro-transfer. Gel purified protein was analysed after ‘in gel’ trypsin digestion by mass spectrometry and N-terminal sequencing, whereas protein transferred onto PVDF membrane was subjected to chemical di-
2.8. Prediction and detection of phosphorylation Prediction of possible phosphorylation sites was performed using NETPHOS 2.0 (Blom et al., 1999) and PHOSPHOBASE 2.0 (Kreegipuu et al., 1999) prediction databases. Immunodetection of phospho-amino acids was performed on Menangle virus NP protein transferred onto polyvinylidene fluoride membrane and probed with rabbit antibody (antigen affinity purified) raised against phospho-tyrosine, phospho-threonine and phospho-serine (Zymed Laboratories Inc., CA). Antiphosphoamino acid antibodies were probed with goat anti-rabbit IgG conjugated to horse-radish peroxidase (HRP). Visualisation of reactive protein bands was performed using an enhanced chemiluminescence HRP substrate kit (Amersham Pharmacia). Mass spectrometry analysis of phosphorylation was performed by manual searching of the data for masses 80 Da higher (corresponding to addition of phosphate), or multiples
Fig. 1. SDS-PAGE separation of proteins from three different preparations (A – C) of RNP isolated from Menangle virus-infected cells (lanes 2, 4 and 6, 15 mg protein; lanes 3, 5 and 7, 5 mg protein). Lane 1 contains Novex Mark 12 and lane 8 Novex SeeBlue™ Plus 2 standard marker proteins (10 ml of supplied sample). The marked (arrow) 61 kDa protein is the putative Menangle virus NP.
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Fig. 2. LC – MS of Menangle virus NP protein digested with trypsin. Conditions for MS analysis are given in Section 2. T1 – T3 denote peptides identified as autolytic products of trypsin. U1 – U3 denote unidentified peptides. Peptide masses and sequence assignment is given in Table 1.
gestion by cyanogen bromide prior to analysis by N-terminal sequencing.
3.1. Peptide mapping and sequence analysis Trypsin was used to generate internal peptides from the NP protein samples excised from polyacrylamide gels. The digests were analysed by liquid chromatography tandem mass spectrometry (a LC –MS of tryptic digest is shown in Fig. 2) and N-terminal amino acid sequencing. No sequence coverage was obtained for a number of small peptides 312–348 and 432– 474, or the large peptide from residues 120 to 180. Both peptides 120– 180 and 312–348 with respective monoiso-
topic masses of 6995.24 and 4313.86 are outside the mass range of the LCQ mass spectrometer. It is possible that the third peptide 432– 474 is too negatively charged and thus suppressed in the MS analysis. No amino acid sequence data was obtained by direct sequencing of gel purified NP suggesting that the protein was blocked at the N-terminus (see above). Amino acid sequencing and mass spectrometry analyses of HPLC-purified peptides derived from ‘in gel’ trypsin digestion generated a large number of peptides of which 40 provided maximum coverage of the NP protein and are included in Table 1. Chemical cleavage with CNBr generated eight peptides and provided confirmatory sequence information for regions
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Table 1 NP peptides identified and confirmed by MS and sequencing analyses Peak
Sequence assignment
Mass found
Mass predictedf
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 T1 17 18 19 20 21 T2 22 23 24 25 26 27 U1 28 U2 T3 29 30 U3 31 32 33 34 35 36 37 38 39 40
Leu-205–Arg-209 Asn-482–Arg-488 Leu-401–Arg-405 Leu-114–Arg-119 Leu-196–Arg-204 Cys-181–Arg-195 Leu-58–Arg-65 Asp-393–Arg-400 Val-37–Arg-48e Leu-475–Arg-481e Ile-90–Arg-101e Ser225–Arg230 Ser-2–Arg-6a Asp-489–Arg-507e Thr-33–Arg-48c His-49–Arg-57 Ser-126–Lys-136 Tyr-209–Arg-217e Trp-281–Lys-295 Gly-406–Arg-420b His-49–Arg-57b Gln-231–Arg-241e Leu-50–Lys-69 Gln-372–Arg-392 Gln-231–Arg-241d Asp-489–Met-519c Gly-406–Arg-420e Gln-372–Arg-392d Leu-401–Arg-420 Unknown Asp-489–Met-519b Unknown Ser-70–Lys-89 Ile-90–Arg-113 Trp-281–Arg-304 Unknown Asp-489–Met-519 Ala-7–Arg-32 Asn-349–Lys-378 Asn-349–Arg-371 Thr-70–Arg-89c Ala-66–Arg-89b Thr-70–Arg-89b Ser-2–Arg-32a Ala-66–Arg-89 Thr-70–Arg-89
531.21 818.42 547.35 700.46 1091.41 1760.63 831.49 850.42 1429.47 886.49 1357.69 676.42 637.31 2075.19 1885.93 1301.47 1153.27 1145.69 1612.99 1573.71 1285.27 1343.75 2162.99 2276.13 1326.55 3524.90 1557.95 2259.27 2086.07 3444.53 3508.91 3462.56 2273.21 2719.55 2745.11 2733.38 3492.53 3069.61 3431.9 2689.61 2213.13 2604.23 2197.05 3688.10 2588.18 2181.21
531.29 818.39 547.32 700.42 1091.60 1760.74 831.47 850.41 1429.69 886.51 1357.67 676.40 637.33 2075.03 1885.96 1301.49 1153.57 1145.64 1612.91 1573.78 1285.50 1343.71 2163.06 2276.12 1326.71 3524.62 1557.78 2259.12 2086.08 – 3508.63 – 2273.16 2719.33 2745.55 – 3492.64 3069.48 3431.71 2689.35 2213.18 2604.41 2197.18 3687.79 2588.42 2181.19
a, N-terminal acetylation; b, one oxidised methionine residue; c, two oxidised methionine residues; d, N-terminal pyroglutamylation; e, confirmed by sequencing; f, predicted masses were determined using the software program MS-DIGEST from the UCSF Mass Spectrometry Facility (http:// prospector.ucsf.edu/ucsfhtml3.4/msdigest.htm).
122–132, 182–185, 253–278, 347–350, 394–416 and 513–518. The sequence coverage obtained from all these peptides was identical with over 69% of that deduced from the nucleotide sequence of the Menangle virus NP gene (Fig. 3).
3.2. Phosphorylation of NP protein NP protein has 18 serine and 25 threonine residues (Bowden et al., 2001), with many of these residues located in predicted phosphorylation-site consensus sequences (16 predicted sites). Detection of phosphoproteins was performed using immunodetection of phosphorylated amino acids with antibodies specifically reacting with phosphotyrosine, phosphothreonine and phosphoserine (P-Tyr, P-Ser, P-Thr). None of the viral proteins reacted with anti-P-Tyr, anti P-Thr and P-Ser antibodies indicating the absence of phosphorylated residues in the NP protein (data not shown). Phospho-proteins from Hendra and Nipah viruses were used as positive controls. Also, liquid chromatography tandem mass spectrometry analysis of the trypsin ‘in gel’ digest of the NP protein did not reveal the presence of any phosphorylated peptide fragments. No unexplained masses corresponding to peptides with the addition of phosphate (+80 Da) were found.
3.3. N-terminal modification Direct N-terminal amino acid sequence analysis of the NP protein immobilised on PVDF membrane yielded no data, thereby indicating that the N-terminus of the protein was chemically blocked. It should be noted that similar results were obtained in experiments when aldehyde-free acetic acid was used in staining procedures suggesting that the NP protein may have its N terminus modified ‘in vivo’. Liquid chromatography tandem mass spectrometry analysis of the N-terminal peptide from the ‘in gel’ trypsin digest revealed that the NP protein has the N-terminal Met cleaved and is indeed acetylated at Ser-2 (Table 1, peptides/peaks c13 and c 38). This indicates that serine is actually the first residue of the mature Menangle virus NP protein. An identical modification has been found in Hendra virus
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Fig. 3. Correlation of amino acid sequence determined for peptides derived from purified NP with deduced amino acid sequence from mRNA sequence obtained by PCR-Select cDNA subtraction cloning (Bowden et al., 2001). The amino acid sequences of peptides derived from the 61 kDa protein in purified RNP that were confirmed by mass spectrometry are underlined while those confirmed by N-terminal sequencing are in bold.
NP protein (B. Shiell and W. Michalski, personal communication).
4. Conclusions In recent years, with improvements in instrumentation and sample handling, mass spectrometry has become the analytical method of choice not only for the identification of proteins, but also for the structural investigation of post-translational modifications. In this paper, we report on identification and comprehensive characterisation of Menangle virus NP protein using peptide mapping (69% coverage), mass spectrometry (61% coverage) and N-terminal sequencing (31% coverage). The application of automated LC– MS – MS methodology using the Finnigan LCQ ESI– MS instrument for analysis of NP protein from the newly emerged Menangle virus provided a comprehensive analysis, in a single chromatographic run on sample derived from a protein band obtained from a single electrophoretic separation. The genome of Menangle virus has recently been characterised and the comparison of nucleotide and deduced amino acid sequences for genes
of Menangle virus with those of members of the family Paramyxoviridae and phylogenetic analyses confirmed that Menangle virus is a member of the genus Rubula6irus. The deduced protein sequence of the NP protein gene exhibited significant homology with the NP proteins of a number of viruses. Database alignment of the partial Menangle virus NP sequence suggested that the virus bore the closest relationship to human parainfluenza virus type 4a and 4b, mumps virus and La Piedad–Michoacan virus, members of the Rubula6irus genus (Bowden et al., 2001). A number of new members of the family Paramyxoviridae that appear to have fruit bats as their native host, have emerged from obscurity and caused significant diseases in man and animals. Hendra and Nipah viruses, which have emerged in Australia and Malaysia, cause acute disease in man and animals and constitute what appears to be a new genus in the subfamily Paramyxovirinae (Wang et al., 2000). Menangle virus caused reproductive failure in pigs and an influenza-like illness in man and is antigenetically related to Tioman virus, also a member of the Rubula6irus genus (Chua et al., 2001). Bats constitute a most abundant vertebrate species with al-
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most 1000 species (Wilson, 1997). They appear to be a rich source of novel viruses, some of which clearly have the potential to cause significant disease in man and domestic animals. Hendra, Nipah, Menangle and Tioman viruses are members of the family Paramyxoviridae. Other uncharacterised members of the same family, Mossman and J viruses have also been isolated from Australian native and introduced rodent species (Campbell et al., 1977; Jun et al., 1977), but their relationship to other members of the Paramyxoviridae is unclear. Detailed study of Mossman and J viruses is warranted to determine their relationship to new and existing members of the Paramyxoviridae and to explore their potential for emergence as new disease agents of man and animals. As zoonotic agents these viruses are rather difficult and costly to purify (requirements of BSL4 facilities) and as a consequence are available to detailed analyses in relatively small quantities. The application of mass spectrometric analysis to such viral proteins offers a powerful and effective approach to rapid identification and characterisation of structural proteins of newly emergent viruses, and its usefulness has been demonstrated in studies of Hendra and Nipah viruses (Wang et al., 1998, 2001; Michalski and Shiell, 1999; Michalski et al., 2000).
Acknowledgements We wish to thank Gary Crameri and Bryan Eaton for the preparation of viral material and stimulating discussions.
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