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Dec 1, 2016 - (r = 0.3), and several examples of posttranscriptional regulation were observed; e.g., proteins related to carbohydrate metabolism...
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Posttranscriptional Regulation in Adenovirus Infected Cells Hongxing Zhao,*,† Anne Konzer,‡ Jia Mi,‡ Moashan Chen,§ Ulf Pettersson,† and Sara Bergström Lind*,‡ †

The Beijer Laboratory, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Rudbeck Laboratory, 751 85 Uppsala, Sweden ‡ Department of Chemistry-BMC, Science for Life Laboratory, Analytical Chemistry, Box 599, Uppsala University, 751 24 Uppsala, Sweden § Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, LaTrobe University, Melbourne, Victoria 3086, Australia S Supporting Information *

ABSTRACT: A deeper understanding of how viruses reprogram their hosts for production of progeny is needed to combat infections. Most knowledge on the regulation of cellular gene expression during adenovirus infection is derived from mRNA studies. Here, we investigated the changes in protein expression during the late phase of adenovirus type 2 (Ad2) infection of the IMR-90 cell line by stable isotope labeling in cell culture with subsequent liquid chromatography−high resolution tandem mass spectrometric analysis. Two biological replicates of samples collected at 24 and 36 h post-infection (hpi) were investigated using swapped labeling. In total, 2648 and 2394 proteins were quantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated >1.6-fold at the two time points. The protein expression was compared with RNA expression using cDNA sequencing data. The correlation was surprisingly low (r = 0.3), and several examples of posttranscriptional regulation were observed; e.g., proteins related to carbohydrate metabolism were up-regulated at the protein level but unchanged at the RNA level, whereas histone proteins were down-regulated at the protein level but up-regulated at the RNA level. The deregulation of cellular gene expression by adenovirus is mediated at multiple levels and more complex than hitherto believed. KEYWORDS: Adenovirus type 2 infection, SILAC, mass spectrometry, RNA sequencing, deregulation of host cell protein and RNA expression, IMR-90 cells



INTRODUCTION Viruses have served as excellent model systems to investigate essential cellular functions and have contributed to the definition of many important principles. On one hand, the host cell activates the cellular antiviral defense system that interferes with virus entry and replication. On the other hand, the virus has evolved mechanisms that suppress the host cell responses and establishes conditions that are favorable for viral progeny production. Human adenovirus (hAd) is an outstanding model for studies of eukaryotic gene structure and expression. They are nonenveloped, icosahedral particles containing a linear, doublestranded DNA molecule. On the basis of hAd gene expression, the replication cycle can be divided into two major phases, an early and a late phase, defined by the start of viral DNA replication. Early viral proteins are involved in the regulation of the cell cycle and suppression of the cellular antiviral response, whereas the viral structural proteins are synthesized during the late phase (for a review see Shenk1). Studies of host cell gene expression during the course of adenovirus type 2 (Ad2) infection in IMR-90 cells show that the infection can be further divided into four periods.2 Each period is characterized by deregulation of specific sets of cellular genes. At 6 hpi (hours post-infection) genes involved in the inhibition of cell growth are © 2016 American Chemical Society

significantly deregulated, at 12 hpi the deregulation concerns gene involved in control of the cell cycle, at 24 hpi genes involved in nucleic acid and protein synthesis are deregulated, and at 36 hpi genes important for maintenance of cellular structure are deregulated. The most dramatic changes in cellular gene expression occurs at 24 hpi, when infection promotes the cell cycle switch from G0 to S-phase. By this time the adenovirus has gained control of the cellular metabolic machinery for its genome replication and expression. Many effects of hAd on host cells early after infection mimic tumorigenesis by promotion of cell growth and inhibition of apoptosis. Most knowledge on the regulation of the host cell gene expression during hAd infection has been derived from studies on mRNA expression and needs to be complemented with temporal studies of protein expression. In addition to transcription, gene expression is regulated at the posttranscriptional level including mRNA processing, export, turnover, translation, posttranslational modification, cleavage and finally transport of proteins to the destinations where they carry out their functions. Mass spectrometry (MS)-based protein detection is a powerful technique and has revolutionized proteomics. Particularly, so-called Received: September 16, 2016 Published: December 1, 2016 872

DOI: 10.1021/acs.jproteome.6b00834 J. Proteome Res. 2017, 16, 872−888

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Journal of Proteome Research

by sonication and lysates were cleared by centrifugation at 16 000g for 5 min. Protein concentration was determined by DC (detergent compatible) protein assay (Bio-Rad, Hercules, California, USA). For all time points, heavy and light labeled samples were mixed in a 1:1 ratio as followed: Light labeled mock sample was mixed with heavy labeled cells infected with Ad2. As a biological replicate, a SILAC swap experiment was performed by mixing light labeled cells infected with Ad2 with heavy labeled mock samples at all time points. Screening experiments were performed for all time points by filter aided sample preparation protocol (FASP) for in-solution digestion of proteins.16 In brief, protein disulfide bonds were reduced with 100 mM dithiothreitol at 37 °C for 30 min prior to sample loading onto a 30 kDa centrifugal filter unit (Millipore, Merck, Germany). Next, samples were washed with urea buffer (8 M urea in 100 mM Tris/HCl pH 8.5) and alkylated with 550 mM iodoacetamide. Lys-C (protein-to-enzyme ratio 100:1) (Wako Chemicals GmbH, Osaka, Japan) was used for protein digestion at 30 °C for 2 h followed by trypsin (protein-to-enzyme ratio 100:1) (Promega Corporation, Madison, WI, USA) at 37 °C overnight. Resulting peptides were eluted, acidified and concentrated by stop and go extraction tips17 before analysis by LC−MS/ MS. Samples harvested at 24 and 36 hpi were subjected to in-depth analysis by SDS-PAGE fractionation and in geldigestion. Mixed samples were loaded on a 4−12% gel (NuPAGE, Bis-Tris gel, Life Technologies, Carlsbad, CA, USA) to separate proteins by molecular weight. After protein staining by Colloidal Coomassie Blue, gel lanes were cut into 10 pieces and subjected to in gel-digestion.18 In brief, gel pieces were washed several times with 50 mM ammonium bicarbonate and dehydrated with absolute ethanol. Next, protein disulfide bonds were reduced with 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide. Protein digestion was performed by trypsin 12.5 ng/μL (Promega Corporation, Madison, WI, USA) in 50 mM ammonium bicarbonate buffer at 37 °C overnight. Resulting peptides were extracted stepwise by acetonitrile and concentrated using stop and go extraction tips17 before analysis by LC− MS/MS.

shotgun/bottom-up liquid chromatography−tandem MS (LC− MS/MS) is used for full proteome analysis as reviewed by Yates et al.3 In addition, a number of alternative upstream steps for sample preparation and fractionation are used to optimize protein detection.4 The most accurate strategy to compare protein abundance levels is to use stable isotope labeling of amino acids in cell culture (SILAC)5 combined with MS.6 By growing two cell populations (such as infected vs uninfected cells) in culture media that are identical except that one of them contains a “light” (e.g., 12C or 14N) or “heavy” (13C or 15N) form of amino acids, different isoforms of amino acids are incorporated into proteins in the two samples. After mixing the samples in a 1:1 ratio with respect to protein amount the samples are subjected to the same sample preparation and LC−MS/MS analysis. Quantitative differences at the protein level between different conditions can thus be measured from the ratios of peptides and proteins with heavy and light labeling. The impact of MS-based proteomics in the field of virology is increasing, as reviewed by Greco et al.7 SILAC in combination with LC−MS/MS have been applied to a number of virus studies.8−14 For hAd, this techniques have been used for analysis of (i) protein degradation after inactivation of hAd by sunlight and UVC light,9 (ii) quantitative changes in the protein composition of the nucleolus during hAd infection,11 (iii) the protein composition of highly purified wild type hAd and mutants8 and (iv) proteome of hAd infected cells by using transcriptomics data.15 In this paper we study the deregulation of cellular protein expression after Ad2 infection in order to identify genes and biological functions that are under posttranscriptional control. SILAC-labeled samples from 24 and 36 hpi of Ad2 were processed and analyzed with high resolving LC−MS/MS. The changes in protein expression were compared with those of cellular RNA expression in order to study consistency and divergence of gene regulation at the RNA and protein levels. Bioinformatic analyses of biological functions and pathways were used to reveal characteristics of regulated proteins.



EXPERIMENTAL SECTION

Cell Culture, Infection and Sample Harvest

NanoLC−MS/MS

A human lung fibroblast cell line (IMR-90), purchased from American Type Culture Collection (ATCC) was cultured in minimum essential medium (without lysine and arginine) (Thermo Fishcer Scientific, Waltham, MA, USA) supplemented with 10% dialyzed fetal bovine serum (Thermo Fischer Scientific), 100 U/mL penicillin and 100 μg/mL streptomycin, L-lysine (0.89 mM) and L-arginine (0.40 mM) (Sigma-Aldrich, St Louis, MO, USA) in light medium or 13C15N-labeled lysine (Lys8, 0.89 mM) and 13C15N-labeled arginine (Arg10, 0.40 mM) (Silantes, Munich, Germany) in heavy medium. Label efficiency was tested in MS experiments after five passages and calculated by SILAC ratio H/L/(SILAC ratio H/L + 1) for all proteins. After six cell doublings labeled cells were either mock infected (only medium) or infected with Ad2 at a multiplicity of infection of 100 in a serum free medium for 60 min. Then the medium was replaced with fresh SILAC-labeled medium and cultured for 2, 6, 12, 24, and 36 h before cells were collected. A biological replicate in form of a swap-labeling experiment was performed. Cells were washed with PBS and directly snap frozen on dry ice.

Reverse-phase chromatography for peptide separation was performed using an Easy nano flow system (Thermo Fisher Scientific, Bremen, Germany), which was coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were separated by precolumn (100 μm ID, 5 μm C18-beads) and analytical columns (75 μm ID, 3 μm C18-beads) (Thermo Fisher Scientific) using a linear gradient for 150 min. In brief, a gradient was set from 4% to 48% acetonitrile with 0.1% formic acid for 138 min at a flow rate of 250 nL/min, followed by 75% acetonitrile for 6 min and 4% acetonitrile for 6 min for re-equilibration. Next, peptide ionization was performed using a nano electrospray ionization source. Peptides were transferred into the mass spectrometer and analyzed in positive ion mode (m/z = 400− 1750) using an automated gain control target of 3 × 106 at a resolution of 70 000. The 10 most intense peaks were isolated for higher-energy collisional dissociation fragmentation (25% normalized collision energy) and MS/MS spectra were generated with an automated gain control target of 5 × 105 at a resolution of 17500. The mass spectrometer worked in the datadependent mode. Raw data were processed using MaxQuant (1.4.1.2)19 and database searches were performed using the implemented

Cell Lysis and Sample Preparation before LC−MS/MS

Collected cells were lysed in SDS buffer (4% SDS in 100 mM Tris/HCl pH 7.6) at 95 °C for 3 min. Next, DNA was sheared 873

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Figure 1. Strategy for investigation of the gene expression at different levels upon Ad2 infection. (A) Experimental setup for RNA sequencing. (B) Workflow for MS-based identification and quantification of proteins.

genome sequences (GRCh38, Ensembl) with TopHat2 software.21 TopHat2 incorporates Bowtie2 (http://bowtie-bio.sourceforge. net/bowtie2/index.shtml) algorithm to perform the alignment. We used default parameters which allowed a maximum of two mismatches when mapping the reads to the human genome. Cufflinks22 was then used to profile gene expression at each time point based on human gene annotation by Ensembl. RNAs witha minimum of 10 FPKM (Fragments per Kilobase of exon per Million fragments mapped) were considered as significantly expressed. Differentially expressed RNAs, compared to mock, were characterized by three statistical values. First, fold change was calculated by the FPKM values in different libraries; second, based on Poison distribution23 p-values were used to present the significances of differentially expressed RNAs; last, using the NOIseq package24 the probability of a differentially expressed RNA was calculated. RNA data for quantified proteins are provided in Supplemental Table S2.

Andromeda search engine. MS/MS spectra were correlated to the Uniprot human database (release 2014−07, 89028 entries) combined with a human adenovirus typ 2 (HAdV2) database (release 2014−08, 481 entries). False discovery rate (FDR) was calculated based on reverse sequences from the target-decoy search and an FDR of 1% was accepted for protein and peptide identification. The following parameters were used for data processing: maximum of two miss cleavages, the mass tolerance was 4.5 ppm for main search and for fragment masses it was up to 20 ppm, trypsin as digesting enzyme, carbamidomethylation of cysteines as fixed modification, oxidation of methionine, acetylation of the protein N-terminus and phosphorylation (STY) as variable modifications. For SILAC labeling, Lys8 and Arg10 were set for heavy label and two ratio counts was the minimum for protein quantification. For protein identification, only peptides with a minimum of 7 amino acids and at least one unique peptide were required. Only proteins with at least two peptides and of them at least one unique peptide were considered as identified and used for further data analysis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (www.proteomexchange.org) via the PRIDE20 partner repository with the data set identifier PXD004096. The data for protein quantification is provided in Supplemental Table S1.

Data handling and Bioinformatics

Normalized SILAC-ratios were used to present the fold change of protein expression and differentially expressed proteins were obtained using a cut off fold change >1.6. Differentially expressed proteins (average of the two replicates) and mRNAs (fold change >1.5 for both) were mapped using gene symbol. Gene ontology functional analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/)25 for both regulated proteins and for protein-RNA correlation groups. Protein accessions or gene symbols were submitted to DAVID analysis as input and the data was processed using the Functional annotation chart. Biological processes (BP5) and KEGG pathways were evaluated. Biological processes that satisfy p-value < 0.05 and FRD < 0.01 were considered.

RNA Extraction, cDNA Library Preparation, Sequencing and Data Analysis

Total RNA from Ad2- or mock-infected IMR-90 cells was extracted using TRIZOL Reagent (Invitrogen/Thermo Fischer Scientific). The quality of the input RNA was controlled by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then total RNA was treated with RiboZero (Epicenter, Madison, WI, USA) to remove rRNA and cDNA libraries were constructed using ScriptSeq v2 RNA-Seq library preparation kit according to the manufacturer’s protocol (Epicenter). The cDNA libraries were sequenced using an Illumina HiSeq 2000 system. After data cleaning, the reads were aligned to human 874

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Figure 2. Protein expression after Ad2 infection. Biological replicates described by number of quantified proteins (n) and Pearson’s correlation (r). Specified groups of regulated proteins are color-coded. (A) 24 hpi. (B) 36 hpi. (C) The correlation of differentially expressed proteins between 24 and 36 hpi of Ad2 infection.



Experimental Design

Two biological replicates with swapped amino acid labeling for the protein expression studies at 24 and 36 h after Ad2 infection were used to validate the reproducibility of the data. Each replicate was fractionated by SDS-PAGE giving 10 samples per time point and per biological experiment. These 40 samples were analyzed once in the LC−MS/MS. Biological replicates with swapped labeling were preferred over technical replicates. The control samples of mock-infected cells were grown in parallel to the Ad2-infection. The controls were prepared for each time point and the number of controls equals to the number of infected replicates. One RNA-sequencing was performed per time point of study. The experimental design is presented in Figure 1.

RESULTS AND DISCUSSION

Study of Cellular Protein Expression during Late Phase of Ad2 Infection Using SILAC-MS Technology

In this study, we investigated the deregulation of host cell gene expression, at RNA and protein levels, during the late phase of Ad2 infection (24 and 36 hpi). Growth arrested human lung fibroblast cells (IMR-90 cell line) were used because of the slow progression of Ad2 infection cycle and because of the possibility to synchronize them in G0 phase of the cell cycle. Compared with, e.g., HeLa cells, which provide extremely efficient replication of Ad2, the complete virus cycle in IMR-90 cells is approximately twice the time under equivalent conditions.2 Thus, there is a wider time window for examination of regulatory mechanisms of cellular gene expression in the used host cells. Previous studies of mRNAs, miRNA and lncRNA expression have revealed that dramatic changes in cellular gene expression in IMR-90 take place at 24 hpi when the infection enters the late phase.2,26,27 At this time, Ad2 has redirected host cellular

Safety Considerations

Chemicals for sample preparation might be toxic and/or carcinogenic and biological material should be handled with great care according to the local laboratory guidelines. 875

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Journal of Proteome Research Table 1. Comparison of Deregulation of Gene Expression at RNA and Protein Levels proteins Total proteins or RNAs quantified Deregulated Up-regulated Down-regulated

RNA

24 hpi

36 hpi

in total

overlap between 24 and 36 hpi

24 hpi

36 hpi

2648 659a (25%)b 515 (78%)e 144 (22%)g

2394 646 (27%)b 497 (77%)e 149 (23%)g

2777 742 569 173

2265 563 443 120

6209 2395c (39%)d 1424 (60%) f 971 (40%)h

6218 2817 (45%)d 1695 (60%) f 1122 (40%)h

a The cutoff of fold change for protein was 1.6-fold. bPercentage of differentially expressed proteins compared with total detected proteins. cThe cutoff of fold change for RNA as 2-fold. dPercentage of differentially expressed RNAs compared with total detected RNAs. ePercentage of upregulated proteins to all deregulated proteins. fPercentage of up-regulated RNAs to all deregulated RNAs. gPercentage of down-regulated proteins to all deregulated proteins. hPercentage of down-regulated RNAs to all deregulated RNAs.

24 and 36 hpi observed at the two time points was expected since it has been shown that the host gene expression remains relatively stable during this period.2 No proteins showed opposite expression patterns between 24 and 36 hpi, whereas 73 proteins showed a change in protein expression, e.g., nonregulated at 24 hpi but deregulated at 36 hpi, or vice versa. This group included proteins that were (i) up-regulated (n = 11, such as PPP6R3, DCTD, and ZCCHV) or down-regulated (n = 9, such as GOLGB1, THSD4, B4DN67) at 24 hpi, but were unchanged at 36 hpi, and (ii) proteins whose expression were not changed at 24 hpi but became up-regulated (n = 38, such as RS15A, EIF5B, SLC16A1, SUMO1, THRAP3) or down-regulated (n = 15, PPP1R12A, IFI16, LUZP1 and MAGED1) at 36 hpi. The most highly up-regulated protein at both 24 and 36 hpi was the heat shock 70 kDa protein 1A/1B (HSPA1A/1B) which reached an 8-fold increase. The pronounced up-regulation of these stress response proteins was also noted in a study by Evans et al.,15 and was expected from other report.30 Furthermore, expression of a group of mini-chromosome maintenance proteins (MCMs) that are involved in the initiation of eukaryotic genome replication increased more than 2-fold. Up-regulation of a large group of eukaryotic translation initiation factors (EIFs) was also noteworthy. Among the most down-regulated proteins were a group of collagens (COLs) and growth factor receptors (such as EGFR, PDGFRB, PDGFRA, CTGF). The top 50 up- and down-regulated proteins at 24 and 36 hpi are summarized in Tables 2A−2D. The average ratios for the swapped experiments are presented and even though the RSD values for a few of the proteins were high, only two proteins showed inconsistency in their regulation (i.e., up-regulated in the first experiment while nonregulated in the replicate (Table 2C)). The increase in stress response30 and the promotion of DNA, RNA and protein synthesis as well as the decrease in cellular structural proteins and growth factor receptors were expected from previous RNA studies.2,26,27,31 In agreement with previous studies at the RNA level, COLs have been reported to be down-regulated upon Ad2 infection.31 Also, proteins that are involved in signaling pathways such as TGF-β, Rho, G-protein, Map kinase, STAT and NF-κB have been reported to be down-regulated at the RNA level.31 Such proteins were, however, not observed among the top downregulated proteins in this study. The down-regulation of growth factor receptors, generally involved in phosphorylation-dependent signaling, might contribute to the rather low level of phosphorylation observed very late after infection.32 Similar to the study by Evans et al.15 we also noted the down-regulation of integrin alpha 3 (ITGA3). Some integrins, however not ITGA3, are known to be hAd receptors.33 Interestingly, some integrin receptors such as ITGAV/ITGB3 and ITGAV/ITGB5 were downregulated in our studies. The down-regulation of hAd receptors is probably part of the cellular response to infection.

metabolism to conditions that are optimal for production of virus progeny. Still, host antivirus responses are not yet completely suppressed. To investigate the protein deregulation after infection the SILAC-MS technology was used. Efficient incorporation of heavy lysine and arginine was observed (97% heavy-labeled peptides) in the cells after five passages before infection. This high protein label efficiency reflects a complete labeling, which is recommended for quantitative proteomics.5 Cell infection was performed in biological replicates as swap experiments meaning that heavy and light labeled cells were Ad2 and mock infected, respectively. Proteins in cell lysates of differently labeled samples were mixed 1:1 and fractionated using SDS-PAGE to increase the number of identified and quantified proteins. The gel was then cut into 10 pieces that were subjected to in-gel enzymatic digestion and subsequent LC−MS/MS analysis (Figure 1) resulting in SILAC protein ratios of Ad2/mock and mock/Ad2. For comparison of both replicates at each time point we calculated reciprocal values of one replicate. Next, Pearson correlation (r) revealed good reproducibility of SILAC data at 24 hpi (r = 0.81) and 36 hpi (r = 0.79) as shown in Figure 2A and 2B. This demonstrates that our strategy is useable for accurate protein quantification to study the host cell protein expression during Ad2 infection. In total, 3507 cellular proteins were identified including 2648 and 2394 proteins, which were quantified in both labeling experiments at 24 and 36 hpi, respectively, at a false discovery rate of 1% (Supplemental Table S1). The number of identified and quantified proteins was in the expected range when compared to similar studies in HeLa cells.15 At 24 and 36 hpi, 27 and 28 viral proteins, respectively, were identified that correspond to 73−75% of the Ad2 proteome, demonstrating that an efficient virus infection has taken place. The complete data set includes screening experiments from three additional, earlier, time points. From these initial experiments, approximately 2−300 proteins were quantified (no in-gel fractionation was used), which was considered too few for a kinetic comparison between time points. It was, however, clear that the 24 and 36 hpi samples revealed the highest numbers of deregulated proteins, which motivates in depth study of these time points for the best chance to discover discordant mRNA and protein expression. Deregulation of Cellular Protein Expression

In agreement with other publications28,29 we used a 1.6-fold cutoff level for selection of differentially expressed proteins. With this criterion, 659 and 645 cellular proteins were differentially expressed at 24 and 36 hpi, respectively (Table 1), which corresponds to approximately 25% of all quantified proteins. Moreover, 86% and 83% of up- and down-regulated proteins at 24 hpi were sustained at 36 hpi as shown in Figure 2C. The high correlation (r = 0.96, Figure 2C) for the deregulated proteins at 876

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Journal of Proteome Research Table 2A. Top 50 Up-Regulated Proteins at 24 hpi Ad2 Infection rank

fasta headers

Uniprot

symbol

fold change proteina

RSD (%)

fold change RNA

groupb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Heat shock 70 kDa protein 1A/1B Heat shock 70 kDa protein 1A/1B Thymidylate synthase Creatine kinase B-type Nucleolar and coiled-body phosphoprotein 1 Probable RNA-binding protein EIF1AD Isoform 2 of Transcription factor BTF3 Fatty acid-binding protein, epidermal PCNA-associated factor DNA replication licensing factor MCM7 Uridine-cytidine kinase 2 Ubiquitin-conjugating enzyme E2 T Ribonucleoside-diphosphate reductase large subunit Isoform 2 of Ran-specific GTPase-activating protein PEST proteolytic signal-containing nuclear protein Cysteine and histidine-rich domain-containing protein 1 Isoform Short of Eukaryotic translation initiation factor 4H Amidophosphoribosyltransferase E3 ubiquitin-protein ligase UHRF1 Purine nucleoside phosphorylase Translationally controlled tumor protein Phosphatidylethanolamine-binding protein 1 Tubulin-specific chaperone A DNA replication licensing factor MCM3 Isoform 2 of Mini-chromosome maintenance complexbinding protein DNA replication licensing factor MCM6 Latexin Pyridoxal phosphate phosphatase Em:AP000351.3 protein BolA-like protein 2 BolA-like protein 2 Isoform 2 of Cyclin-dependent kinase 2 Ubiquitin-like protein 5 L-aminoadipate-semialdehyde dehydrogenasephosphopantetheinyl transferase Isoform 3 of Protein PRRC2C ACBP_Acyl-CoA-binding protein Thioredoxin BAG family molecular chaperone regulator 3 Thioredoxin domain-containing protein 17 von Willebrand factor A domain-containing protein 5A Isoform 2 of Glyoxalase domain-containing protein 4 DNA replication licensing factor MCM4 Hypoxanthine-guanine phosphoribosyltransferase YTH domain-containing family protein 2 Aldose reductase Nuclear fragile X mental retardation-interacting protein 2 Isoform 2 of CLIP-associating protein 1 Translation machinery-associated protein 7 SH3 domain-binding glutamic acid-rich-like protein Nicotinamide phosphoribosyltransferase

P08107 P08107 P04818 P12277 Q14978 E9PQD0 P20290−2 Q01469 Q15004 P33993 Q9BZX2 Q9NPD8 P23921 P43487−2 Q8WW12 Q9UHD1 Q15056−2 Q06203 Q96T88 P00491 P13693 P30086 E5RIW3 P25205 Q9BTE3−2

HSPA1B HSPA1A TYMS CKB NOLC1 EIF1AD BTF3 FABP5 KIAA0101 MCM7 UCK2 UBE2T RRM1 RANBP1 PCNP CHORDC1 EIF4H PPAT UHRF1 PNP TPT1 PEBP1 TBCA MCM3 MCMBP

8.2 8.2 6.5 5.7 5.7 5.6 5.2 5.0 4.4 4.3 4.1 4.1 4.0 3.9 3.8 3.7 3.7 3.7 3.6 3.4 3.4 3.3 3.3 3.2 3.2

2 2 29 12 87 18 47 42 11 10 28 29 19 6 17 13 7 9 14 3 14 5 4 19 21

2.1 1.6 11.9 11.8 6.7 3.8 2.3 7.1 33.5 14.3 2.0 13.4 12.3 8.5 1.7 4.4 2.0 4.3 12.7 5.2 1.1 2.4 2.6 20.0 4.1

UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUu UUn UUu UUu UUu UUu

Q14566 Q9BS40 Q96GD0 Q6ICJ4 Q9H3K6 Q9H3K6 P24941−2 Q9BZL1 Q9NRN7

MCM6 LXN PDXP GSTT2B BOLA2 BOLA2B CDK2 UBL5 AASDHPPT

3.2 3.2 3.1 3.1 3.1 3.1 3.1 3.1 3.0

22 2 51 4 6 6 9 28 21

6.7 −1.5 29.5 2.8 5.0 2.4 7.8 2.0 1.5

UUu UUn UUu UUu UUu UUu UUu UUu UUn

Q9Y520−3 P07108 P10599 O95817 Q9BRA2 O00534 Q9HC38−2 P33991 P00492 Q9Y5A9 P15121 Q7Z417 Q7Z460−2 Q9Y2S6 O75368 P43490

PRRC2C DBI TXN BAG3 TXNDC17 VWA5A GLOD4 MCM4 HPRT1 YTHDF2 AKR1B1 NUFIP2 CLASP1 TMA7 SH3BGRL NAMPT

3.0 3.0 3.0 3.0 2.9 2.9 2.9 2.9 2.9 2.9 2.9 2.8 2.8 2.8 2.8 2.8

1 4 9 9 16 60 8 29 13 9 9 47 28 27 17 19

−2.3 1.6 2.4 1.0 1.6 −1.5 3.6 14.3 4.4 2.2 1.3 2.5 −1.4 2.5 −1.2 1.1

UUd UUu UUu UUn UUu UUd UUu UUu UUu UUu UUn UUu UUn UUu UUn UUn

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

rank 36 hpi 1 2 4 10 c c

12 3 c

15 39 17 6 29 c

23 17 13 c

25 46 c

36 9 24 19 c c c

33 34 c c c

c c

30 c

45 c c

50 c

43 c c c c c c

Average ratio of the two SILAC experiments with swapped labeling. The first two capital letters designate the regulation observed in the two protein expression analyses, while the lower case indicates the change in RNA expression (U/u, up-regulated; D/d, down-regulated; and N/n, nonregulated). cProtein not among the top 50 up-regulated proteins at 36 h.

a

b

to the study. For up-regulated proteins, the most significant functional categories at 24 hpi were proteins involved in cellular carbohydrate and nucleoside metabolism. Cellular genes involved in carbohydrate metabolism, e.g., the hexose metabolism, were not reported among the most significantly deregulated

The biological functions of the deregulated proteins and their involvement in cellular pathways were revealed using the DAVID tool (Figure 3 and Supplemental Table S3). An additional bioinformatics tool, Ingenuity Pathway Analysis (IPA), was tested but did not provide additional information 877

DOI: 10.1021/acs.jproteome.6b00834 J. Proteome Res. 2017, 16, 872−888

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Journal of Proteome Research Table 2B. Top 50 Down-Regulated Proteins at 24 hpi Ad2 Infection rank

fasta headers

Uniprot

symbol

fold change proteina

RSD (%)

fold change RNA

groupb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Isoform 2 of CD166 antigen Chondroitin sulfate proteoglycan 4 Collagen alpha-1(I) chain Epidermal growth factor receptor Plasminogen activator inhibitor 1 Metalloproteinase inhibitor 3 Tissue factor Thrombospondin-1 Collagen alpha-2(I) chain Platelet-derived growth factor receptor beta Vasorin Integrin beta-5 Collagen alpha-1(XII) chain Cell surface glycoprotein MUC18 Isoform 2 of Leucine-rich repeat-containing protein 17 Histone H1.3 Insulin-like growth factor-binding protein 5 FH1/FH2 domain-containing protein 1 Trophoblast glycoprotein Neuropilin-1 Sodium-coupled neutral amino acid transporter 2 Cadherin-6 DNA topoisomerase 2-alpha Platelet-derived growth factor receptor alpha Collagen alpha-1(V) chain Fibronectin Basal cell adhesion molecule Isoform 3 of Collagen triple helix repeat-containing protein 1 Thyrotropin-releasing hormone-degrading ectoenzyme C-type mannose receptor 2 Connective tissue growth factor Isoform 5 of Growth arrest-specific protein 6 Integrin alpha-3 Isoform V1 of Versican core protein CD59 glycoprotein Integrin alpha-2 Endosialin Reversion-inducing cysteine-rich protein with Kazal motifs Isoform 3 of Tissue-type plasminogen activator Isoform Gamma of Poliovirus receptor Tenascin Isoform 3 of Integrin alpha-V Histone H1.5 Cadherin-13 Matrix metalloproteinase-14 Prolow-density lipoprotein receptor-related protein 1 Protein-lysine 6-oxidase Isoform 2 of EGF-like repeat and discoidin I-like domaincontaining protein 3 Transforming growth factor-beta-induced protein ig-h3 Isoform 2 of Laminin subunit alpha-4

Q13740−2 Q6UVK1 P02452 P00533 P05121 P35625 P13726 P07996 P08123 P09619 Q6EMK4 P18084 D6RGG3 P43121 Q8N6Y2−2 P16402 P24593 Q9Y613 Q13641 E9PEP6 Q96QD8 P55285 P11388 P16234 P20908 P02751 P50895 Q96CG8−3 Q9UKU6 Q9UBG0 P29279 Q14393−5 P26006 P13611−2 E9PNW4 P17301 Q9HCU0 O95980 P00750−3 P15151−3 P24821 P06756−3 P16401 P55290 P50281 Q07954 P28300 O43854−2

ALCAM CSPG4 COL1A1 EGFR SERPINE1 TIMP3 F3 THBS1 COL1A2 PDGFRB VASN ITGB5 COL12A1 MCAM LRRC17 HIST1H1D IGFBP5 FHOD1 TPBG NRP1 SLC38A2 CDH6 TOP2A PDGFRA COL5A1 FN1 BCAM CTHRC1 TRHDE MRC2 CTGF GAS6 ITGA3 VCAN CD59 ITGA2 CD248 RECK PLAT PVR TNC ITGAV HIST1H1B CDH13 MMP14 LRP1 LOX EDIL3

−6.1 −5.1 −4.8 −4.7 −4.7 −4.5 −4.1 −4.1 −4.0 −4.0 −3.8 −3.8 −3.5 −3.5 −3.5 −3.5 −3.3 −3.3 −3.2 −2.9 −2.9 −2.9 −2.8 −2.8 −2.8 −2.8 −2.8 −2.7 −2.7 −2.7 −2.6 −2.5 −2.5 −2.5 −2.5 −2.5 −2.5 −2.4 −2.4 −2.4 −2.3 −2.3 −2.3 −2.3 −2.3 −2.3 −2.2 −2.2

13 23 23 14 22 9 13 28 20 26 41 23 24 18 28 14 17 38 5 11 19 14 6 38 28 13 10 41 42 27 61 30 28 37 10 5