Proteomic analysis of purified Newcastle disease virus particles
- Xiangpeng Ren†1, 2,
- Chunyi Xue†2,
- Qingming Kong2,
- Chengwen Zhang2,
- Yingzuo Bi3 and
- Yongchang Cao2Email author
© Ren et al.; licensee BioMed Central Ltd. 2012
Received: 10 November 2011
Accepted: 9 May 2012
Published: 9 May 2012
Newcastle disease virus (NDV) is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles.
In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52). The presence of five selected cellular proteins (i.e., β-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin) associated with the purified NDV particles was validated by Western blot or immunogold labeling assays.
The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.
Newcastle disease (ND) is a contagious fatal viral disease affecting most species of birds, which was classified as a list A infectious disease by the World Organization for Animal Health. Newcastle disease virus (NDV) as the etiological agent for ND is a nonsegmented single-stranded negative sense RNA virus that belongs to the genus Avulavirus within the Paramyxoviridae family . NDV is endemic to many countries, most notably in domestic poultry due to their high susceptibility, and has caused tremendous economic consequences to the poultry industry throughout the world. NDV virion contains at least six structural proteins, Haemagglutinin Neuraminidase (HN), Fusion protein (F), Matrix protein (M), Nucleocapsid protein (NP), Phosphate protein (P) and Large protein (L) [2–5]. HN and F are the two surface glycoproteins of viral envelope membrane, whereas NP、P、L and membrane-associated M are inner components of NDV virions [2–5]. F protein, which is considered to be the key virulence determinant of the virus, mediates the fusion process between viruses and cell membranes [6–8]. HN is a multifunctional virion protein, which plays roles in helping membrane fusion, cell tropism determination and viral pathogenicity [9–11]. M lies beneath the viral membrane and surrounds the ribonucleoprotein (RNP) complex . The RNP complex consists of the viral RNA coated with NP and bound by the polymerase complex that contains P and L .
It has been reported that many host proteins might be packaged into the enveloped virions along with the viral components during the virus life cycle, but the role of these cellular proteins in viral infection are not fully understood [14, 15]. Identification of the protein composition of the infectious virions has important implications for understanding the interaction of viruses with host cells, which provides valuable information for elucidating viral replication, tropism and virulence .
Due to enhanced proteomic techniques based on two-dimensional gel electrophoresis (2-DE) separation and Mass spectrometry (MS) combined with database searching for identification, virion proteomics (the protein composition of the purified virus particles) becomes a useful tool in global evaluation of interaction between viruses and their hosts through identifying cellular proteins in virions . Numerous host proteins have been found that incorporate into the membranes or inside the envelopes of the virions using virion proteomic approaches. Herpes virus, an enveloped DNA virus which is a leading cause of human viral diseases, is currently the best studied virus group. Among this group are human cytomegalovirus (HCMV) , murine cytomegalovirus (MCMV) , Epstein-Barr virus (EBV) , Kaposi’s sarcoma-associated herpesvirus (KSHV) [20, 21], rhesus monkey rhadinovirus (RRV) , Marek’s disease virus (MDV)  and murine gammaherpesvirus 68 (MHV68) . Moreover, virion proteomics have been performed for other enveloped DNA viruses, such as vaccinia virus (VV) [25, 26], gigantic mimivirus , White spot syndrome virus (WSSV) [28, 29] and Singapore grouper iridovirus (SGIV) .
Compared with enveloped DNA viruses, only a few enveloped RNA viruses have been analyzed by virion proteomics, potentially because of the relatively simpler structures and lower number of proteins encoded by RNA viruses. The most well studied RNA virus is retrovirus human immunodeficiency virus (HIV). Proteomic analysis revealed that HIV virions contain a high number of host cell proteins [31, 32]. Severe acute respiratory syndrome (SARS) coronavirus has also been analyzed by virion proteomics [33, 34]. In addition, 36 host-encoded cellular proteins have been found to incorporate into influenza virus (IV) virions . Other enveloped RNA viruses which have been proteomically analyzed were vesicular stomatitis virus (VSV) , infectious bronchitis virus (IBV)  and porcine reproductive and respiratory syndrome virus (PRRSV) . Virion proteomics have been used extensively to analyze the composition of a variety of virions, leading to a more complete picture of the viral particle.
However, to the best of our knowledge there is no mention of incorporation of host proteins in the enveloped-virus NDV so far. In this study, we selected the widely used NDV vaccine strain La Sota, utilized 2-DE/MS approaches to conduct a comprehensive proteomic analysis of purified NDV particles. Our analysis resulted in the identification of five virus-encoded structural proteins and thirty incorporated host proteins. Furthermore, the presence of five selected cellular proteins in the purified NDV particles was verified by Western blot or immunogold labeling detection.
Purification of NDV virions
Virion proteomic analysis requires large quantity of virions for preparation of highly purified virus particles. Therefore, the choice of host system used for virus growth is an important consideration. Since specific pathogen free (SPF) embryonated chicken eggs are the preferred host system for growth of NDV and the chicken genome is already well annotated which would benefit the identification of cellular proteins, the 9-day-old SPF embryonated chicken eggs were selected as the host system for NDV propagation in this study.
The allantoic fluid (AF) with enrichment of NDV virions harvested at 108 h post-infection was clarified by differential centrifugation in order to remove the contamination of nuclei, mitochondria, lysosomes, peroxisomes from the chicken embryo. The virus was concentrated and firstly purified through a 20% (W/V) sucrose cushion before further purified over a non-linear 20%-60% sucrose-TNE (Tris-buffered saline including 50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4) gradient. The high density opalescent virus band was observed at 40%–50% sucrose-TNE gradients.
Proteomic analysis of purified NDV particles
Identification and functional classification of NDV-associated proteins
Virus-encoded structural proteins and cellular proteins associated with purified NDV particles identified by MALDI-TOF/TOF MS
Reported in other viruses
S100 calcium-binding protein A6
Calcium ion binding
S100 calcium-binding protein A11
Calcium ion binding
Ras small GTPase
tumor protein, translationally-controlled 1
Calcium ion binding
tumor protein D52
Calcium ion binding
YWHAE 14-3-3, zeta polypeptide
chromatin modifying protein 5
chromatin modifying protein 2A
chromatin modifying protein 4B
chromatin modifying protein 4 C
YWHAE 14-3-3, theta polypeptide
YWHAE 14-3-3, epsilon polypeptide
syntenin;syndecan binding protein
tropomyosin 1 alpha
capping protein muscle Z-line, alpha 2
Annexin A8-like 1
Actin, gamma 1 propeptide;
ARP2 Actin -related protein 2 homolog
ARP3 Actin -related protein 3
similar to type I hair keratin KA31
Heat shock protein 70
CAP-GLY containing linker protein 2
heat shock 90 kDa aa protein 1, alpha
To better understand the implications of cellular proteins identified in NDV particles, these proteins were functionally categorized with biological processes according to Uniprot knowledgebase (Swiss-Prot/TrEMBL) and Gene Ontology Database. The identified thirty cellular proteins were composed of nine cytoskeletal proteins, two molecular chaperones, four chromatin modifying proteins, three enzymes-binding proteins, two calcium-binding proteins, two metabolism proteins, two signal transduction-associated proteins, two tumor-associated proteins (also characterized as calcium-binding proteins) and four uncharacterized proteins (Table 1). We firstly identified two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52) from the purified NDV particles.
Validation of cellular proteins by western blot
Validation of cellular proteins by electron microscopy and immunogold labeling
There is compelling evidence that enveloped virions carry multiple host proteins both internally and externally during infection . To date, no studies have been carried out on the incorporation of cellular proteins in NDV virions. In this study, we obtained highly purified NDV particles by sucrose gradients ultracentrifugation. Virion-associated proteins were identified by 2-DE/MS proteomic analysis followed by Western blot and electron microscopy. A total of five viral proteins and thirty host proteins were successfully detected. Our study provided strong evidence that cellular proteins were incorporated into the enveloped viruses.
The present study identified all the structural constitutes of NDV virions except L protein. F and HN are two major glycosidoproteins located on the surface of membrane, which are easy to detect in intact virions. M protein is also easy to identify because it is the most abundant structural protein produced throughout the process of the virus infection. The RNP complex of NDV particle contains three inner protein components, the major structural subunit (NP) and two associated proteins (P and L) binding together to RNA genome. Both P and NP protein were successfully obtained due to their comparatively higher expression level and smaller molecular weight; whereas the identification of L protein by MS was a difficult task, possibly due to its low-abundant in the virion and large molecular weight (about 220 kD).
Of the thirty host cellular proteins associated with purified NDV particles we have identified, a significant number of proteins have also been reported to be present in virions of other virus families, such as herpes viruses, poxviruses and retroviruses [14–16]. Considering that these studies were performed independently using different cell types and different mass spectrometry methods, this similarity is probably not an issue of contamination. The most likely explanation is that these viruses all share some fundamental feature and that these host proteins are involved in the processes associated with that common trait. Enveloped viruses enter the cell via a membrane fusion manner and exit by budding. Therefore, one hypothesis would be that these common incorporated host proteins play a role in the entry and release stages of the virus life cycle.
Enveloped viruses acquire their membranes through budding from the host cell, thus cytoskeleton proteins may be integrated inside the virions because of their propinquity to viral assembly and budding sites. Our virion proteomics identified 9 host cytoskeleton system proteins in purified NDV particles, which were the most abundant group of cellular proteins, including Tropomyosin 1 alpha, actin, actin -related protein 3 ARP3, tubulin alpha-1, annexin A2, ezrin, CAP-GLY domain containing linker protein 2, capping protein (Actin filament) muscle Z-line and KIAA0174. Numerous viral proteins interact with cytoskeleton elements. Available evidences indicate that host cytoplasm cytoskeleton components are involved in virus transport in cells, especially in the stages of virus entry and release . Several studies have also indicated that cytoskeleton proteins such as Tubulin and Actin are required for viral gene expression [46, 47] and are involved in several virus budding processes . Interestingly, actin was originally thought as a cellular contaminant, but later demonstrated to be an internal component of the measles virus [49, 50]. In a number of viruses, such as HIV and moloney murine leukemia virus (MMLV), actin is important during their budding [51–53]. For influenza virus, actin plays indispensable roles during the endocytosis of the virus into polarized epithelia . An association of M with cytoskeleton elements has been reported , which indicates an essential function of actin in the replication cycle of coronavirus IBV. As for NDV, early studies have suggested that the cellular cytoskeletal framework actively participated in the structural and functional assembly of NDV transcriptive complex . Therefore, cytoplasm cytoskeleton-associated proteins might take part in the assembly and budding process of newly formed NDV virions, contribute to the transportation of the virus to the correct location of host cell, and also participate in assembling the RNP complex.
Annexins are a well-known multigene family of Ca2+ regulated phospholipid-binding and membrane binding proteins with diverse functions. The presence of annexin A2 is thought to support viral binding, fusion and replication [57–61]. In the present study, cellular annexin A2 was also identified in purified NDV virions, which has been found to be endogenously associated with HCMV, HIV, IV virions, and herpes simplex virus 1 [31, 35, 40, 62]. The exact role of annexin A2 in NDV life cycle needs to be further investigated.
Heat-shock proteins (HSP), known as molecular chaperones, has been identified in a number of envelope viruses. Several viruses require host molecular chaperones for entry, replication, and assembly, as well as other steps in viral production [63, 64]. In this study, we identified two molecular chaperones incorporated into purified NDV particles, HSP70 and HSP90. HSP70 interacts with various viral proteins and may be involved in the assembly of adenovirus , enterovirus , vaccinia virus  and hantaan virus . Virion-associated HSP70 might participate in early events of infection, uncoating the viral capsid in a manner similar to its role in the uncoating of clathrin cages. HSP70 and HSP90 have been shown to interact with hepatitis B virus reverse transcriptase and to facilitate the initiation of viral DNA synthesis from hepatitis B virus pregenomic RNA [66, 67]. HSP90, which can cooperate with other proteins such as p23 and HSP70, has 2–4 copies existing internally in a duck liver virus particle, and might be related to interaction between virus polymerase . Besides, it has been proposed that HSP90 is a major host factor for viral replication of many RNA viruses , implying a important role of HSP90 in NDV replication.
In this study, NDV virion proteomic analysis revealed four chromatin modifying proteins, including chromatin modifying protein 2A, 4B, 4 C and 5. It was reported that they can be expressed in chicken bursal lymphocytes, and may be associated with regulating a variety of gene expression in lymphocytes . Chromatin modifying proteins have also been found in KSHV by virion proteomics , providing a number of clues and potential links to understanding the mechanisms regulating the replication, transcription, and genome maintenance of KSHV. Therefore, NDV virion might modulate the gene expression of host cells through binding with chromatin modifying proteins for better propagation.
According to our investigation, three enzymes-binding proteins were identified, including tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; epsilon polypeptide and theta polypeptide, which have also been found expressed in chicken bursal lymphocytes , and may be related to the metabolic pathways during embryo development . Meanwhile, two calcium-binding proteins, calcium-binding protein A6 and A11 were associated with the purified NDV particles. The calcium-binding proteins play a vital role in the regulation of cellular growth and signal transduction pathways; however, their effect on virus infection remains to be investigated [31, 35, 38].
Among the indentified cellular proteins in our study, three have not yet been reported in other viruses, including two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52), which have not been described to be present in other virions of quite diverse virus families. Previous work has identified syntenin of the shrimp Penaeus monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection through its interaction with alpha-2-macroglobulin (alpha2M), which plays an important role in the immune defense mechanisms of viral infections of shrimps . Ras small GTPase is a very important host signaling mediator, regulating the replication of viruses [73, 74]. The molecular mechanisms of these two signaling mediators are largely unknown.
Our virion proteomic analysis of purified NDV particles revealed the presence of five viral structural proteins and successfully identified thirty incorporated cellular proteins. It is reasonable to speculate that the incorporated cellular proteins in NDV virions may play roles in virus replication and virulence. Future experiments involving RNAi knockdown of these host proteins coding genes will help to address these questions. Indeed, a better understanding of cellular proteins in NDV virions may provide novel targets for the design of antiviral drugs as well as vaccines.
Propagation and purification of NDV
NDV La Sota strain (Beijing Merial Vital Laboratory Animal Technology Co, Ltd, Beijing, China) were propagated in 9-day-old specific pathogen free (SPF) embryonated eggs (Beijing Merial Vital Laboratory Animal Technology Co, Ltd, Beijing, China) at 37°C. The allantoic fluid (AF) with enrichment of NDV virions harvested at 108 h post-infection was clarified by differential centrifugation at 4°C, first centrifugated at 4,000 × g for 15 min and then the supernatant was centrifugated at 12,000 × g for 30 min. The viral supernatant was concentrated and firstly purified at 31,000 rpm through 5.5 ml of 20% (W/V) sucrose in TNE buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4) for 2 h in a 70Ti rotor (Beckman Coulter, Optima™ L-100XP Preparative ultracentrifuge) at 4°C. Condensed and firstly purified virus pellet was then resuspended in TNE buffer and loaded on a preformed sucrose density gradient (20%, 30%, 40%, 50%, and 60% W/V) in TNE buffer for further purification. After centrifugation at 24,100 rpm for 2 h at 4°C in a SW41 rotor (Beckman Coulter, Optima™ L-100XP Preparative ultracentrifuge), the purified virus band between 40%-50% sucrose gradient was collected, diluted in approximately 1 ml of TNE buffer, and finally centrifuged at 24,100 rpm for 2 h at 4°C in a SW41 rotor to exclude the residuary sucrose. In order to get high purified NDV particles, the collected banded viruses were purified for a second time according to the same purification procedure. The purified virus pellet was stored at −80°C for further use.
Validation of purified NDV particles by electron microscope and SDS-PAGE
Highly purified virus (3 μl) was adsorbed to Formavar-supported, carbon-coated nickel grids (230 mesh) for 2 min at room temperature (RT). The grids were negatively stained with 2% phosphotungstic acid and examined under a JEM-1400 electron microscope (JEM-100CX-II, JEOLLTD, Japan) operated at 120 kV.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was also performed to validate the purified NDV particles. Proteins from the purified virus (15 μg) were denatured at 100°C for 10 min in 1× (SDS-PAGE) sample buffer and were then separated by SDS-PAGE. Coomassie Blue R250 was used for protein staining.
Two-dimensional gel electrophoresis (2-DE) separation of proteins of purified NDV particles
The purified NDV particles were dissolved in 500 μl virus lysis buffer (7 M Urea, 2 M thiourea, 2% Triton X-100, 100 mM DTT, 0.2% IPG buffer pH 3–10) and incubated at 4°C for 1 h. After lysing by sonication (pulse durations of 2 s on and 3 s off) in an ice bath for 5 min, the lysates were clarified by centrifugation at 12,000 × g for 30 min at 4°C. The supernatant was collected and the concentration was determined by 2-DE Quant kit (Amersham, USA). The viral protein samples were then aliquoted and stored at −80°C for further analysis.
The first-dimension separation was performed using 18 cm Ready Strip IPG strips (non-linear, pI 3–10, GE Healthcare) for isoelectric focusing (IEF). The IPG strips were rehydrated with 400 μl rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 65 mM DTT, 0.2% IPG buffer pH 3–10) containing 150 μg protein for 12 h at 20°C by a passive rehydration method. IEF was carried out at 20°C on an Ettan IPGphor III electrophoresis unit (GE Healthcare), and performed as follows: 100 V, linear, 100 Volt-Hours (Vhs); 200 V, Gradient, 200 Vhs; 500 V, linear, 500 Vhs; 1,000 V, linear, 1000 Vhs; 4,000 V, Gradient, 4,000 Vhs; 8,000 V, linear, 32,000 Vhs. The IPG strips were incubated for 15 min with gentle shaking in an equilibration buffer (6 M urea, 30% glycerol, 2% SDS and 0.375 M Tris–HCl, pH 8.8) with 1% (w/v) DL-Dithiothreitol (DTT) followed by additional equilibration for 15 min in SDS equilibration buffer containing 2.5% iodoacetamide (IAA).
The second-dimensional separation was carried out by using 5%-15% continuous gradient SDS-PAGE in Tris: glycine buffer (192 mM glycine, 25 mM Tris, 0.1% SDS, pH 8.3) at 140 V for about 10 h. The gels were stained by the modified silver staining method compatible with MS  and scanned at a resolution of 600 dpi using the Image scanner (Amersham Pharmacia Biotech). Spot detection, spot matching, and quantitative intensity analysis were performed using Image Master 2D Platinum 5.0 according to the manufacture’s protocol (GE Healthcare).
In-gel tryptic digestion
The protein spots on the silver-stained gels were excised and transferred into 0.5 ml Eppendorf tubes, washed three times with ddH2O, destained with 15 mM potassium ferricyanide (K3Fe(CN)6, Amresco) and 50 mM sodium thiosulfate (NaS2O3, Amresco) in 50 mM NH4HCO3. After hydrating with 100% acetonitrile (ACN, Wako) and drying in a SpeedVac concentrator (Thermo Savant, USA) for 20 min, the gels were incubated with 12.5 ng/μl trypsin (Sequenceing grade, Promega) at 37°C overnight. The supernatant was collected and transferred into a 200 μl microcentrifuge tube, while the gels were extracted once with extraction buffer (67% ACN containing 5% trifluoroacetic acid (TFA, Wako)) at 37°C for 1 h. The supernatant of the gel spots were combined and then completely dried thoroughly in SpeedVac.
MALDI-TOF/TOF MS, MS/MS analysis and database searching
Protein digestion extracts were resuspended with 5 μl of 0.1% TFA, and then the peptide samples were mixed (1:1) with a matrix consisting of a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid (α-CCA, Sigma) in 50% ACN containing 0.1% TFA. Digested proteins (0.8 μl) of each sample were spotted onto stainless steel target plates and allowed to air-dry at RT. Peptide mass spectra were obtained on an Applied Biosystem Sciex 4800 MALDI-TOF/TOF Plus mass spectrometer (Applied Biosystems, Foster City, CA). Data were acquired in positive MS reflector using a CalMix5 standard to calibrate the instrument (ABI 4800 Calibration Mixture). Mass spectra were obtained from each sample spot by accumulation of 900 laser shots in an 800–3500 mass range. For MS/MS spectra, the 5–10 most abundant precursor ions per sample were selected for subsequent fragmentation and 1200 laser shots were accumulated per precursor ion.
Combined MS and MS/MS spectra were submitted to MASCOT searching engine (V2.1, Matrix Science, London, UK) by GPS Explorer software (V3.6, Applied Biosystems) for proteins identification. Parameters for searches were as follows: trypsin as the digestion enzyme, one missed cleavage site, partial modification of cysteine carboamidomethylated and methionine oxidized, none fixed modifications, MS tolerance of 60 ppm, MS/MS tolerance of 0.25 Da. MASCOT protein score in IPI_CHICKEN (V3.49) database (based on combined MS and MS/MS spectra) of greater than 57 (p ≤ 0.05) or in NCBInr database of greater than 67 (p ≤ 0.05) was accepted.
Validation of cellular proteins by western blot
Mouse monoclonal antibodies against actin (MAB1501), HSP90 (05–594) and NDV (HN14f) were purchased from Millipore. Rabbit polyclonal antibodies against annexin A2 (ab40943) and tubulin alpha-1 (ab4074) were products of Abcam Corparation. The critical challenge of virion proteomics was to prove that the host proteins were really an integral part of the virions and are not just non-specifically attached to the outside of the virions or derived from the contaminants. To address this question, we performed control experiment. Extracts from 13-day-old SPF embryonated eggs were designed as a positive control; AF from 13-day-old SPF embryonated eggs performed with the same protocol as the purification of NDV virions was used as a negative control.
The highly purified NDV particles were suspended in 1 × loading buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 100 mM DTT) and denatured by heating at 100°C for 5 min. The viral protein samples were then separated at 120 V on linear 5%-15% SDS-PAGE with 5% stacking gels in Tris: glycine buffer for about 3 h. After separated by SDS-PAGE, the viral proteins were transferred onto a polyvinylidene fluoride membrane (PVDF, P/N 66485, BioTrace, Pall Corporation). The membrane was blocked in freshly prepared 5% bovine serum albumin (BSA) with 0.05% Tween-20 for 2 h at RT with constant agitation. The PVDF membrane was washed three times with Tris buffered saline buffer (TBS) plus 0.2% Tween-20 and incubated with properly diluted primary antibodies for 2 h at RT. Following three washes with TBS, the secondary antibody conjugated to horseradish peroxidase (HRP) (00001–14, Proteintech Group, Inc) was added for 1 h at RT. The chemiluminescence system (AR1022, Boster Bio-Technology Co. LTD) was used for detection of antibody-antigen complexes.
Protease treatment of NDV virions
Purified virus particles equivalent to 50 μg protein was incubated with bromelain (BB0243, BBI) at 0.2 mg/ml in 50 mM DTT (pH 7.2) in Dulbecco’s phosphate buffered saline (PBS) at 37°C for 15 min. After incubation, the samples were directly centrifuged to equilibrium in 11.5 ml non-linear 20%-60% sucrose-TNE gradients at 24,100 rpm for 2 h at 4°C in a SW41 rotor (Beckman Coulter, Optima™ L-100XP Preparative ultracentrifuge). Condensed virus was diluted with TNE buffer, followed by sedimentation at 24,100 rpm for 2 h at 4°C in a SW41 rotor to remove the sucrose and were then subjected to immunogold labeling and electron microscopy analysis.
Validation of cellular proteins by electron microscopy and immunogold labeling
Rabbit polyclonal antibody against chicken IgG (15 nm Gold) (ab41500), goat polyclonal against rabbit IgG (5 nm Gold) (ab27235) and goat polyclonal against mouse IgG (10 nm Gold) (ab27241) were purchased from Abcam. Protease treated NDV particles were suspended in PBS (pH 7.4) and then were collected onto 230-mesh formwar-coated nickel grids and adsorbed on the grids for 5 min. The virus particles were fixed in 2% paraformaldehyde for 5 min at RT, after treating with Triton X-100 (0.2%) in PBS (pH 7.4) for 5 min; the sample was blocked with 5% BSA in PBS-Tween 20 (pH 7.4) for 30 min at RT. All grids were then blocked with blocking buffer (5% BSA, 5% normal serum, 0.1% cold water skin gelatin, 10 mM phosphate buffer, 150 mM NaCl, pH 7.4) for 30 min. After washing with PBS, immobilized virions were incubated for 1.5 h with 50 μg/ml primary antibody (in 1% BSA), and washed three times for 5 min in PBS/1% BSA. Anti-rabbit or anti-mouse immunoglobulin G coupled to 10 nm colloidal gold particles was used as the secondary antibody and virions were incubated in it for 40 min at RT. The unbound antibodies were removed, the grids were thoroughly washed and negatively stained with 2% sodium phosphotungstate (pH 6.5) for 1 min. Negatively stained virions were examined on a scan and transmission electron microscope.
Newcastle disease virus
Sodium dodecylsulfate polyacrylamide gel electrophoresis
Matrix-assisted laser desorption/ionization time of flight mass spectrometry
Specific pathogen free
Bovine serum albumin
Tris-buffered saline including 50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4
This work was supported by the grants from State Public Industry Scientific Research Programs (nyhyzx07-038, 2007GYJ019) and Science Technology Strategic Plan (2009B020201008, 2009B090600101) of Guangdong, People’s Republic of China. We acknowledge Mr. Shuiming Li for technical assistance in MALDI-TOF/TOF MS/MS at Peking University. We would like to thank Dr George Dacai Liu for critical discussion, comments and revision of the manuscript.
- Alexander DJ: Newcastle disease and other avian paramyxoviruses. Rev Sci Tech 2000, 19: 443–462.Google Scholar
- Bikel I, Duesberg PH: Proteins of Newcastle disease virus and of the viral nucleocapsid. J Virol 1969, 4: 388–393.PubMed CentralGoogle Scholar
- Evans MJ, Kingsbury DW: Separation of Newcastle disease virus proteins by polyacrylamide gel electrophoresis. Virology 1969, 37: 597–604. 10.1016/0042-6822(69)90277-3View ArticleGoogle Scholar
- Scheid A, Choppin PW: Isolation and purification of the envelope proteins of Newcastle disease virus. J Virol 1973, 11: 263–271.PubMed CentralGoogle Scholar
- Chambers P, Samson AC: A new structural protein for Newcastle disease virus. J Gen Virol 1980, 50: 155–166. 10.1099/0022-1317-50-1-155View ArticleGoogle Scholar
- Nagai Y, Klenk HD, Rott R: Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 1976, 72: 494–508. 10.1016/0042-6822(76)90178-1View ArticleGoogle Scholar
- Chen L, Gorman JJ, McKimm-Breschkin J, Lawrence LJ, Tulloch PA, Smith BJ, Colman PM, Lawrence MC: The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion. Structure 2001, 9: 255–266. 10.1016/S0969-2126(01)00581-0View ArticleGoogle Scholar
- Peeters BP, de Leeuw OS, Koch G, Gielkens AL: Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol 1999, 73: 5001–5009.PubMed CentralGoogle Scholar
- Crennell S, Takimoto T, Portner A, Taylor G: Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat Struct Biol 2000, 7: 1068–1074. 10.1038/81002View ArticleGoogle Scholar
- Takimoto T, Taylor GL, Connaris HC, Crennell SJ, Portner A: Role of the hemagglutinin-neuraminidase protein in the mechanism of paramyxovirus-cell membrane fusion. J Virol 2002, 76: 13028–13033. 10.1128/JVI.76.24.13028-13033.2002PubMed CentralView ArticleGoogle Scholar
- Huang Z, Panda A, Elankumaran S, Govindarajan D, Rockemann DD, Samal SK: The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence. J Virol 2004, 78: 4176–4184. 10.1128/JVI.78.8.4176-4184.2004PubMed CentralView ArticleGoogle Scholar
- Coleman NA, Peeples ME: The matrix protein of Newcastle disease virus localizes to the nucleus via a bipartite nuclear localization signal. Virology 1993, 195: 596–607. 10.1006/viro.1993.1411View ArticleGoogle Scholar
- Hamaguchi M, Yoshida T, Nishikawa K, Naruse H, Nagai Y: Transcriptive complex of Newcastle disease virus I. Both L and P proteins are required to constitute an active complex. Virology 1983, 128: 105–117. 10.1016/0042-6822(83)90322-7View ArticleGoogle Scholar
- Maxwell KL, Frappier L: Viral proteomics. Microbiol Mol Biol Rev 2007, 71: 398–411. 10.1128/MMBR.00042-06PubMed CentralView ArticleGoogle Scholar
- Cantin R, Methot S, Tremblay MJ: Plunder and stowaways: incorporation of cellular proteins by enveloped viruses. J Virol 2005, 79: 6577–6587. 10.1128/JVI.79.11.6577-6587.2005PubMed CentralView ArticleGoogle Scholar
- Viswanathan K, Fruh K: Viral proteomics: global evaluation of viruses and their interaction with the host. Expert Rev Proteomics 2007, 4: 815–829. 10.1586/147894126.96.36.1995View ArticleGoogle Scholar
- Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, Pasa-Tolic L, Wang D, Camp DG, Rodland K, Wiley S, et al.: Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J Virol 2004, 78: 10960–10966. 10.1128/JVI.78.20.10960-10966.2004PubMed CentralView ArticleGoogle Scholar
- Kattenhorn LM, Mills R, Wagner M, Lomsadze A, Makeev V, Borodovsky M, Ploegh HL, Kessler BM: Identification of proteins associated with murine cytomegalovirus virions. J Virol 2004, 78: 11187–11197. 10.1128/JVI.78.20.11187-11197.2004PubMed CentralView ArticleGoogle Scholar
- Johannsen E, Luftig M, Chase MR, Weicksel S, Cahir-McFarland E, Illanes D, Sarracino D, Kieff E: Proteins of purified Epstein-Barr virus. Proc Natl Acad Sci USA 2004, 101: 16286–16291. 10.1073/pnas.0407320101PubMed CentralView ArticleGoogle Scholar
- Bechtel JT, Winant RC, Ganem D: Host and viral proteins in the virion of Kaposi’s sarcoma-associated herpesvirus. J Virol 2005, 79: 4952–4964. 10.1128/JVI.79.8.4952-4964.2005PubMed CentralView ArticleGoogle Scholar
- Zhu FX, Chong JM, Wu L, Yuan Y: Virion proteins of Kaposi’s sarcoma-associated herpesvirus. J Virol 2005, 79: 800–811. 10.1128/JVI.79.2.800-811.2005PubMed CentralView ArticleGoogle Scholar
- O’Connor CM, Kedes DH: Mass spectrometric analyses of purified rhesus monkey rhadinovirus reveal 33 virion-associated proteins. J Virol 2006, 80: 1574–1583. 10.1128/JVI.80.3.1574-1583.2006PubMed CentralView ArticleGoogle Scholar
- Liu HC, Soderblom EJ, Goshe MB: A mass spectrometry-based proteomic approach to study Marek’s Disease Virus gene expression. J Virol Methods 2006, 135: 66–75. 10.1016/j.jviromet.2006.02.001View ArticleGoogle Scholar
- Bortz E, Whitelegge JP, Jia Q, Zhou ZH, Stewart JP, Wu TT, Sun R: Identification of proteins associated with murine gammaherpesvirus 68 virions. J Virol 2003, 77: 13425–13432. 10.1128/JVI.77.24.13425-13432.2003PubMed CentralView ArticleGoogle Scholar
- Chung CS, Chen CH, Ho MY, Huang CY, Liao CL, Chang W: Vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles. J Virol 2006, 80: 2127–2140. 10.1128/JVI.80.5.2127-2140.2006PubMed CentralView ArticleGoogle Scholar
- Resch W, Hixson KK, Moore RJ, Lipton MS, Moss B: Protein composition of the vaccinia virus mature virion. Virology 2007, 358: 233–247. 10.1016/j.virol.2006.08.025View ArticleGoogle Scholar
- Renesto P, Abergel C, Decloquement P, Moinier D, Azza S, Ogata H, Fourquet P, Gorvel JP, Claverie JM: Mimivirus giant particles incorporate a large fraction of anonymous and unique gene products. J Virol 2006, 80: 11678–11685. 10.1128/JVI.00940-06PubMed CentralView ArticleGoogle Scholar
- Li Z, Lin Q, Chen J, Wu JL, Lim TK, Loh SS, Tang X, Hew CL: Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes. Mol Cell Proteomics 2007, 6: 1609–1620. 10.1074/mcp.M600327-MCP200View ArticleGoogle Scholar
- Huang C, Zhang X, Lin Q, Xu X, Hu Z, Hew CL: Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466. Mol Cell Proteomics 2002, 1: 223–231. 10.1074/mcp.M100035-MCP200View ArticleGoogle Scholar
- Song W, Lin Q, Joshi SB, Lim TK, Hew CL: Proteomic studies of the Singapore grouper iridovirus. Mol Cell Proteomics 2006, 5: 256–264.View ArticleGoogle Scholar
- Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, Bess JW, Sowder RC, Barsov E, Hood BL, Fisher RJ, et al.: Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol 2006, 80: 9039–9052. 10.1128/JVI.01013-06PubMed CentralView ArticleGoogle Scholar
- Saphire AC, Gallay PA, Bark SJ: Proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins. J Proteome Res 2006, 5: 530–538. 10.1021/pr050276bView ArticleGoogle Scholar
- Ying W, Hao Y, Zhang Y, Peng W, Qin E, Cai Y, Wei K, Wang J, Chang G, Sun W, et al.: Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus. Proteomics 2004, 4: 492–504. 10.1002/pmic.200300676View ArticleGoogle Scholar
- Zeng R, Ruan HQ, Jiang XS, Zhou H, Shi L, Zhang L, Sheng QH, Tu Q, Xia QC, Wu JR: Proteomic analysis of SARS associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectroemtric analysis. J Proteome Res 2004, 3: 549–555. 10.1021/pr034111jView ArticleGoogle Scholar
- Shaw ML, Stone KL, Colangelo CM, Gulcicek EE, Palese P: Cellular proteins in influenza virus particles. PLoS Pathog 2008, 4: e1000085. 10.1371/journal.ppat.1000085PubMed CentralView ArticleGoogle Scholar
- Moerdyk-Schauwecker M, Hwang SI, Grdzelishvili VZ: Analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach. Virol J 2009, 6: 166. 10.1186/1743-422X-6-166PubMed CentralView ArticleGoogle Scholar
- Kong Q, Xue C, Ren X, Zhang C, Li L, Shu D, Bi Y, Cao Y: Proteomic analysis of purified coronavirus infectious bronchitis virus particles. Proteome Sci 2010, 8: 29. 10.1186/1477-5956-8-29PubMed CentralView ArticleGoogle Scholar
- Zhang C, Xue C, Li Y, Kong Q, Ren X, Li X, Shu D, Bi Y, Cao Y: Profiling of cellular proteins in porcine reproductive and respiratory syndrome virus virions by proteomics analysis. Virol J 2010, 7: 242. 10.1186/1743-422X-7-242PubMed CentralView ArticleGoogle Scholar
- Si H, Verma SC, Robertson ES: Proteomic analysis of the Kaposi’s sarcoma-associated herpesvirus terminal repeat element binding proteins. J Virol 2006, 80: 9017–9030. 10.1128/JVI.00297-06PubMed CentralView ArticleGoogle Scholar
- Padula ME, Sydnor ML, Wilson DW: Isolation and preliminary characterization of herpes simplex virus 1 primary enveloped virions from the perinuclear space. J Virol 2009, 83: 4757–4765. 10.1128/JVI.01927-08PubMed CentralView ArticleGoogle Scholar
- Macejak DG, Luftig RB: Association of HSP70 with the adenovirus type 5 fiber protein in infected HEp-2 cells. Virology 1991, 180: 120–125. 10.1016/0042-6822(91)90015-4View ArticleGoogle Scholar
- Macejak DG, Sarnow P: Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells. J Virol 1992, 66: 1520–1527.PubMed CentralGoogle Scholar
- Jindal S, Young RA: Vaccinia virus infection induces a stress response that leads to association of Hsp70 with viral proteins. J Virol 1992, 66: 5357–5362.PubMed CentralGoogle Scholar
- Ye L, Liu Y, Yang S, Liao W, Wang C: Increased expression of Hsp70 and co-localization with nuclear protein in cells infected with the Hantaan virus. Chin Med J (Engl) 2001, 114: 535–539.Google Scholar
- Radtke K, Dohner K, Sodeik B: Viral interactions with the cytoskeleton: a hitchhiker’s guide to the cell. Cell Microbiol 2006, 8: 387–400. 10.1111/j.1462-5822.2005.00679.xView ArticleGoogle Scholar
- Burke E, Dupuy L, Wall C, Barik S: Role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus. Virology 1998, 252: 137–148. 10.1006/viro.1998.9471View ArticleGoogle Scholar
- Bukrinskaya A, Brichacek B, Mann A, Stevenson M: Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton. J Exp Med 1998, 188: 2113–2125. 10.1084/jem.188.11.2113PubMed CentralView ArticleGoogle Scholar
- Arthur LO, Bess JW, Sowder RC, Benveniste RE, Mann DL, Chermann JC, Henderson LE: Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science 1992, 258: 1935–1938. 10.1126/science.1470916View ArticleGoogle Scholar
- Tyrrell DL, Norrby E: Structural polypeptides of measles virus. J Gen Virol 1978, 39: 219–229. 10.1099/0022-1317-39-2-219View ArticleGoogle Scholar
- Lamb RA, Mahy BW, Choppin PW: The synthesis of sendai virus polypeptides in infected cells. Virology 1976, 69: 116–131. 10.1016/0042-6822(76)90199-9View ArticleGoogle Scholar
- Ott DE, Coren LV, Kane BP, Busch LK, Johnson DG, Sowder RC, Chertova EN, Arthur LO, Henderson LE: Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 1996, 70: 7734–7743.PubMed CentralGoogle Scholar
- Ott DE, Coren LV, Johnson DG, Kane BP, Sowder RC, Kim YD, Fisher RJ, Zhou XZ, Lu KP, Henderson LE: Actin-binding cellular proteins inside human immunodeficiency virus type 1. Virology 2000, 266: 42–51. 10.1006/viro.1999.0075View ArticleGoogle Scholar
- Nermut MV, Wallengren K, Pager J: Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy. Virology 1999, 260: 23–34. 10.1006/viro.1999.9803View ArticleGoogle Scholar
- Sun X, Whittaker GR: Role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells. Cell Microbiol 2007, 9: 1672–1682. 10.1111/j.1462-5822.2007.00900.xView ArticleGoogle Scholar
- Wang J, Fang S, Xiao H, Chen B, Tam JP, Liu DX: Interaction of the coronavirus infectious bronchitis virus membrane protein with beta-actin and its implication in virion assembly and budding. PLoS One 2009, 4: e4908. 10.1371/journal.pone.0004908PubMed CentralView ArticleGoogle Scholar
- Hamaguchi M, Nishikawa K, Toyoda T, Yoshida T, Hanaichi T, Nagai Y: Transcriptive complex of Newcastle disease virus II. Structural and functional assembly associated with the cytoskeletal framework. Virology 1985, 147: 295–308. 10.1016/0042-6822(85)90132-1View ArticleGoogle Scholar
- Raynor CM, Wright JF, Waisman DM, Pryzdial EL: Annexin II enhances cytomegalovirus binding and fusion to phospholipid membranes. Biochemistry 1999, 38: 5089–5095. 10.1021/bi982095bView ArticleGoogle Scholar
- Ma G, Greenwell-Wild T, Lei K, Jin W, Swisher J, Hardegen N, Wild CT, Wahl SM: Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection. J Exp Med 2004, 200: 1337–1346. 10.1084/jem.20041115PubMed CentralView ArticleGoogle Scholar
- LeBouder F, Morello E, Rimmelzwaan GF, Bosse F, Pechoux C, Delmas B, Riteau B: Annexin II incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin. J Virol 2008, 82: 6820–6828. 10.1128/JVI.00246-08PubMed CentralView ArticleGoogle Scholar
- Derry MC, Sutherland MR, Restall CM, Waisman DM, Pryzdial EL: Annexin 2-mediated enhancement of cytomegalovirus infection opposes inhibition by annexin 1 or annexin 5. J Gen Virol 2007, 88: 19–27. 10.1099/vir.0.82294-0View ArticleGoogle Scholar
- Ryzhova EV, Vos RM, Albright AV, Harrist AV, Harvey T, Gonzalez-Scarano F: Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages. J Virol 2006, 80: 2694–2704. 10.1128/JVI.80.6.2694-2704.2006PubMed CentralView ArticleGoogle Scholar
- Wright JF, Kurosky A, Pryzdial EL, Wasi S: Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts. J Virol 1995, 69: 4784–4791.PubMed CentralGoogle Scholar
- Mayer MP: Recruitment of Hsp70 chaperones: a crucial part of viral survival strategies. Rev Physiol Biochem Pharmacol 2005, 153: 1–46. 10.1007/s10254-004-0025-5View ArticleGoogle Scholar
- Maggioni C, Braakman I: Synthesis and quality control of viral membrane proteins. Curr Top Microbiol Immunol 2005, 285: 175–198. 10.1007/3-540-26764-6_6Google Scholar
- Chappell TG, Welch WJ, Schlossman DM, Palter KB, Schlesinger MJ, Rothman JE: Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell 1986, 45: 3–13. 10.1016/0092-8674(86)90532-5View ArticleGoogle Scholar
- Hu J, Seeger C: Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase. Proc Natl Acad Sci USA 1996, 93: 1060–1064. 10.1073/pnas.93.3.1060PubMed CentralView ArticleGoogle Scholar
- Liu K, Qian L, Wang J, Li W, Deng X, Chen X, Sun W, Wei H, Qian X, Jiang Y, He F: Two-dimensional blue native/SDS-PAGE analysis reveals heat shock protein chaperone machinery involved in hepatitis B virus production in HepG2.2.15 cells. Mol Cell Proteomics 2009, 8: 495–505. 10.1074/mcp.M800250-MCP200PubMed CentralView ArticleGoogle Scholar
- Hu J, Toft DO, Seeger C: Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. EMBO J 1997, 16: 59–68. 10.1093/emboj/16.1.59PubMed CentralView ArticleGoogle Scholar
- Okamoto T, Nishimura Y, Ichimura T, Suzuki K, Miyamura T, Suzuki T, Moriishi K, Matsuura Y: Hepatitis C virus RNA replication is regulated by FKBP8 and Hsp90. EMBO J 2006, 25: 5015–5025. 10.1038/sj.emboj.7601367PubMed CentralView ArticleGoogle Scholar
- Caldwell RB, Kierzek AM, Arakawa H, Bezzubov Y, Zaim J, Fiedler P, Kutter S, Blagodatski A, Kostovska D, Koter M, et al.: Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis. Genome Biol 2005, 6: R6. 10.1186/gb-2005-6-4-p6PubMed CentralView ArticleGoogle Scholar
- Agudo D, Gomez-Esquer F, Diaz-Gil G, Martinez-Arribas F, Delcan J, Schneider J, Palomar MA, Linares R: Proteomic analysis of the Gallus gallus embryo at stage-29 of development. Proteomics 2005, 5: 4946–4957. 10.1002/pmic.200402056View ArticleGoogle Scholar
- Tonganunt M, Phongdara A, Chotigeat W, Fujise K: Identification and characterization of syntenin binding protein in the black tiger shrimp Penaeus monodon. J Biotechnol 2005, 120: 135–145. 10.1016/j.jbiotec.2005.06.006View ArticleGoogle Scholar
- Harmon B, Ratner L: Induction of the Galpha(q) signaling cascade by the human immunodeficiency virus envelope is required for virus entry. J Virol 2008, 82: 9191–9205. 10.1128/JVI.00424-08PubMed CentralView ArticleGoogle Scholar
- Zheng Y, Li J, Johnson DL, Ou JH: Regulation of hepatitis B virus replication by the ras-mitogen-activated protein kinase signaling pathway. J Virol 2003, 77: 7707–7712. 10.1128/JVI.77.14.7707-7712.2003PubMed CentralView ArticleGoogle Scholar
- Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ: A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 2000, 21: 3666–3672. 10.1002/1522-2683(200011)21:17<3666::AID-ELPS3666>3.0.CO;2-6View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.