Comparative Proteomic Analysis of Wild-Type and SAP Domain

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Comparative Proteomic Analysis of Wild-Type and SAP Domain-Mutant Foot-and-Mouth Disease Virus (FMDV)-Infected Porcine Cells Identifies the Ubiquitin-Activating Enzyme UBE1 Required for Virus Replication Zixiang Zhu, Fan Yang, Keshan Zhang, Weijun Cao, Ye Jin, Guoqing Wang, Ruoqing Mao, Dan Li, Jianhong Guo, Xiangtao Liu, and Haixue Zheng J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00310 • Publication Date (Web): 10 Sep 2015 Downloaded from http://pubs.acs.org on September 15, 2015

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Journal of Proteome Research

Comparative Proteomic Analysis of Wild-Type and SAP Domain-Mutant Foot-and-Mouth Disease Virus (FMDV)-Infected Porcine Cells Identifies the Ubiquitin-Activating Enzyme UBE1 Required for Virus Replication

Zixiang Zhu, Fan Yang, Keshan Zhang, Weijun Cao, Ye Jin, Guoqing Wang, Ruoqing Mao, Dan Li, Jianhong Guo, Xiangtao Liu and Haixue Zheng*

State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China

1

ACS Paragon Plus Environment


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ABSTRACT: Leader protein (Lpro) of foot-and-mouth disease virus (FMDV) manipulates the activities of several host proteins to promote viral replication and pathogenicity. Lpro has a conserved protein domain SAP that is suggested to subvert interferon (IFN) production to block antiviral responses. However, apart from blocking IFN production, the roles of the SAP domain during FMDV infection in host cells remain unknown. Therefore we identified host proteins associated with the SAP domain of Lpro by a high-throughput quantitative proteomic approach [isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with liquid

chromatography/electrospray

ionization

tandem

mass

spectrometry

(LC-ESI-MS/MS)]. Comparison of the differentially regulated proteins

in

rA/FMDVmSAP- versus rA/FMDV-infected SK6 cells revealed 45 down-regulated and 32 up-regulated proteins that were mostly associated with metabolic, ribosome, spliceosome and ubiquitin-proteasome pathways. The results also imply that the SAP domain has a similar function to SAF-A/B besides its potential PIAS function. One of the identified proteins UBE1 was further analyzed, and displayed a novel role for the SAP domain of Lpro. Over-expression of UBE1 enhanced the replication of FMDV, and knockdown of UBE1 decreased FMDV replication. This shows that FMDV manipulates UBE1 for increased viral replication, and the SAP domain was involved in this process. KEYWORDS: foot-and-mouth disease virus, SAP domain, iTRAQ, quantitative proteomics, pathway analysis

2


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INTRODUCTION Foot-and-mouth disease (FMD) is an important infectious disease of ruminants1. FMD virus (FMDV) is the etiological agent of FMD, which belongs to the aphthovirus genus of the Picornaviridae family. The genome of FMDV is a single-stranded positive-sense RNA, approximately 8.3 kb in length. The viral RNA encodes a polyprotein, which is post-translationally processed, and generates mature viral proteins. Two different start codons separated by 84 nucleotides are located in viral mRNAs, and produce two forms of leader protein (Lpro), termed Labpro and Lbpro, respectively2, 3. Lpro is a virus-encoded proteinase that has functions to antagonize the host innate immune response4-6. Lpro is a well-characterized papain-like proteinase7, 8, that autocatalytically self-cleaves from the viral polyprotein precursor. It is well known that Lpro can cleave the host translation initiation factor eIF4G, which results in shut-off of host cap-dependent mRNA translation without interfering with the viral cap-independent protein synthesis; a characteristic of most picornavirus infections9, 10. In

addition,

Lpro

plays

an

important

role

in

inhibiting

nuclear

factor

(NF)-κB-dependent gene expression by degradation of NF-κB11. Moreover, Lpro subverts interferon (IFN) production to block antiviral responses10, and type I IFN transcription induced by double-stranded RNA can be inhibited by Lpro through decreasing IFN regulatory factor 3/7 protein levels5. Various studies have indicated that Lpro plays critical roles in FMDV evasion from the host immune response6, 12. A conserved putative protein domain within FMDV Lpro known as the SAP domain [scaffold-attachment factor (SAF)-A/B, apoptotic chromatin-condensation 3



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inducer in the nucleus (ACINUS) and PIAS (protein inhibitor of activated signal transducer and activator of transcription) domain] has been found by de los Santos et al.11. In eukaryotic cells, the SAP domain is implicated in SAF-A/B-, ACINUS-, and PIAS-associated functions13-15. SAF-A/B are scaffold attachment factors involved in nucleic acid binding and chromosomal organization16-18, ACINUS is related to apoptosis occurrence19 and PIAS can regulate innate immune responses and suppress virus-triggered and IFN-stimulated transcription20. Lpro inhibits type I IFN induction, and the SAP domain in Lpro is involved in blocking IFN-induced responses, resulting in downregulation of several IFN-stimulated genes (ISGs) 5, 12, 21, 22, which implies that the SAP domain of FMDV has a potential function of PIAS to suppress IFN-stimulated transcription. However, several novel mechanisms apart from IFN-related responses that are regulated by this SAP domain remain unclear; moreover, whether the SAP of FMDV has the function of SAF-A/B and ACINUS is still unknown. To further understand the novel specific functions of the SAP domain and the regulatory network activated by the SAP domain of Lpro in FMDV-infected porcine cells, we compared the in vitro characteristics of the rA/FMDV and rFMDVmSAP (mutations of two amino acids in SAP domain of rA/FMDV) strains in different cell lines. To date, quantitative proteomics approaches have become a tool to analyze host cellular responses during virus infection. Hence, we further compared and analyzed the host protein profiles of rA/FMDV- and rA/FMDVmSAP-infected SK6 cells that were deficient in type I IFN production23. A high-throughput quantitative proteomics 4


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approach, isobaric tags for relative and absolute quantitation (iTRAQ) was used in conjunction with the liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method. iTRAQ has been widely applied in proteomic studies due to its accurate quantification

24

. iTRAQ was developed to bind to amines instead of cysteines25, 26,

which was primarily to improve proteome coverage27. It is apparent that iTRAQ is a robust and favorable technique for proteomics analysis

27

. Here, using iTRAQ,

cellular proteins were identified and quantified to seek differentially expressed proteins in porcine cells infected with rA/FMDV or rA/FMDVmSAP virus. Seventy-seven differentially expressed proteins were determined. No obvious alteration of IFN-related proteins was observed. Bioinformatics analysis of pathways and gene ontology showed significant alteration of several proteins involved in cellular metabolism, proteolysis and immune response pathways. One of the identified proteins UBE1 was determined to promote FMDV replication and SAP domain was involved in this process. In conclusion, this study uses the quantitative proteomics to analyze systematically the host protein profile and pathways induced by the SAP domain of FMDV, which may provide valuable insights into the novel functions of the domain and the molecular mechanisms of FMDV pathogenesis.

MATERIALS AND METHODS Viruses, Cells, Plasmid and Transfection SK6, PK15 and BHK-21 cells were purchased from the Cell Bank of Type Culture 5



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Collection of Chinese Academy of Sciences (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) with 1% antibiotic/antimycotic (A/A) was used as the cell culture media. All cells were maintained at 37°C under 5% CO2. The porcine UBE1 cDNA was cloned and inserted into pcDNATM3.1/myc-His A vector (Invitrogen) to construct a Myc-tagged UBE1 plasmid (Myc-UBE1). LipofectamineTM2000 (Invitrogen) was used as the transfection reagents. A chimeric virus, named rA/FMDV, was a potential vaccine strain constructed by our lab previously28. rA/FMDVmSAP strain was generated from the full-length rA/FMDV infectious clone by SAP mutation in Lpro as described by de los Santos et al 11

(Figure 1).

Infection The prepared cell monolayers were washed three times with phosphate-buffered saline (PBS) and then challenged with FMDV at a multiplicity of infection (MOI) of 1 at 37°C. The inoculum was removed after 1 h adsorption, and the cells were maintained in DMEM supplemented with 1% FBS (virus growth media) at 37°C. For mock infection, the procedure was performed similar to the viral infection using PBS as the inoculum. The cells for proteomic analysis were harvested at 6 h post-infection (hpi).

Determination of Viral Titers by Plaque-Forming Unit (PFU) 6


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BHK-21 or SK6 cells were used to determine the viral titers by plaque assay, the cells were seeded in six-well plates for 12 h, and infected with a graded increasing concentration of viruses. 1% LMP agarose (Invitrogen) in DMEM was added to the cells after 1 h viral adsorption. The detailed detection method was performed as previously described29. Plaques were counted and the viral titers were determined, and all assays were performed in triplicate.

Challenge with the rA/FMDV and rA/FMDVmSAP Strains To test and compare the pathogenicity, all pigs were challenged by intradermal inoculation in the heel bulb, with high dose (5107 TCID50) of rA/FMDV or rA/FMDVmSAP strains. Clinical signs were observed and recorded each day. Heparinized and clotted blood was collected daily after virus infection, and nasal swabs were also collected. Detection of viral RNA in the blood and nasal swabs, and the criteria for determining the positive samples, were as previously described28. Besides, serum samples were collected at intervals until 25 days post-challenge for detection of neutralizing antibodies. The antibody titers were calculated by virus neutralization test (VNT), as previously described30. All animal experiments were performed in compliance with the standard guidelines of the Gansu Animal Experiments Inspectorate and the Gansu Ethical Review Committee [License no. SYXK (GAN) 2010-003].

Protein Extraction 7



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The cells were harvested at 6 hpi and the cell pellets were lysed in a cold lysis buffer (7M Urea, 2MThiourea, 4% CHAPS, 40mM Tris-HCl, pH 8.5) containing 1mM PMSF and 2mM EDTA (final concentration). The whole cell lysates were sonicated for 15 cycles of 1 s on and 1 s oﬀ and then centrifuged for 15 min at 25 000 g at 4°C. The concentration of the protein was determined through the Bradford method. Samples of aliquots were stored at 80°C until used in proteomic detection.

Tryptic Digestion and iTRAQ Labeling For tryptic digestion and iTRAQ labeling, 100µg protein was digested with 10 µL trypsin (0.5µg/µL) at 37°C for 4 h. An equal volume of trypsin was added for an additional 8 h digestion. The peptide was collected by vacuum centrifugation. After centrifugation, the peptide pellet was dissolved in 0.5 M triethylammonium bicarbonate. The prepared peptides were then labeled with different iTRAQ tags for 2 h at room temperature. iTRAQ 113 and 116 tags were used respectively to label the rA/FMDV- and rA/FMDVmSAP-infected samples. The labeled peptides were thoroughly mixed and the mixtures were dried through vacuum centrifugation.

Fractionation by Strong Cation Exchange (SCX) Chromatography The Shimadzu LC-20AB HPLC Pump system and a 4.6 × 250 mm Ultremex SCX column carrying 5-μm particles (Phenomenex) were used for SCX chromatography. The labeled peptide sample was resuspended in 4 mL Buffer A [25 mM NaH2PO4 in 25% Acetonitrile (ACN), pH 2.7] and transferred to the column. Buffer A was firstly 8


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used to elute the peptides for 10 min, then 5–35% Buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 2.7) for 9 min, and finally 35–80% Buffer B for 1 min. The elution flow rate was set to 1 mL/min and fractions were collected every 1 min, and the whole process was monitored by measuring absorbance at 214 nm. Ten fractions were collected and desalted by Strata X C18 column (Phenomenex). Each fraction was concentrated by vacuum freeze-drying.

LC-ESI-MS/MS Identification by LTQ-Orbitrap Higher-energy Collisional Dissociation (HCD) Each fraction was reconstituted with 40 L Buffer A [2% ACN, 0.1% formic acid (FA)] to reach a peptides concentration of 0.5 g/L. The dissolved fractions were centrifuged at 20 000 g for 10 min to remove insoluble impurities. The Shimadzu LC-20AD nanoHPLC and C18 columns were used for reversed phase separation. The supernatant was loaded at 15 μL/min for 4 min, then the gradient was run at 400 nL/min starting from 2 to 35% Buffer B (98% ACN, 0.1% FA) for 44-min, 35 to 80% Buffer B for 2 min; subsequently, 80% Buffer B for 4 min; and returning to 2% for 1 min. The peptides were detected and identified by nanoelectrospray ionization and MS/MS in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. The resolution of the Orbitrap was set to 60000. The mass resolution for HCD was 60000. The peptides were chosen by an HCD operating mode with collision energy setting of 45%. The ion fragments were detected in the LTQ. The detailed procedure was 9



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performed as described previously31. The MS scan recorded the range between 350 and 2000 m/z.

Bioinformatics Analysis of Proteomics Data Protein identification and quantiﬁcation for iTRAQ analysis was carried out using Mascot 2.3.02 (MatrixScience, London, UK). The NCBInr database (release June 30, 2013) containing pig (Sus scrofa) sequences (31786 sequences) was selected as the analytical database, and the derived files were screened using Mascot 2.3.02. The following parameters were used for the searches: the search type was MS/MS ion search. As for the search, trypsin was used as a specific enzyme; a maximum of one missed cleavage was permitted and Gln->pyro-Glu (N-term Q), Oxidation (M), iTRAQ8plex (Y) were selected as variable modifications; carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K) were set as fixed modifications; the mass tolerance for the peptide and fragment was set to 10 ppm and 0.05 Da respectively. The unique peptides were used for protein quantitation by mascot, and the normalization method was set as summed intensities. The protein ratio was calculated by averaged peptide ratio of the proteins identified with at least two unique peptide hits. The differentially expressed proteins in the two samples were determined based on a minimum of 1.2-fold change of the quantification and student test p

Comparative Proteomic Analysis of Wild-Type and SAP Domain

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