Proteomics Analysis of Differential Expression of Chicken Brain Tissue

May 3, 2010 - of proteins to H5N1 avian influenza virus with neurovirulence infection ... when the host was infected by the neurovirulent avian influe...
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Proteomics Analysis of Differential Expression of Chicken Brain Tissue Proteins in Response to the Neurovirulent H5N1 Avian Influenza Virus Infection Wei Zou,† Jianjiang Ke,† Anding Zhang,†,‡ Mingguang Zhou,† Yonghong Liao,† Jiping Zhu,† Hongbo Zhou,†,‡ Jiagang Tu,† Huanchun Chen,†,‡ and Meilin Jin*,†,‡ State Key Laboratory of Agriculture Microbiology, Huazhong Agriculture University, Wuhan 430070, P. R. China, and Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agriculture University, Wuhan 430070, P. R. China Received November 23, 2009

A certain H5N1 avian influenza virus has gained the ability to cause the classic central nervous system dysfunction in poultry and migratory birds. This study presents the proteomics analysis on the change of proteins to H5N1 avian influenza virus with neurovirulence infection in chicken brain tissue. By using 2-DE, coupled with MALDI-TOF MS/MS, we identified a set of differentially expressed cellular proteins, including 18 up-regulated proteins and 13 down-regulated proteins. The most significant changes were found in cytoskeleton proteins, proteins associated with the ubiquitin-proteasome pathway, and neural signal transduction proteins. Some identified proteins such as CRMP and SEP5 were found to participate in the pathogenesis progress of Parkinson’s and Huntington’s diseases, which also developed encephalitis accompanied with CNS dysfunction. The obtained data can provide insight into the virus-chicken brain tissue interaction and reveal the potential mechanism of the neuropathogenesis when the host was infected by the neurovirulent avian influenza virus. Keywords: avian influenza virus • proteomics • neurovirulence • neuropathogenesis

1. Introduction The highly pathogenic avian influenza (HPAI) H5N1 virus has spread across five continents posing significant threat to poultry breeding, causing human death and enormous economic loss. As of December 30, 2009, the HPAI H5N1 avian influenza virus has caused 467 human-infected cases, and among them, 282 died (http://www.who.int/csr/disease/ avian_influenza/country/en/). The neurovirulence of HPAI in waterfowl was first reported in 2004 by Webster. Migratory birds were killed in the outbreak of HPAI in the parks of Hong Kong in 2002, and the birds displayed the dysfunction in their central neural system (CNS).1 The viruses with neurovirulence were then isolated successively in Japan, Thailand, and China. Some viruses isolated in the outbreak of avian influenza in west China also caused disorder of CNS in the migratory waterfowl.2 However, the investigation about these viruses was restricted to the virulence and the genome sequences. The reported data in vitro have demonstrated that avian influenza virus could transmit through neural axons, and the apoptosis and proinflammatory reaction could be triggered when primary mouse microglia and astrocyte were infected by human H1N1 and avian H5N1 influenza viruses.3,4 However, the mechanism of how the virus caused the neuro* Corresponding author. Phone: +86-27-87286905. Fax: +86-27-87282608. E-mail: [email protected]. † State Key Laboratory of Agriculture Microbiology. ‡ College of Veterinary Medicine. 10.1021/pr100080x

 2010 American Chemical Society

pathogenesis, the interaction of the host with the virus, and how the protein changed when the host was infected were still unknown. Proteomics has the potential to reveal differences between pathologic and healthy tissues or cells in terms of a single protein, the protein post-translational modification, and protein-protein interaction. It could be also beneficial to the comprehensive investigation, such as to the analysis on signal pathways and biological progress. Proteomics methods have frequently been applied to studying the brain, brain development, and other nerve tissues in respect to neural diseases.5,6 Meanwhile, the alterations of cellular proteins in human cell lines infected by H9N2 have been studied by Ning Liu.7 Genomics and proteomics were also carried out to reveal the change of molecular signature in influenza virus-inflected macaques,8 and a proteomics approach has been adopted to study cellular proteins involved in the process of H1N1 virus infection in 293T and MDCK cells.9 However, few studies reported the change of tissue proteins in vivo and the neuropathogenesis when the host was infected by avian influenza virus. To better understand the molecular and cellular basis of H5N1 avian influenza virus infection and the neuropathology caused by the virus in poultry, chicken brain tissue was chosen to be investigated in the present study. First, the model of encephalitis of chickens infected by HPAI was constructed, and then a proteomics approach was applied to study the changed patterns of the chicken brain tissue proteins upon the infection Journal of Proteome Research 2010, 9, 3789–3798 3789 Published on Web 05/03/2010

research articles of the H5N1 virus, A/duck/Hubei/hangmei01/2006, which exhibited the neurovirulence in both chickens and ducks.10 The findings are believed to contribute to the comprehension of the interaction of the H5N1 virus and chickens and the progress of the neuropathogenesis. Besides, they can also help to search the potential protein targets for further studies.

2. Materials and Methods 2.1. Animal, Virus Infection, and Sample Preparation. Eighteen 4-week old SPF chickens were inoculated intranasally with 100 µL of PBS containing 106 EID50 virus of the HPAI virus A/duck/Hubei/hangmei01/2006. Six chickens were raised separately as a control group and only inoculated intranasally with 100 µL of PBS. The chickens were euthanized when they displayed apparent central nerve system dysfunction symptoms or were dying. Half brain tissues of all the 24 chickens including the cerebrum, cerebellum, and brainstem were harvested immediately after slaughter, chopped into pieces, separated to several parts with aluminum foil, and frozen in liquid nitrogen, and then kept at -80 °C for subsequent proteome and viral titers analysis. Another half brain tissue was fixed in 10% neutral buffered formalin for pathology, immunohistochemistry, and apoptosis analysis. The frozen brains were separately placed in liquid nitrogen and grounded thoroughly to a very fine powder. The brain tissue homogenate and 10-fold serial diluting solution were inoculated in a 9-day-old embryonated SPF egg for 72 h at 37 °C. The allantoic fluid was then harvested and tested for hemagglutination activity. The virus titer of tissue homogenate was determined by calculating the 50% egg infectious dose (EID50) per milliliter of tissue homogenates, using the method of Reed and Muench.1 The lower limit of virus detection was 10 EID50 per mL. Simultaneously, about 100 mg of tissue powder of the brain tissue samples collected on the fifth day post inoculation (dpi) was transferred to sterile tubes containing 1 mL of sample lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 2% IPG Buffer, pH 4-7, a piece of proteinase inhibitor cocktail (BBI, Canada)), and the mixture was ultrasonicated 9 times (10 s per time), then incubated for 60 min on ice with occasional vortexing and centrifuged at 20 000g for 45 min at 4 °C. The supernatant was collected, and the protein concentration was determined by the PlusOne 2-D Quant Kit (GE Healthcare Bio-Sciences, Uppsala, Sweden). All experiments with the H5N1 viruses, including animal studies, were performed in a biosafety level-3 laboratory. 2.2. Pathology, Immunohistochemistry, and Apoptosis Analysis of the Chicken Brain Tissues. Samples of cerebrum, cerebellum, and brain stem were fixed in 10% neutral buffered formalin and routinely processed into paraffin. Sections of 2-3 mm thickness were cut for hematoxylin and eosin staining for histopathologic evaluation. Duplicate 4 mm sections of the fixed tissues were prepared, and one was immunohistochemically stained by first microwaving the sections in 1 mM ethylenediaminetetra-acetic acid solution (pH 8.0) for antigen exposure. Then, a 1:1000 dilution of a mouse-derived monoclonal antibody (1G4) specific for a type A influenza virus nucleoprotein was applied and allowed to incubate for 2 h at 37 °C. The primary antibody was then detected by the application of biotinylated goat antimouse IgG secondary antibody using a biotin-streptavidin detection system (BOSTER, China). Diaminobenzidine (DAB) and aminoethylcarbazole served as the substrate chromagen, and hematoxylin was used as a counterstain. The other section was detected with an indirect TUNEL labeling assay for cell apoptosis analysis of the brain 3790

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Zou et al. tissues with the in situ cell death detection kit (Roche, German) according to the manufacturer’s protocol of the kit. 2.3. 2-DE and Image Analysis. IEF was performed with the IPGphor II system (GE Healthcare, USA) and the Immobiline DryStrip IPG strips of 18 cm (pH 4-7). The prepared chicken brain tissue protein samples (150 mg/strip) were mixed with rehydration buffer (7 M urea, 2 M thiourea, 2% w/v CHAPS, 1% w/v DTT, 0.5% v/v IPG buffer pH 4-7, 0.002% w/v bromophenol blue). The protein samples were focused for a total of 70 kV · h. After IEF, six gels were run as follows: the IPG strips were equilibrated with 10 mg/mL of DTT and 40 mg/ mL of iodoacetamide, respectively, for 15 min in equilibration buffer (6 M urea, 2% w/v SDS, 30% v/v glycerol, 0.002% w/v bromophenol blue, 50 mM Tris-HCl, and pH 8.8). After equilibration, the second dimension electrophoresis was performed on a 10% SDS polyacrylamide gel using a EttanTM DALTSix electrophoresis unit (GE Healthcare). Proteins of one gel were visualized by staining with silver, and gel evaluation and data analysis were carried out using the ImageMaster v 6.01 program (GE Healthcare). 2.4. In-Gel Digestion of Proteins. The protein spots were manually excised from the silver-stained gels and then transferred to V-bottom 96-well microplates loaded with 100 µL of 50% ACN/25 mM ammonium bicarbonate solution per well. After being destained for 1 h, gel plugs were dehydrated with 100 µL of 100% ACN for 20 min and then thoroughly dried in a SpeedVac concentrator (Thermo Savant, USA) for 30 min. The dried gel particles were rehydrated at 4 °C for 45 min with 2 µL/well of trypsin (Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate and then incubated at 37 °C for 12 h. After trypsin digestion, the peptide mixtures were extracted with 8 µL of extraction solution (50% ACN/0.5% TFA) per well at 37 °C for 1 h prior to MALDI-TOF MS analysis. 2.5. MALDI-TOF MS/MS Analysis and Database Search. The protein sample with an equivalent matrix solution was applied to the MALDI-TOF target and prepared for MALDITOF MS/MS analysis according to the procedure.11 HCCA was used as the matrix. MALDI-TOF spectra were calibrated using trypsin autodigestive peptide signals and matrix ion signals. MALDI analysis was performed by a fuzzy logic feedback control system (Ultraflex aMALDI TOF/TOF system Bruker, Karlsruhe, Germany) equipped with delayed ion extraction. Peptide masses were searched against the NCBI database using the MASCOT program (http://www.matrixscience.com). 2.6. Real-Time RT-PCR. Specific primers suited to target genes were designed according to the corresponding gene sequences of tandem MS (MS/MS)-identified proteins and the available gene information in the GenBank library by using the Lasergene sequence analysis software (DNAStar, Madison). The infected and control chicken brain tissues were homogenated with ice-cold PBS and then lysed with Trizol reagent (Invitrogen, USA). Total tissue RNA was extracted using the RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol. RNA concentrations were measured by a spectrophotometer (260 nm/280 nm). After reversing the RNA to cDNA, the real-time RT-PCR was performed in the 7500 RealTime PCR System (Applied Biosystems, CA, USA) in a total volume of 20 µL containing 100 ng of cDNA template, 1 × SYBR Premix Ex Taq (Perfect Real Time; TaKaRa), and 200 nM of each primer. After initial denaturation at 95 °C for 30 s, the amplification was carried out through 40 cycles, each consisting of denaturation at 95 °C for 15 s, primer annealing at 58 °C for 30 s, and DNA extension at 72 °C for 30 s. Melting curves were

Proteomics Analysis of Chicken Brain Tissue Proteins

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Figure 1. Pathology staining of the chicken brain tissues infected by AIV. (a) The edema of the cerebral pia mater and the turgescence of the meningeal vessels, HE, bar ) 50 µm. (b) Perivascular cuffing, considerable satellitosis, and neuronophagia, HE, bar ) 25 µm. (c) Neutrophil infiltration, HE, bar ) 25 µm. (d) Myelin degeneration, HE, bar ) 25 µm.

obtained, and quantitative analysis of the data was performed using the 7500 System SDS software v1.3.1 in a relative quantification (ddCt) study model (Applied Biosystems). 2.7. Western Blot Analysis. Samples of infected and control chicken brain tissues were lysed for total protein extraction, and the protein concentration was determined as described above. Equivalent amounts of cell lysates (80 µg) were subjected to 12% SDS-PAGE and then transferred to nitrocellulose membranes (Amersham Biosciences). After blocking with 5% nonfat dried milk, the membranes were incubated, respectively, with mouse monoclonal antibodies (mAbs) to β-tubulin (clone TBU2.1, BOSTER, China), swine vimentin (clone V9, BOSTER, China), actin (clone AC-15, BOSTER, China), goat antibody to CRMP-2 (AB9892, Chemicon, USA), and rabbit antibody to SNAP25 (BA2175, BOSTER, China). The membranes were then separately incubated with horseradish peroxidase (HRP)conjugated antimouse IgG, HRP-conjugated antigoat IgG, and HRP-conjugated antirabbit IgG (BOSTER, China), then visualized with the ECL western-blotting detection system (Amersham). Each reaction was performed in triplicate.

3. Results 3.1. Classic Nonsuppurative Encephalitis of HPAI-Infected Chicken Brain Tissues, the Immunohistochemical Staining and Apoptosis Analysis of the Brain Tissues. Having been euthanized, the brain tissue samples were collected from the chickens displaying classic CNS dysfunction at three phases: (1) from the 3 infected chickens on the fourth day post inoculation (dpi); (2) from 12 chickens on the fifth dpi; (3) from 2 chickens on the sixth dpi and the last chicken with the clinical symptom of emotional suppression though it did not display classic CNS dysfunction. The control group chickens were euthanized on the fifth day. The virus titers of infected chicken brain tissues collected in three phases are displayed in the Supporting Information (Table s1), and the control chicken brain tissue homogenates did not cause the death of the embryonated SPF eggs. The

chickens infected by the HPAI displayed the same CNS dysfunction symptoms as those shown by the field ducks. The HPAI-infected chicken brain tissues developed classic nonsuppurative encephalitis. The cerebral pia mater was severely edematose with the turgescence of the meningeal vessels (Figure 1a). Different degrees of neuronal degeneration, inflammatory reactions such as neutrophil infiltrates, considerable satellitosis, and neuronophagia could be observed (Figure 1b, 1c). Severely perivascular cuffing and degeneration of myelin was accompanied by HPAI infection (Figure 1b, 1d). Immunohistochemical staining displayed that viral antigens were detected mainly in the glial cells in the cerebrum and cerebellum (Figure 2a, 2b). The antigen location could hardly be detected in neurons in the cerebrum. Meanwhile, the glial cells of the cerebrum and cerebellum also displayed apoptosis when the brain was infected by the avian influenza virus (Figure 2c, 2d). The apoptosis was more abundant in the cerebellum than the cerebrum. Both of the molecular layers and stratum granulosum of the cerebellum were found to have stigmatic apoptosis. 3.2. Two-Dimensional Gel Electrophoresis Profiles of HPAIInfected Chicken Brain Tissues. To obtain the profile of the changed proteins, the proteins of HPAI-infected chicken brain tissues collected on the fifth dpi and the control chicken brain tissues were extracted for 2-DE analysis. The spots of detectable protein ranged from 1400 to 1650 on 18 cm 2-D gels (pI 4-7) loaded with 150 µg of total tissue proteins per gel. On the basis of the average intensity ratios of protein spots, a total of 40 protein spots were found to be changed in HPAI-infected brains, including 23 significantly up-regulated protein spots (ratio infection/control g2, p e 0.05) and 17 significantly downregulated protein spots (ratio infection/control e0.5, p e 0.05) (Figure 3). 3.3. Mass Spectrum Identification of Differentially Expressed Proteins. To identify the differentially expressed proteins in HPAI-infected chicken brain tissues in 2-DE gels, a total of 40 differential spots with a threshold greater than 2-fold Journal of Proteome Research • Vol. 9, No. 8, 2010 3791

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Figure 2. Immunohistochemical staining of the chicken brain tissues and in situ detection of apoptosis in the cerebrum and cerebellum. The brain sections of the infected chicken brain tissue show a positive signal (brown stain) in cell nuclei. (a) Viral antigen is demonstrated in glial cells of the cerebrum. Immunohistochemistry for influenza A nucleoprotein. DAB chromogen, hematoxylin counterstain, bar ) 50 µm. (b) Viral antigen is demonstrated in glial cells of the cerebellum. Immunohistochemistry for influenza A nucleoprotein. DAB chromogen, hematoxylin counterstain, bar ) 50 µm. (c) Apoptosis of glial cells in the cerebrum, bar ) 50 µm. (d) Apoptosis of glial cells in the cerebellum, bar ) 25 µm.

Figure 3. Representative 2-D gel obtained from chicken brain tissues infected/uninfected by AIV. The proteins (150 µg) were first separated in a linear gradient of pH 4-7, followed by separation in an SDS-PAGE (12%) and silver staining. (a) Protein gel of the chicken brain tissues infected by the virus. (b) Protein gel of the chicken brain tissues uninfected by the virus. Numbers 1 to 31 mean the protein spots number.

were excised and subjected to in-gel trypsin digestion and the subsequent MALDI-TOF/TOF identification. Thirty one differentially expressed spots were successfully identified (Table 1). According to annotations from UniProt knowledgebase (SwissProt/TrEMBL) and Gene Ontology Database, the identified cellular proteins were involved in the cytoskeleton, the neural signal transduction, the ubiquitin-proteasome pathway (UPP), and the macromolecular biosynthesis metabolism. Eighteen up-regulated spots corresponded to the following 18 proteins: 1 viral protein (HPAI hemagglutinin protein (HA)), 2 cytoskeleton proteins, 5 cellular signal transduction proteins, 6 molecular biosynthesis proteins, and 4 other proteins (Figures 3 3792

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and 4). Thirteen down-regulated spots were mainly involved in UPP, signal transduction, intermediate filament (IF) proteins, and other functions as well (Figures 3 and 4). Notably, most of the proteins involved in UPP, metabolism, and biosynthesis were down-regulated during HPAI infection (Figure 4 and Table 1). 3.4. Transcriptional Profiles of Differentially Expressed Proteins during HPAI Infection. The transcriptional alterations of nine selected genes of the HPAI-infected chicken brain tissues were analyzed by the mRNA transcript of the histone H5 gene as a control housekeeping gene. In general, the trends of the change of the nine genes’ mRNA abundance in the

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Proteomics Analysis of Chicken Brain Tissue Proteins

Table 1. Summary of Proteins Differentially Expressed in Chicken Brain Tissues Infected by the Avian Influenza Virus spot IDa

NCBI no.b

1 2 3 4 5

71897287 6572956 5074843 46397724 82194891

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

1710781 211235 73852955 71897193 82197919 118114540 135474 135464 71895411 45384370 45383177 3122036 45383179 822227598 45382251 50754375 118102801 71896779 118082941 50754481 57525333 82082513 57530041 25091747

30 31

82081029 114326309

matched peptidesc

coverage (%)d

6.56/68.5 5.05/21.1 4.66/23.5 4.66/23.5 4.84/28.4

9 11 9 14 12

17 40 37 67 67

89 82 60 94 59

RPSA CKB HA SPT5 ACTG1 CD163 TUBB2 TUBB3 COPG HSPA8 CRMP2A CRMP-2 CRMP1 IGF2BP1 UCHL3 UQCRC1 GFAP ARF6 BICD1 PDHB PMSC2 ATP5B IMMT FUSE

4.80/33.1 5.78/42.5 6.5/64.3 6.35/31.3 5.31/42.1 5.56/56.5 4.78/50.1 4.78/52.9 5.32/98.7 5.47/71.0 6.03/73.9 5.96/62.7 6.58/62.8 9.21/63.6 4.91/26.5 6.58/53.4 5.45/48.7 8.99/17.2 6.74/13.4 5.95/39.3 5.72/49.0 5.59/56.7 5.72/79.5 6.46/81.1

8 14 10 12 11 11 16 12 7 12 16 20 13 16 16 9 23 5 19 9 19 9 12 7

35 40 34 42 36 27 38 27 11 20 33 46 29 28 28 23 45 51 24 37 45 21 20 10

95 85 89 61 77 53 79 98 81 86 78 131 86 62 58 58 111 68 79 67 95 49 63 57

TBCD VIM

5.74/115.1 5.0/53.1

16 12

16 43

74 89

protein name

abbr.

phosphoglucomutase 2 NLI-interacting factor isoform T3 P: similar to prosomal p27k protein synaptosomal-associated protein tyrosine, 3-monooxygenase/tryptophan, 5-monooxygenase activation protein, γ polypeptide 40S ribosomal protein SA B-creatine kinase (brain type) hemagglutinin septin 5 Actin P: similar to CD163 molecule tubulin β-2 or 7 tubulin β-3 or 2c coatomer protein complex, subunit γ Hsp70 dihydropyrimidinase-like 2 dihydropyrimidinase-related protein 2 collapsin response mediator, protein 1 insulin-like growth factor 2, mRNA-binding protein 1 ubiquitin carboxyl-terminal esterase L3 P: similar to ubiquinol-cytochrome c reductase P: similar to glial fibrillary acidic protein R ADP-ribosylation factor-like 6 similar to bicaudal-D P: similar to pyruvate dehydrogenase β proteasome (prosome, macropain) 26S subunit, ATPase, 2 ATP synthase subunit β, mitochondrial inner membrane protein, mitochondrial (mitofilin) far upstream element-binding protein 2(FUSE), zipcode-binding protein tubulin-folding cofactor D vimentin

PGM2 NIFIT3 PP27K SNAP25 YWHAG

theoretical pI/MW (kD)

MASCOT scoree

a Spot ID represents the protein spot number on the 2-DE gels.a) b Accession numbers according to the NCBInr database. c Number of peptides identified by MS/MS is given by MASCOT. d Coverage (%) means the number of amino acids spanned by the assigned peptides divided by the protein sequence length. e MOWSE score is -10 log(p), where p is the probability that the observed match is a random event. Based on the NCBInr database using the MASCOT searching program as MS/MS data. Scores greater than 53 are significant (p < 0.05).

infected and uninfected brain tissues were similar to the changed patterns of their corresponding proteins in 2-DE gels. Of these genes, the mRNA abundance of actin, tubulin, SNAP25, CRMP2, and septin5 was up-regulated, while gene vimentin, TBCD, IGF2Bp1, and PMSC2 were down-regulated (Figure 5). These data provided transcriptional information complementary to the differentially expressed proteins detected by proteomics analysis. 3.5. Western Blot Validation. To further confirm the alterations of protein expression during HPAI infection, five proteinssactin, vimentin, β-tubulin, SNAP25, and CRMP2swere selected for Western blot analysis. Equal amounts of HPAIinfected and control brain tissue proteins were examined by Western blot analysis with specific antibodies to actin, vimentin, β-tubulin, SNAP25, and CRMP2. In Western blot analysis, the actin, tubulin, SNAP25, and CRMP2 demonstrated upregulation; the vimentin was down-regulated (Figure 6). These data agreed with the expression changes by the 2-DE analysis.

4. Discussion The chickens displayed obvious CNS dysfunction symptoms, such as head shaking, violent tremors, and loss of balance. Pathology analysis of the infected chicken brain tissues revealed that the chickens have developed classic nonsuppurative encephalitis, coupled with the symptoms of neutrophil infil-

trates, considerable satellitosis, neuronophagia, and the perivascular cuffing. The edema of cerebral pia mater and the turgescence of the meningeal vessels tend to raise the pressure in the cerebrum and then lead to the CNS dysfunction of chickens. Previous studies have focused on comparative proteomics to screen the differentially expressed proteins associated with host cellular pathophysiological processes of virus infection, and some have adopted the proteomics technology to explore the mechanism of neurodegeneration and neuronal dysfunction when neurons were infected by the West-Nile-Virus and the rabies virus.12,13 The present study provides a comprehensive overview on the altered protein expression of chicken brain tissue responding to H5N1 virus infection. It proves that the function of identified proteins involves cytoskeleton organization, signal transduction, UPP, stress response, macromolecular biosynthesis, and metabolism (Table 1). So far, due to the limitation of the 2-D method to identify low abundance proteins, few studies could identify the viral proteins in proteome research about the influenza virus. The present study identified the HPAI HA protein in the infected chicken brain tissue (Table 1 and Figure 4), demonstrating that the brain tissues were infected by the virus. The identified differential proteins participating in different biological progresses were discussed as follows. Journal of Proteome Research • Vol. 9, No. 8, 2010 3793

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Figure 4. Enlarged regions of differentially expressed protein spots. a) Represents protein spot number and abbreviation of protein name; refer to Table 1.

Figure 5. Transcript alteration of the differentially expressed proteins in AIV-infected chicken brain tissues. Total RNA of brain tissues infected/uninfected by AIV was measured by real-time RT-PCR analysis. Samples were normalized with the chicken histone H5 gene as the control housekeeping gene. Gene symbols indicating different genes refer to Table 1.

1. Alteration of the Cytoskeleton Protein. The cytoskeleton protein system is closely related to maintaining cell morphology, regulating the progress of protein synthesis, enabling cellular motion, and playing important roles in both intracellular transport and cell division. The obtained data have strongly indicated the important role of the cytoskeleton system in the progress of HPAI infection in chicken brain tissue. In the identified microfilament-associated and microtubule-associated proteins, actin and tubulin were up-regulated, whereas the intermediate filament (IF) proteins (VIM, GFAP) were down-regulated (Table 1, Figure 4). Actin, one of the most highly conserved proteins in eukaryotic cells, constitutes microfilament which is one of the three main components of the cytoskeleton. The existing evidence displays that the actin protein can regulate gene transcription in the virus-induced signaling. Due to the binding activity with nascent mRNAs, actin is supposed to be involved in anchoring, transporting, and topological positioning of mRNAs.14 The recognization of 3794

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Figure 6. Western blot confirmation of representative proteins in AIV-infected chicken brain tissues. Control 1, control 2, and control 3 represent the brain tissue protein of chickens uninfected by AIV. Sample 1, sample 2, and sample 3 represent the brain tissue protein of chickens infected by AIV.

Proteomics Analysis of Chicken Brain Tissue Proteins the actin cytoskeleton in the virus-induced signaling is believed to be associated with actin that facilitates transporting viruses or viral components between the nucleus and cell periphery and the efficient spreading of progeny virus particles.15 The down-regulation of VIM was also observed in chicken embryo fibroblast (CEF) infected by the infectious bursal disease virus (IBDV), indicating that this change was not specific for HPAIinfected chicken brain tissue. However, the role of the expression change of cytoskeleton proteins in chicken brain tissues infected by the H5N1 virus particle requires further study. 2. Neural Signal Transduction Associated Protein. Collapsin response mediator proteins (CRMPs) are candidates for key intracellular components of the guidance mechanism of the growth cone. The mutation of Unc33 (CRMP’s homologue) in Caenorhabditis elegans results in a ubiquitous impairment of the formation of neural circuits and severely uncoordinated locomotion.16,17 In this study, three different isoforms (CRMP1, CRMP2, and CRMP2A) were identified up-regulated in HPAI infected brain tissue (Figure 4), and the up regulation of CRMP2 was also confirmed by RT-PCR and Western blot analysis (Figures 5 and 6). CRMP2 has been identified as a member of the signal pathway mediated by Semaphorin3A (Sema3A),17 and the semaphorins constitute the major family of axon guidance cues in the central as well as in the peripheral nervous system.18 Previous studies demonstrate that sequential phosphorylation of CRMPs by cyclin-dependent kinase 5 (cdk5, a proline-directed serine/threonine kinase whose activity is the highest in postmitotic neurons) and GSK3β was an important progress of Sema3A signaling.19,20 Meanwhile, it has been documented that cdk5 also plays a pivotal role in the progress of apoptosis of neuron cells.21,22 So, it is possible that the activation of CRMP2 by cdk5 could be related to the neuron cells apoptosis in chicken brain tissues. Synaptosomal-associated protein of 25 kDa (SNAP-25) is a SNARE protein that participates in the regulation of neuronal exocytosis and regulates neurotransmission. It was identified that SNAP25 was up-regulated when infected by HPAI. Unlike our research, SNAP25 was found down-regulated in mice infected by the rabies virus.13 It has been reported that interaction of SNAP-25 with the synaptic vesicle protein, synaptotagmin I, an important calcium sensor regulating neurotransmitter release,23 was essential for the calciumdependent triggering of membrane fusion and for controlling of fusion pore during the progress exocytosis.24,25 Besides, SNAP-25 has been proved to be involved in modulating various ion channels including N-type and P/Q-type voltage-gated calcium channels (VGCCs).26,27 Therefore, SNAP-25 represents a multifunctional protein involved in the control of exocytosis and regulating ion channels by multiple interactions. The expression of septin 5 was identified significantly upregulated when the chicken brain tissue was infected by HPAI. Septin 5, a parkin substrate, is a vesicle and membrane associated protein that plays a significant role in inhibiting exocytosis and affects the motor dysfunction in the transgenic mice.28 An increased septin 5 level has been shown to exert an inhibitory effect on exocytosis and acts as a negative regulator, as shown by the inhibition of dopamine (DA) release in PC12 cells.29 The principal cause of a familial Parkinson’s disease is due to the loss of function mutations of parkin, and a prevailing hypothesis is that the loss of parkin activity results in accumulation of septin 5, which causes neuron-specific toxicity in SN-DAergic neurons.28 Specifically, Son has demonstrated that the accumulation of septin 5 was also associated with

research articles motor dysfunction of transgenic mice which displayed severe progressive deterioration of rotarod performance and subtle decreases in motor coordination at 2-4 months of age.28 Therefore, it is possible that this pathological mechanism could be related to the neurovirulence of the influenza virus and the neuropathogenesis of chickens. 3. UPP Disorder in HPAI Infected Chicken Brain Tissue. During influenza virus infection, protein synthesis switchs from cellular to viral protein. The degradation of some intercellular proteins is mediated by the UPP, which is critical for diverse cellular functions. Three UPP-linked proteins were identified as differentially expressed proteins after the HPAI infection: the up-regulated PP27K and the down-regulated UCHL3, PSMC2 (Figure 4). Apoptosis was observed in the cerebrum, especially in the molecular layer and the stratum granulosum of the cerebellum in the infected brain tissues. These data indicated that HPAI infection resulted in functional disorders of the UPP system as a “cell cleaner” and apoptosis of neural cells. UCHL3 has also been found to be down-regulated in EB virus and enterovirus 71-infected cells,30,31 but it was UCHL1 that was observed to be down-regulated in CEF infected by IBDV,32 demonstrating that different viruses disturb cellular UPP differently. 4. Macromolecular Biosynthesis and Metabolism Associated Proteins. Ten proteins related to protein transcription, biosynthesis, and metabolism were identified as differentially expressed. PGM2, RPSA, and COPG were proved to be upregulated, and IGF2BP1, ARF6, BICD1, PDHB, ATP5B, IMMT, FUSE, and TBCD were involved in down-regulation (Figure 4). IGF2BP1, a member of the insulin-like growth factor-II (IGFII) mRNA-binding protein (IMP) family, is identical to the mouse c-myc coding region determinant binding protein (CRDBP) and the chicken β-actin mRNA-binding protein-1 (ZBP-1). So far, it has been reported that there are at least 5 RNA targets for IMP-1, including IGF-II, c-myc, β-actin, tau, and H19.33,34 IMP-1 plays an important role in affecting stability, localization, and translation of its target RNAs. Chicken ZBP-1/IMP-1 binds to the “zipcode” segment in the 3′-UTR of β-actin mRNA and localizes the mRNA to the lamellipodia of fibroblasts.33 It was also observed that HIV-1 assembly was interfered in 293T cells when IMP1 was overexpressed. The ADP-ribosylation factor (ARF) was initially identified as a cofactor required for the cholera toxin-catalyzed ADPribosylation of the R subunit of the trimeric G protein. The coatomer complex (COP) was identified as a soluble complex of about 700 kDa containing polypeptides of 160, 110, 98, and 61 kDa, designated R, β, γ, and δCOP, respectively.35 Bicaudal D (BICD) protein was initially identified as a member of the dynein pathway in Drosophila. These three proteins were proved to be associated with protein transportation and modification in the Golgi membrane. The previous studies have shown that ARF is required for binding of the coatomer protein to the Golgi membrane and modulating Golgi membrane transportation. They point out that the γ-subunit of the COP participates in the Cdc42-mediated changes in the actin cytoskeleton, cell morphology, or malignant transformation.36 The C-terminal domain of the BICD protein is responsible for interaction with membranes via Rab6, and the N-terminal domain binds cytoplasmic dynein.37 Meanwhile, by binding dynein with the N-terminal domain, the BICD is capable of inducing microtubule minus end-directed transport and cellular organelles relocation.38 BICD regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor Journal of Proteome Research • Vol. 9, No. 8, 2010 3795

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Figure 7. Functional network analysis of the differentiated identified protein. (a) The association of differentiated identified protein with neural signal transduction. (b) The association of differentiated identified protein with neurological disease of the nervous system. Shaded proteins are the proteins identified by proteomics analysis, and others are those associated with the regulated proteins based on the pathway analysis. Nodes represent proteins, with their shape representing the functional class of the proteins, and different lines and arrows indicate the biological relationship between the nodes.

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Proteomics Analysis of Chicken Brain Tissue Proteins 39

complex. The coatomer complex is important in protein intracellular trafficking because of its ability to assemble at Golgi membranes under the direction of the GTP-binding protein ARF. 5. Relationship of Differentiated Proteins with the Neurological Disease Pathway. The relationship was analyzed among the differentiated proteins, and the analysis was also performed on the relationship of these proteins in the neurological disease pathway with Ingenuity Pathways Analysis (Ingenuity Systems). Many identified proteins were associated with neural development, proliferation, and neural signal transduction and the cell-cell interaction signal pathway, such as SEP5, SNAP25, YWHAG, ACTG1, and TUBB2C (Figure 7a). Some proteins participated in the pathological progress of Huntington’s disease, such as CRMP1, UCHL3, PMSC2, and 26s proteasome, and Hsp70 assembled around the HTT protein networks in the neurological disease signaling pathway in the neuropathological progress (Figure 7b). This result may possibly indicate that these proteins also participate in the neuropathological progress of the chicken brain tissues when infected by HPAI.

Concluding Remarks In summary, the chickens infected by the HPAI A/duck/ hubei/hangmei01/2006 displayed severe CNS dysfunction, and the chicken brain tissues developed classic nonsuppurative encephalitis. By comparison with the uninfected brain tissues, a number of altered proteins in the chicken brain tissues infected by the virus were identified. Most of the altered proteins demonstrated to be the key factors in the cytoskeleton system, signal transduction, UPP, molecular biosynthesis, and metabolism. Meanwhile, some proteins were related to neuron signal transduction, axon function, and morphology. Especially, some proteins participated in the pathogenetic progress of Parkinson’s and Huntington’s diseases which developed encephalitis accompanied with CNS dysfunction as well. The data presented here give a fundamental understanding of progress during the infection of the H5N1 influenza virus in chicken brain tissue. Further investigation is suggested to focus on the altered proteins to facilitate the understanding of the mechanism of the neurovirulence of the HPAI and the progress of neuropathogenesis of the brain tissue to HPAI infection.

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Acknowledgment. This work was supported by 973 program (2010CB534002, 2005CB523003) and National S & T major project (2008ZX10004-103, 2009ZX10004-109). We thank Ms. Haiya Wu at Department of Experimental Mouse Genetics, Helmholtz Centre for Infection Research & University of Veterinary Medicine Hannover, Braunschweig, Germany, for assistance in Ingenuity Pathways Analysis, and we thank Professor Yanxiu Liu of Foreign Language School, Huazhong Agriculture University, for her help with English.

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Supporting Information Available: The virus titers of the chicken brain tissues. This material is available free of charge via the Internet at http://pubs.acs.org.

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