Identification of mumps virus protein and lipid composition by mass spectrometry
© Brgles et al. 2016
Received: 17 July 2015
Accepted: 5 January 2016
Published: 14 January 2016
Mumps virus is a negative-sense, single stranded RNA virus consisting of a ribonucleocapsid core enveloped by a lipid membrane derived from host cell, which causes mumps disease preventable by vaccination. Since virus lipid envelope and glycosylation pattern are not encoded by the virus but dependent on the host cell at least to some extent, the aim of this work was to analyse L-Zagreb (L-Zg) mumps virus lipids and proteins derived from two cell types; Vero and chicken embryo fibroblasts (CEF). Jeryl Lynn 5 (JL5) mumps strain lipids were also analysed.
Virus lipids were isolated by organic phase extraction and subjected to 2D-high performance thin layer chromatography followed by lipid extraction and identification by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Virus samples were also subjected to gel electrophoresis under denaturating conditions and protein bands were excised, in-gel trypsinized and identified by MS as well as tandem MS.
Results showed that lipids of both mumps virus strains derived from Vero cells contained complex glycolipids with up to five monosaccharide units whereas the lipid pattern of mumps virus derived from CEF was less complex. Mumps virus was found to contain expected structural proteins with exception of fusion (F) protein which was not detected but on the other hand, V protein was detected. Most interesting finding related to the mumps proteins is the detection of several forms of nucleoprotein (NP), some of which appear to be C-terminally truncated.
Differences found in lipid and protein content of mumps virus demonstrated the importance of detailed biochemical characterization of mumps virus and the methodology described here could provide a means for a more comprehensive quality control in vaccine production.
Mumps virus is a nonsegmented, negative stranded RNA virus of the family Paramyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus that causes mumps disease. Mumps virions are pleomorphic particles ranging from 100 to 800 nm in size , consisting of a helical ribonucleocapsid core surrounded by a host cell-derived lipid envelope. Full-length genomic RNA consisting of 15384 nucleotides contains 7 tandemly linked transcription units that encode open reading frames for the nucleoprotein (NP), phosphoprotein (P), V protein, I protein, matrix (M) protein, fusion (F) protein, small hydrophobic (SH) protein, hemagglutinin-neuraminidase (HN) protein, and the large (L) protein . Due to the RNA editing by insertion of G nucleotides, P gene results in three mRNA transcripts corresponding to P, V and I proteins . Nucleoprotein packages RNA into a nucleocapsid that is used as a template for transcription and genome replication by RNA polymerase which is composed of L and P protein [4, 5]. V protein was found to inhibit INF-β induced antiviral state [6–8]. SH protein is considered to be a membrane protein  with possible function in blocking TNF-α-mediated apoptosis pathway . On the virus surface are HN and F proteins that are glycosylated by the host cell machinery. HN is an attachment protein that binds receptor, and the role of F protein is to drive fusion of virus and the cell membrane resulting in virus entry . Virus envelope is acquired from the lipids of the host cell during budding process and as well as protein glycosylation pattern, it is not encoded directly by the virus. For paramyxoviruses, as well as for many other viruses (retroviruses, alphaviruses, rabdoviruses, orthoviruses), it has been shown that they bud predominantly from the surface of the infected cells. However, virus budding can also occur from intracellular membranes, e.g. human immunodeficiency virus type-1 (HIV-1) [12, 13]. Lipid composition of several viruses has been investigated so far such as HIV [14–17], murine leukemia virus , influenza virus , hepatitis C virus , Singapore grouper iridovirus , semliki forest virus [21–23], and vesicular stomatitis virus [21, 24]. These investigations revealed in some cases resemblance of the virus membrane to plasma membrane of the host cell, but in some cases also significant differences which have been described as a consequence of virus budding from lipid rafts. So, whether virus buds from a distinct membrane region and whether this is to some extent virus directed still remains an open question. Mumps virus proteins have been investigated in 1970s and 1980s using technology available at that time. Most of the mumps proteins were tentatively detected, underwent limited characterization, and were found to be similar to other paramyxoviruses but were mostly not confirmed unambiguously [25–32].
Mass spectrometry (MS) is a powerful tool in the field of biochemistry and is emerging as a core component of fundamental discoveries also in the field of virology . Combination of mass spectrometry and a relatively simple but efficient method high-performance thin layer chromatography (HPTLC) has been shown as a very convenient method for lipid analysis for not too complex lipidomes [34, 35].
Virus proteome and lipidome are expressed virus features that to some extent depend on the host cell and these must have an impact on virus characteristics such as infectivity and stability. Considering live virus vaccines, these features possibly affect vaccine performance. Therefore, methods characterizing both virus proteins and lipids could be a tool in raising the level of viral vaccines quality control. The aim of this research was to analyse lipid and protein pattern of the mumps virus derived from two cell lines (Vero and chicken embryo fibroblasts (CEF)) by means of mass spectrometry. Genome of L-Zagreb (L-Zg) mumps virus has been fully characterized  and here we report results on L-Zg mumps virus lipids and proteins for the first time in a truly holistic approach. Obtained data indicate that lipid profile of mumps virus depends on the host cell line and interestingly viruses derived from Vero cells contain glycolipids with up to five monosaccharide units. Mumps virus proteome was found to contain more than one nucleoprotein form whose function remains to be determined.
Results and discussion
Mumps virus proteins
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 . 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  and in case of radioactively labelled glycoproteins in purified mumps virions . 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  same as found in our work on mumps virus.
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 , thought to form tetramers as a mature protein [56–58] and was detected as dimer  and dimer and tetramer in addition to monomer form  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 .
Mumps virus lipids
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.
Presented data on mumps virus lipids and proteins indicate the presence of host cell proteins in mumps virions as found for other viruses. Interestingly, three NP forms were detected, two of which appear to be C-terminally truncated NP proteins of yet unknown function. Lipid analysis of mumps virus showed lipid pattern to be host dependent and viruses derived from Vero cells were found to contain glycolipids with up to five monosaccharide units. Presented results demonstrate virus variability and impact of these differences on virus characteristics remains to be determined.
Cell lines and viruses
Vero cells (African green monkey kidney cells) were purchased from ATCC and maintained in Minimum Essential Medium with Hank’s salts (MEM-H) (AppliChem) supplemented with 10 % foetal calf serum (FCS) (Moregate) and 50 μL/mL neomycin (Gibco-Life Technologies). Chicken embryo fibroblasts (CEF) were prepared from the 11-day old embryonated chicken eggs as described . L-Zg mumps virus strain was obtained from the Institute of Immunology Inc., Zagreb, Croatia. The virus strain JL5 was kindly provided by B. K. Rima (Queen's University of Belfast, United Kingdom).
Both viruses, L-Zg and JL5, were grown in Vero cells and CEFs in MEM-(H) with 2 % FCS at m.o.i. 0.0001. After 24 h the medium was replaced with medium without serum and virus was further grown until the cytopathic effect was observed. Then the medium was collected and centrifuged at 3000 × g to remove large-sized contaminants, and after that viruses were ultracentrifuged at 142,000 × g for 2 h and the pellet was resuspended in phosphate-buffered saline.
Electrophoresis under denaturating conditions was performed using 8 cm 4–12 % Bis-Tris precast gels, using MES running buffer and Novex Sharp Standard, in an XCell Sure Lock system from Invitrogen (Carlsbad) according to manufacturer’s instructions. Western blot detection was performed using guinea pig serum against mumps virus which was cultured in Vero cells and purified by ultracentrifugation. ECL Prime Western Blotting Detection Reagent was used for detection, according to the manufacturer’s instructions (GE Healthcare). Detection of protein bands was performed using acidic Coomassie Brilliant Blue R250 solution. Protein bands were excised from the gel, trypsinized and peptides isolated and purified for MS analysis as described . Experiments were performed with at least two sample replicates.
Lipid isolation was performed according to Bligh and Dyer . Lipids were dried in vacuo and separated by 2D-HPTLC (silica plates, 5 × 5 cm, particle size 4–8 μm, Merck) using chloroform/methanol/conc. ammonia 10:5:1 (V/V/V) for first dimension and after air-drying by means of chloroform/methanol/acetic acid/water 15:3:2:0.6 (V/V/V) for second dimension. Lipids were detected using primuline solution (0.05 % solution of primuline in acetone/water 8:2 (V/V)) and 365 nm UV light. Lipid spots were scratched from plates and 250 μL of chloroform/methanol/water 10:10:0.1 (V/V/V) was added in order to extract the lipids and extraction procedure was repeated two times. Obtained extracts were pooled and dried in vacuo. Lipids were dissolved in 4 μL of 20 mg/mL 2,4,6-trihydroxyacetophenone solution in methanol and applied to a stainless steel MALDI (matrix-assisted laser desorption/ionization) -target. Ricinus oil (0.5 μL in 1 mL of methanol with addition of small amount of NaCl) was used for mass calibration of the MS instruments. Experiments were performed at least with three separate samples.
MALDI MS analysis
Measurements were performed either on an AXIMA TOF2 instrument (Shimadzu - Kratos Analytical) or an UltrafleXtreme (Bruker Daltonik) in positive, reflectron ion mode. AXIMA TOF2 is equipped with a 20 Hz nitrogen laser (337 nm) and was operated in the positive ion mode applying an accelerating voltage of 20 keV. Analyses by means of an UltrafleXtreme (with a 2 kHz SmartBeam solid state laser (355 nm)) were performed at an acceleration voltage of 8 kV in the positive mode.
Raw data generated on AXIMA TOF2 instruments were converted into mzXML files and processed using mMass . Data generated on UltrafleXtreme were processed using Flexanalysis (3.4.70) and Biotools (3.2_SR4). Identification searches were performed against NCBInr database, “other viruses”, with the following fixed parameters: precursor ion mass tolerance and product ion mass tolerance of ± 0.6 Da and ± 1.2 Da for AXIMA TOF2, ± 200 ppm and ± 200 ppm for UltrafleXtreme (respectively), two missed trypsin cleavages, carbamidomethylation of Cys and with variable modification: oxidation of Met, ammonia loss from N-terminal Cys, N-acetylation, deamidation, and phosphorylation. Data on lipids were analysed using LIPID MAPS Structure Database (LMSD) .
The authors thank Sophie Fröhlich for her invaluable advices with the Ultraflextreme measurements, the Institute of Immunology for providing L-Zg mumps strain and Bertus K. Rima for providing JL5 strain, and Jelena Ivančić Jelečki for helpful discussion. The UltrafleXtreme mass spectrometer was made available by an IP grant of the Vienna University of Technology (to GA). This work was supported by the Bilateral Cooperation Grant Austria-Croatia (2012/2013 to MMD and MB), by the Ministry of Science, Education and Sports of the Republic of Croatia (grant No. 021-0212432-3123 to M.Š), and by Croatian Science Foundation (grant No. 8193 to MB). Further support is provided by the Shimadzu TU Wien CEE fellowship grant and the Austrian Federal Ministry for Transport, Innovation and Technology (FFG project 826132/GENIE granted to MMD).
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