Quantitative Analysis of Lysine Acetylation in Vero Cells Infected With Peste Des Petits Ruminants Virus Xuelian Meng ( [email protected] ) Lanzhou Veterinary Research Institute Xueliang Zhu Lanzhou Veterinary Research Institute Rui Zhang Lanzhou Veterinary Research Institute Zhidong Zhang Lanzhou Veterinary Research Institute Research Keywords: Post-translational modication, Acetylation, Protein-protein interaction, Peste des petits ruminants Posted Date: June 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-560607/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 Title page 2 3 The accession number: 4 5 6 Quantitative analysis of lysine acetylation in Vero cells infected 7 with peste des petits ruminants virus 8 Xuelian Meng*, Xueliang Zhu, Rui Zhang, Zhidong Zhang* 9 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of 10 Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural 11 Sciences, Xujiaping 1, Yanchangpu, Chengguan District, Lanzhou 730046, China 12 13 14 Xuelian Meng: [email protected] 15 Xueliang Zhu: [email protected] 16 Rui Zhang: [email protected] 17 Zhidong Zhang: [email protected] 18 19 *Correspondence: Xuelian Meng; [email protected]; Tel.: +86-0931-8374122 20 Zhidong Zhang; [email protected] 21 22 1 1 Quantitative analysis of lysine acetylation in Vero cells infected 2 with peste des petits ruminants virus 1 3 Xuelian Meng*, Xueliang Zhu, Rui Zhang, Zhidong Zhang* 4 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of 5 Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural 6 Sciences, Xujiaping 1, Yanchangpu, Chengguan District, Lanzhou 730046, China 7 8 Abstract: 9 Background:Peste des petits ruminants virus (PPRV) is a negative-stranded RNA virus belonging to 10 the Paramyxoviridae family and causes acute, highly contagious disease in small ruminants. Lysine 11 acetylation plays central role in regulating gene expression. However, the extent and function of 12 lysine acetylation in host cells during PPRV infection remains unknown. 13 Methods: Lysine acetylation of PPRV-infected Vero cells was tested and differentially expressed 14 lysine acetylation was found. The acetylated peptides were enriched using specific antibody and 15 labeled with demethylation. Proteins with acetylation sites were identified. Subsequently, intensive 16 bioinformatics analysis of succinylome of PPRV-infected Vero cells was were performed. 17 In this study, intensive proteomic quantification analysis of the proteome and acetylome of 18 PPRV-infected Vero cells was performed using dimethylation labeling-based quantitative proteomics. 19 Results: We identified 4729 cellular proteins and 1068 proteins with 2641 modification sites 20 quantifiable detected by mass spectrometry, of which 304 proteins with 410 acetylation sites were 21 significantly acetylated in response to PPRV infection. Bioinformatics analyses revealed that the Xuelian Meng, Xueliang Zhu, Rui Zhang, Zhidong Zhang State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Yanchangpu, Chengguan District, Lanzhou 730046, China E-mail addresses: [email protected] (X. Meng), [email protected] (Z. Zhang) 2 1 differentially acetylated proteins mainly participated in carbohydrate catabolic and DNA metabolic 2 process, and were associated with multifarious functions, suggesting that intracellular activities were 3 extensively changed after PPRV infection. Protein-protein interaction (PPI) network of the identified 4 proteins further indicated that a variety of chaperone and ribosome processes were modulated by 5 acetylation. 6 Conclusions: To our knowledge, this is the first study on acetylome in host cell infected with PPRV. 7 It provides an important baseline to future study the roles of acetylation in the host response to PPRV 8 replication. 9 Keywords: Post-translational modification, Acetylation, Protein-protein interaction; Peste des petits 10 ruminants 11 12 Background 13 Peste des petits ruminants virus (PPRV), the causative agent of Peste des petits ruminants (PPR), 14 belongs to the genus Morbillivirus in the family Paramyxoviridae, which also includes measles 15 virus (MV), rinderpest virus (RPV), canine distemper virus (CDV), marine mammalian 16 morbilliviruses ( phocine distemper virus (PDV), porpoise morbillivirus (PMV), dolphin 17 morbillivirus (DMV)) and feline morbillivirus (FeMV) [1]. PPRV has a negative, non-segmented 18 single-stranded genomic RNA, encoding six structural proteins ( nucleocapsid protein, N; 19 phosphoprotein, P; matrix protein, M; fusion protein, F; haemagglutinin protein, H; and large 20 polymerase protein, L) in the 3’ to 5’ direction and two non-structural proteins (V and C proteins). 21 PPRV was first reported in the Ivory Coast, West Africa, in 1942 and is currently endemic in Africa, 22 the Middle East and Asia affecting global trade and causing significant economic losses [2]. 23 Following the successful eradication of rinderpest, the World Organization of Animal Health (OIE) 24 and the Food and Agriculture Organization (FAO) have proposed a plan for global eradication of PPR 25 by 2030 [3]. 3 1 The interaction between host and virus is a complex dynamic competitive process. As a kind of 2 obligate intracellular parasites, viruses depend on their ability to "hijack" host cellular functions to 3 facilitate their replication and inhibit host antiviral defenses. On the contrary, in order to maintain 4 normal physiological functions, the host utilizes the nonspecific and specific immune based antiviral 5 responses to resist viral invasion, inhibit virus replications, or eliminate virus particles. In previous 6 studies, host cellular response was deciphered by clustered regularly interspaced short palindromic 7 repeat (CRISPR) [4-6] small interfering RNA (siRNA) [7] and transcriptomic and proteomic 8 analyses [8]. 9 Today, it is well known that protein post-translational modifications (PTMs) affect significant diverse 10 functions of proteins via modulating biological processes, protein activity, cellular location and 11 protein-protein interaction (PPI) by transferring modified groups to one or more amino acid residues. 12 To date, more than 450 protein modifications including over 200 PTMs have been identified [9] to be 13 dynamic and reversible protein processing events and play key roles in the response to the 14 pathogenesis and development of diseases [10-13]. PTMs have become a hot topic in viral infection. 15 Some PTMs including phosphorylation, acetylation and succinylation have been shown to potently 16 regulate innate immunity and inflammation in response to viruses infection [14, 15]. 17 Of the 20 amino acid residues, lysine is one of the most frequent targets of covalent modifications 18 because it can accept di erent types of chemical groups [16-21]. Among the lysine PTMs, lysine 19 acetylation is widespreadff and one of the most well-studied PTM in both prokaryotes and eukaryotes 20 [9, 22-25]. Lysine acetylation is highly conserved in organisms ranging from bacteria to human and 21 is particularly important [23, 24, 26, 27]. Lysine acetylation impacts protein functions in multiple 22 cellular processes including enzyme activity, chromatin structure, localization and PPI.[28] 23 Accumulating evidence highlights that lysine acetylation is an important molecular toggle of 24 protein function [22, 29-31] and is a key regulatory point in mechanisms of both host antiviral 25 response and virus replication [28, 32, 33]. However, the extent and function of lysine acetylation 4 1 in host cells during PPRV infection have not yet been reported. 2 In this study, we investigated the acetylome in Vero cells (an African green monkey kidney cell line) 3 infected with PPRV. By combining dimethylation labeling, HPLC fractionation and antibody-affinity 4 enrichment with LC-MS/MS analysis, we systematically analyzed the quantitative comparison of the 5 global proteome and acetylome in Vero cells with or without PPRV infection, and calculated the 6 regularity of sequence features around the acetylated sites. We successfully quantified 4729 proteins 7 and 2641 lysine acetylation sites in 1068 proteins with diverse molecular functions, biological 8 processes and subcellular localizations. Altogether, the results provided the first extensive dataset on 9 lysine acetylation in Vero cells infected with PPRV, and novel insights into the infection mechanism 10 of PPRV. 11 Methods 12 Cell, virus and infection 13 Vero cells were maintained in authors’ laboratory, and cultured in DMEM medium (Sigma Aldrich, 14 St Louis, MO, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 15 μg/ml streptomycin at 37 °C in 5% CO2 incubator. The PPRV vaccine strain Nigeria 75/1 was 16 cultured in our laboratory and passaged in Vero cells as previously described [34]. Vero cells were 17 infected with PPRV at 1 MOI of or mock-infected with phosphate-buffered saline (PBS, 0.01M, 18 pH7.4) at 37°C for 1 h. The MOI was confirmed according to the viral titers of Vero cell line. After 19 adsorption, the virus inoculum was removed, and the fresh medium was added to wells and 20 incubated. In order to determine the sampling time point, the cells were