Identification of mumps virus protein and lipid composition by mass spectrometry

Investigation of the most abundant mumps virus proteins was performed on L-Zg mumps virus grown on two cell substrates, CEF and Vero cells. Purified mumps virus was subjected to electrophoresis under denaturating conditions. Results show that proteins of mumps virus derived from two cell types, CEF and Vero, are similar (Figs. 1 and 2, respectively, Additional file 1: Table S1). There are more host cell proteins detected in samples derived from Vero cells but this is probably a result of limitation of the applied purification and detection techniques. Disruption of the cell caused by virus infection is variable, and is expected to have an impact on the purity of the virus. Problem of the virus isolation procedure and ability to clearly discern contaminating from interacting or constituting host cell proteins in virions has been recognized as controversial [37]. Virus purification procedure employed here consists of collecting supernatant from cells with visible cytopathic effect and removal of cell parts by low speed centrifugation followed by pelleting of viruses by ultracentrifugation. Analysis of ultracentrifugation purification procedure was found to result in 10 % recovery (n = 23) of infectivity in the pellet (determined by 50 % cell culture infective dose assay) whereas amount of the infective virus in the supernatant was below 0.01 % (manuscript in preparation). Low speed centrifugation step may not be fully effective in removal of cell proteins that are incorporated in the small parts of the cellular membrane generated during cytophatic effect caused by the viral infection of the cell, resulting in their copurification with virus particles during ultracentrifugation. Unfortunately, copurification of parts of cellular membranes with virions cannot be avoided even by density gradient purification due to the possibility of similar sedimentation coefficient. However, host cell proteins can be incorporated into virions simply by being fortuitously present at the site of budding or preferentially incorporated into virions thereby influencing viral biology and pathogenesis [38] and host cell proteins in virions have been reported for various viruses [37‐44]. During our work, several virus preparations were purified and found to vary regarding amount of cell proteins. Host cell proteins that were detected are fibronectin, clathrin, actin, histones, heat shock proteins (HSP), annexins, tubulin, ribosomal protein, myosin, and all these proteins were reported to be detected in other viruses also [37‐44]. Some early papers on sucrose gradient purified mumps virus particles report that mumps virions contain actin [25, 28, 29]. For respiratory syncytial virus, also a paramyxovirus, association with HSP70, HSP90 and actin among other host cell proteins has been reported [44]. Role of actin in virus morphogenesis of several viruses is described [45] as well as its role in respiratory syncytial virus maturation [46] therefore similar function could be expected in mumps.

Out of nine proteins coded by seven mumps genes, only four, HN, NP, M and P, were identified in mumps virus grown in CEF, whereas in mumps virus grown in Vero, additionally L and V proteins were detected (identification data are listed in Additional file 1: Table S1). HN protein was detected in two and NP in three bands.

Proteins not detected and identified in any of our tested samples in purified virus preparations were F, SH and I protein. I protein is not considered as a structural protein so its presence in purified virus preparations is not expected. SH protein was detected in virus infected cells and is considered to be a membrane protein [9, 10] but has not yet been detected in mumps virions. F protein is synthesized as a precursor molecule F₀ that is processed by host cell proteases to yield active F protein composed of two disulphide-linked polypeptides [47]. Processing of the F protein has been well studied on cell lysates and F protein was detected by immunoprecipitation of the disrupted virus by anti-F antibodies [31] and in case of radioactively labelled glycoproteins in purified mumps virions [26]. However, by means of the techniques applied here we could not detect the F protein so far. Possible reasons for the lack of F protein detection could be its very low expression level, glycosylation level of the peptides (not covered by the applied MS method), low extraction efficiency from the gel matrix or peptide ion suppression, or the combination of all these factors. Interestingly, unsuccessful detection of F protein was reported for measles protein (also a member of Paramyxoviridae) and also multiple NP forms were confirmed [48] same as found in our work on mumps virus.

In mumps virus derived from CEF, NP has been detected in three bands and the band with the highest apparent molecular mass is most intensive and corresponds to theoretical (calculated from amino-acid sequence) NP molecular mass around 61 kDa, whereas the other two have lower molecular mass, around 55 and 48 kDa (Fig. 1). Peptide mass fingerprint (PMF) spectra of all three NP forms were analysed (Fig. 3, full peptide list in Additional file 1: Table S2) and it can be seen that spectra of NP bands with lower apparent molecular masses are missing C-terminal peptides. The same was found for mumps virus grown in Vero (Fig. 2). Presence of two NP bands in Fig. 2(a), instead of three as in Fig. 2(b) is presumably due to the limitation of the CBB detection and the present sample amount. Again, analysis of PMF was performed (Additional file 1: Figure S1, Table S3) and the C-terminal tryptic peptide was only present for the NP band of highest molecular mass, the same as with the CEF sample. Additionally, in Fig. 2(b) NP was also detected above 260 kDa marker and this is most likely an aggregated form of NP. The lowest of these protein bands, at 48 kDa, in addition to NP contained also P protein peptides and this molecular mass corresponds to the theoretical mass of P protein of 42 kDa. It should be stressed that a peptide’s occurrence in mass spectra can be influenced by various factors; trypsinization efficiency, selectivity during peptide isolation, MS inherent difficulties like ion suppression by presence of other peptides and selective desorption/ionization [49, 50]. Indeed, PMFs of these three NP bands do not contain completely identical sets of NP peptides. The lowest band contains peptides corresponding to P protein and their presence could possibly suppress some NP peptides. Nevertheless, when molecular masses of these NP proteins are calculated from sequences covered by the MS detected peptides, these data are in line with the experimentally obtained apparent molecular masses of NP bands. For the lowest NP band peptides detected in the PMF spectrum showed peptide coverage up to amino acid 400 (Fig. 3(b)). For the NP band with apparent molecular mass of 55 kDa detected peptide coverage ends with amino acid 525. Only for the NP band with apparent molecular mass of 61 kDa peptide from the C-terminus has been detected so this would indicate that NP bands with lower molecular masses are C-terminally truncated NP protein forms. Additionally, mumps virus proteins were detected also using anti-mumps serum in Western blot. The anti-mumps serum recognized three protein bands between 50 and 60 kDa and these correspond to MS identified NP forms (Additional file 1: Figure S2). Detection of only NP by the anti-mumps serum is probably a result of immunodominance of the NP as the most abundant mumps protein. There are reports on two forms (66 and 61 kDa) of mumps NP (Enders strain) detected with NP-specific monoclonal antibodies [51]. The 61 kDa form was speculated to be a product of cell proteases activity [51]. There is also one report on a truncated NP form spanning amino-acid sequence 331–498, found in cells infected with influenza virus [52]. Occurrence of this truncated NP follows kinetics of full length NP and there are no other NP forms detected with antibodies against the N-terminal part of NP protein. In addition, site at which proteolytic action should occur to result in this truncated form is not characteristic for any known endoprotease so this truncated form is reported as a likely result of transcription of one of the open reading frames [52]. Another example of truncated NP is in human influenza virus where the NP was found susceptible to cleavage by host cell caspase resulting in removal of the N-terminal peptide, and both NP forms have been found in infected cells and virions [53, 54]. The role of N-terminal caspase cleavage of influenza NP correlates with the host origin of the virus and is speculated to be a molecular determinant for host range [55]. The source and role of here detected NP forms that seem to be C-terminally truncated remains to be determined. It is known that the C-terminal part of NP in the subfamily of Paramyxovirinae is largely unstructured varying widely in length and sequence composition [4]. Open reading frames can result in alternate NP forms but the longest such form for mumps virus would have only 88 amino acids so this option is excluded. Other possibilities are editing of transcripts by RNA polymerase or hydrolysis by some cell protease.

M protein was detected in two very closely neighbouring gel bands under non-reducing conditions around 40 kDa corresponding nicely to the theoretical molecular mass of M protein. One band of a slightly higher apparent molecular mass was detected under reducing conditions. MS sequence analysis of M protein-derived peptides confirmed a protein modification, the acetylation of the N-terminus. M protein is one of the three most intensive proteins of mumps virus detected under experimental conditions used here, in addition to HN and NP, which is expected since it is a structural protein linking nucleocapsid to the lipid envelope.

According to literature HN protein is a disulphide bonded oligomer [2], thought to form tetramers as a mature protein [56‐58] and was detected as dimer [32] and dimer and tetramer in addition to monomer form [31] in purified mumps virus samples. Here, HN protein was identified in two protein bands (around 70 and 160 kDa) that roughly correspond to theoretical monomer and dimer forms of HN taking into consideration that the molecular mass of HN calculated from amino-acid sequence is 64 kDa and that HN is glycosylated resulting in higher molecular mass. Electrophoresis under reducing conditions resulted in more intensive gel bands for the monomer in comparison to non-reducing conditions (Fig. 1) but the dimer did not disappear completely. These results indicate that in mumps virus particle a part of HN is present as a monomer and part as a disulphide bridged dimer.

In addition to P protein that was detected in protein band together with smallest NP form (in both CEF and Vero samples), L protein was also detected (in Vero samples), both at apparent molecular masses corresponding to the theoretical ones. V protein was also detected (in Vero samples) but as a faint band. V protein has not been shown as a part of mumps virion so far but this could be a result of limitation of the techniques used, it was only detected in cell lysate [30].

Lipid classes present in two mumps virus strains, L-Zg and Jeryl Lynn 5 (JL5), grown in two cell lines, CEF and Vero, were analysed as well as lipids present in non-infected cells. Lipids were isolated by organic extraction, separated by two-dimensional high-performance thin layer chromatography (2D-HPTLC), visualised by primuline staining, extracted from the excised chromatographic plate material and analysed by MALDI MS. This methodology is relatively straightforward and provides wealth of data, however it is only a qualitative approach providing a global view on present lipid classes which does not detect lipids with masses below m/z 500.

Representative 2D-HPTLC separations of mumps virus lipids and lipids isolated from cells are given in Fig. 4. It can be seen that mumps virus derived from CEF cells has a lower number of present lipid classes compared to viruses grown in Vero cells especially regarding glycolipids. Similar results were observed for total cell lipids. The overall lipid composition of mumps virus samples is characterized by lipids also found for other viruses such as phosphatidylcholine, phosphatidylethanolamine, sphingomyeline, phosphatidic acid, glycerophosphocholine, phosphatidylinositol and glycolipids [14, 15, 17‐24, 59]. Interestingly, in virus samples derived from Vero cells we detected glycolipids with up to five monosaccharide units (the most polar compounds detected on HPTLC plates). Representative mass spectra and PSD spectra of glycolipids are given in Fig. 5. The importance of glycosylation in virulence and immune interactions on the level of glycoproteins is quite well known [60‐62] but the impact of glycolipids on virus properties has not been studied yet and their relevance could be equally important. Some reports on lipidomes of other viruses did not detect any glycolipids [17], some detected only glycolipids with one or two monosaccharide moieties [14, 15, 19, 20] but there are no reports on such complex virus glycolipids as detected in this study for mumps virus derived from Vero cells. The reason for this might be the methodology applied in other studies possibly omitting the separation. Or such glycolipids were actually absent since all reports discuss different cell lines possibly lacking these glycolipids. Difference in glycolipid content for different types of cells is demonstrated by our results showing that e.g. CEF do not contain glycolipids in contrast to Vero cells. One could presume that such complex glycolipids on the viral surface result in significant differences during e.g. cell infection and this we plan to investigate in our future work.