Quantitative Analysis of Lysine Acetylation in Vero Cells Infected With Peste Des Petits Ruminants Virus

: 8 Background: Peste des petits ruminants virus (PPRV) is a negative-stranded RNA virus belonging to 9 the Paramyxoviridae family and causes acute, highly contagious disease in small ruminants. Lysine 10 acetylation plays central role in regulating gene expression. However, the extent and function of 11 lysine acetylation in host cells during PPRV infection remains unknown. 12 Methods: Lysine acetylation of PPRV-infected Vero cells was tested and differentially expressed 13 lysine acetylation was found. The acetylated peptides were enriched using specific antibody and 14 labeled with demethylation. Proteins with acetylation sites were identified. Subsequently, intensive 15 bioinformatics analysis of succinylome of PPRV-infected Vero cells was were performed. 16 In this study, intensive proteomic quantification analysis of the proteome and acetylome of 17 PPRV-infected Vero cells was performed using dimethylation labeling-based quantitative proteomics. 18 Results: We identified 4729 cellular proteins and 1068 proteins with 2641 modification sites 19 quantifiable detected by mass spectrometry, of which 304 proteins with 410 acetylation sites were 20 significantly acetylated in response to PPRV infection. Bioinformatics analyses revealed that the differentially acetylated proteins mainly participated in carbohydrate catabolic and DNA metabolic 1 process, and were associated with multifarious functions, suggesting that intracellular activities were 2 extensively changed after PPRV infection. Protein-protein interaction (PPI) network of the identified 3 proteins further indicated that a variety of chaperone and ribosome processes were modulated by 4 acetylation. 5 Conclusions: To our knowledge, this is the first study on acetylome in host cell infected with PPRV. 6 It provides an important baseline to future study the roles of acetylation in the host response to PPRV 7 replication. 8

The interaction between host and virus is a complex dynamic competitive process. As a kind of 1 obligate intracellular parasites, viruses depend on their ability to "hijack" host cellular functions to 2 facilitate their replication and inhibit host antiviral defenses. On the contrary, in order to maintain 3 normal physiological functions, the host utilizes the nonspecific and specific immune based antiviral 4 responses to resist viral invasion, inhibit virus replications, or eliminate virus particles. In previous 5 studies, host cellular response was deciphered by clustered regularly interspaced short palindromic 6 repeat (CRISPR) [4-6] small interfering RNA (siRNA) [7] and transcriptomic and proteomic 7 analyses [8]. 8 Today, it is well known that protein post-translational modifications (PTMs) affect significant diverse 9 functions of proteins via modulating biological processes, protein activity, cellular location and 10 protein-protein interaction (PPI) by transferring modified groups to one or more amino acid residues. 11 To date, more than 450 protein modifications including over 200 PTMs have been identified [9] to be 12 dynamic and reversible protein processing events and play key roles in the response to the 13 pathogenesis and development of diseases [10][11][12][13]. PTMs have become a hot topic in viral infection.
14 Some PTMs including phosphorylation, acetylation and succinylation have been shown to potently 15 regulate innate immunity and inflammation in response to viruses infection [14,15]. 16 Of the 20 amino acid residues, lysine is one of the most frequent targets of covalent modifications 17 because it can accept different types of chemical groups [16][17][18][19][20][21]. Among the lysine PTMs, lysine 18 acetylation is widespread and one of the most well-studied PTM in both prokaryotes and eukaryotes 19 [9,[22][23][24][25]. Lysine acetylation is highly conserved in organisms ranging from bacteria to human and 20 is particularly important [23,24,26,27]. Lysine acetylation impacts protein functions in multiple 21 cellular processes including enzyme activity, chromatin structure, localization and PPI. [28] 22 Accumulating evidence highlights t h at lysine acetylation is an important molecular toggle of 23 protein function [22,[29][30][31] and is a key regulatory point in mechanisms of both host antiviral 24 response and virus replication [28,32,33]. However, the extent and function of lysine acetylation 25 in host cells during PPRV infection have not yet been reported. 1 In this study, we investigated the acetylome in Vero cells (an African green monkey kidney cell line) 2 infected with PPRV. By combining dimethylation labeling, HPLC fractionation and antibody-affinity   Protein extraction 24 The harvested cell samples were washed twice with cold phosphate-buffered saline (PBS). Then, 25 each sample was sonicated on ice in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail, 3 μM 1 TSA, 50 mM NAM and 2 mM EDTA). The resulting supernatants were centrifuged with 12,000 rpm 2 for 10 min at 4 °C to remove the cell debris. The protein concentration was determined with BCA kit 3 according to the manufacturer's instructions.

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Trypsin digestion and dimethylation labeling 5 The protein solution was reduced with 5 mM dithiothreitol for 30 min at 56 °C and subsequently 6 alkylated with 11 mM iodoacetamide for 15 min at room temperature in dark. For tryptic digestion, 7 the protein samples were diluted with urea concentration of less than 2M by adding 100 mM 8 NH4CO3. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion 9 overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion at 37 °C. Then, peptide 10 was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide samples were   The enriched peptides were dissolved in solvent A (0.1% Formic Acid in 2% acetonitrile (ACN)), 3 directly loaded onto a home-made reversed-phase analytical column (1.9 μm particles, 120 A pore, 4 15-cm length, 75 μm i.d.). The gradient included an increase from 9% to 25% solvent B (0.1% 5 Formic Acid in 90% ACN) for 24 min, followed from 25% to 40% for 10 min, and reaching to 80% 6 in 3 min, then maintained at 80% for the last 3 min on an EASY-nLC 1000 UPLC (Ultra 7 Performance Liquid Chromatography) system at a constant flow rate of 700 nL/min. 8 The resulting peptides were ionized and subjected to tandem mass spectrometry (MS/MS) in Q 9 Exactive™ Plus (Thermo Scientific) coupled online to the UPLC using NanoSpray Ionization (NSI) 10 source. The electrospray voltage applied was 2.0 kV. Intact peptides were detected at a resolution of   Carbamido-methylation of cysteine (Cys) was specified as fixed modification, and oxidation on 24 methionine, acetylation on protein N-terminal and acetylation on lysine were specified as variable 25 modifications. The false discovery rate (FDR) and the minimum score for modified peptide were set 1 at <1% and >40, respectively. the minimum peptide length was set at 7. All other parameters in 2 MaxQuant were set to default values. Genes and Genomes (KEGG) database (http://www. genome.jp/kegg/) was used to annotate and map 10 the pathways. GO, protein domain and KEGG pathway enrichment analysis were performed using 11 the DAVID bioinformatics resources 6.8. Wolfpsort (https://wolfpsort.hgc.jp/), a subcellular 12 localization predication software was used to predict subcellular localization. Amino acid sequence 13 motifs (within ± 10 residues of the acetylated or succinylated sites) were analyzed by motif-X.
14 Motif-based clustering analyses were also performed, and cluster membership was visualized using a 15 heat map. Functional interaction network analysis was performed using the STRING database 16 (v.11.0), with a high confidence threshold of 0.7, and visualized by Cytoscape 3.7.1.

Basic information on quantitative proteomic analysis of PPRV-infected Vero cells 19
To explore and identify the acetylated host proteins or pathways involved in the PPRV replication 20 process, we chose 24 h p.i. as the time point for quantitative proteomic analysis according the result 21 of western blotting (Additional file 1: Figure S1), and conducted a comprehensive proteome analysis 22 to detect acetylated proteins between the infected and control samples. To assess repeatability among 23 the 3 biological replicates, Pearson's correlation coefficients were generated and calculated between 24 two samples, separately, and repeatability ranged from 0.78 to 0.81 for the global proteome ( Fig.   25 1A). 1 The distribution of mass error was close to zero and most of them were less than 5 ppm (Fig. 1B).
2 Consistent with the characteristics of tryptic peptides, most peptides were in the size range from 7 to 3 21 amino acids (Fig. 1C). In total, 5596 proteins were identified by LC-MS/MS, of which 4729 4 could be quantified. Based on the infection/control ratio, differential expression analysis revealed 5 that 65 proteins were found up-regulated and 619 were down-regulated in infected group 6 respectively (fold-change >1.5, P <0.05) (Fig. 2, Additional file 2: Table S1). To elucidate the 7 function and distribution of differentially abundant proteins (80%) in Vero cells response to PPRV 8 infection, GO classification, protein domain, protein annotation, and subcellular localization analyses 9 were performed. The results showed that most proteins located in the cytoplasm and nucleus, and 10 mainly involved in binding, catalytic activity, cellular and metabolic processes. Detail information 11 was shown in Fig. 3, Figure S2 and Table S2.  Global detection of lysine acetylated sites on PPRV-infected Vero cellular proteins 7 Lysine acetylation can alter the structure or function of proteins involved in diverse biological 8 processes. To obtain a comprehensive view of the level of protein acetylation in PPRV-infected Vero 9 cell, enriched analysis of acetylated peptides was performed to identify the proteins with differential 1 acetylated sites using LC-MS/MS. The near-zero distribution of mass error and that the errors were 2 predominantly < 0.02 Da, indicated a high degree of accuracy for identification of the modified 3 peptides (Additional file 1: Figure S3A). Most of the enriched lysine-acetylated peptide lengths were 4 in the range of 7-21 segments, which are consistent with cutting by trypsin at lysine residue sites 5 (Additional file 1: FigureS2B). Repeatability among the 3 biological replicates ranged from 0.56 to 6 0.61 for the acetyl-proteome ( Additional file 1: Figure S3C). 7 In total, 3229 lysine-acetylated sites belonging to 1315 proteins were identified, of which 2641 8 modification sites in 1068 proteins were quantifiable (Additional file 2: Table S3). Among these 9 proteins, about 686 (52.16%) included a single acetylation site, 267 (20.30%) included two sites, 124 10 (9.43%) included three sites, 72 (5.48%) included four sites, and 166 (12.62%) included five or more 11 than five sites (Fig. 4). 107 lysine acetylation sites were found on histone proteins, including 13 sites 12 on H1, 12 sites on H2A, 13 sites on H2B, 9 sites on H3 and 8 sites on H4. These results provide a 13 comprehensive overview of the acetylation events in PPRV-infected Vero cell. HspA5, HspA9, Hsp90AB1 and Hsp90B1, were acetylated at 8, 3, 2, 3 and 3 sites, respectively. Two 3 different sites were acetylated in six paralogous subunits of chaperonin TRiC (also called CCT). * The differentially acetylated sites and proteins were quantified in the 3 biological replicates.

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Functional, subcellular localization and COG classification of differential acetylated proteins 7 To better understand the potential functions of lysine acetylation in PPRV-infected cells, all DAcPs 8 were classified by GO functional classification analysis based on their biological process, molecular 9 function, subcellular localization and COG/KOG categories (Fig. 5, Additional file 2: Table S5).  domain analysis were performed for all the identified proteins with differential acetylated sites ( Fig.   9 7, Additional file 2: Table S6). 10 The GO enrichment result of acetylated proteins was showed in Fig. 7A. The cellular components 1 mainly enriched in MCM complex. In the molecular function category, unfolded protein binding and 2 helicase activity were found to be significantly enriched (Fig. 7A). The KEGG database was used to 3 identify the pathway involved in these DAcPs. Interestingly, there were only two identified enriched 4 processes (single-organism carbohydrate catabolic process and DNA metabolic process) (Fig. 7A). 5 These acetylated proteins were involved in protein processing in endoplasmic reticulum (ER) (Fig.   6 7B). 17 of the DAcPs were involved in protein processing in ER, and 12 of the 17 proteins were

Protein-protein interaction network analysis of differentially acetylated proteins 14
To better understand how lysine acetylation regulates diverse metabolic processes and cellular 15 functions, we assembled the PPI networks of the identified modified proteins. In total, 147 DAcPs 16 were mapped to the protein network database. The global network graph of these interactions was 17 shown in Fig. 8 and Table S7. As indicated in Fig. 8, five highly connected subnetworks, ribosome,   worthy to make an intensive investigation to explore the precise biological function of these 9 acetylated proteins in PPRV infection.

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Quantitative proteomics was carried out to profile the acetylome of PPRV-infected Vero cells. We    Enrichment analysis of proteins related to PPRV in Vero cell.

Figure 4
Pro le of identi ed acetylated sites and proteins in PPRV-infected Vero cell   Representative PPI networks of acetylated proteins