JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_cont_3_4_6ul.csvhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_2.csvhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_1.csvhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_cont_1_2_6ul.csvhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_infect_1_2_6ul.csvhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_3.csvhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_cont_3_4_6ul.rawhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_infect_3_4_6ul.rawhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_2.rawhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_1.rawhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_3.rawhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_infect_1_2_6ul.rawhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_cont_1_2_6ul.rawhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_2.mgfhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_cont_3_4_6ul.mgfhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_infect_3_4_6ul.mgfhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_cont_1_2_6ul.mgfhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_3.mgfhttps://storage.jpostdb.org/JPST001292/files/210323Elite_Ingel_infect_1_2_6ul.mgfhttps://storage.jpostdb.org/JPST001292/files/QE_CoV2_phostag_magnet_1.mgfprimaryOK200ProteomicsYayoi KimuraCellular Organismshttps://repository.jpostdb.org/entry/JPST001292Yokohama City UniversityjPOSTPhosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. SIGNIFICANCE: In this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model.Phosphopeptide enrichment using Phos-tag technology reveals functional phosphorylation of the nucleocapsid protein of SARS-CoV-2.Ino Yoko Y, Nishi Mayuko M, Yamaoka Yutaro Y, Miyakawa Kei K, Jeremiah Sundararaj Stanleyraj SS, Osada Makoto M, Kimura Yayoi Y, Ryo Akihide ACOVID19, SARS-CoV-2 Viruses, 2019 Novel Coronavirus, β-CoV, beta-CoV, protein complex, COVID-19 Viruses, nucleocapsid, Proteins, COVID-19, SARS-CoV-2, Gene, 2019 novel coronavirus, protein, Coronavirus 2, SARS CoV 2 Virus, protein-containing complex, COVID19 Virus, COVID19 Viruses, Severe Acute Respiratory Syndrome Coronavirus 2, Nucleocapsids, native protein, SARS, SARS-CoV-2 Virus, Protein, Novel Coronavirus, Gene Products, Virus, core, Coronavirus, β-coronavirus, protein aggregate, 2019-nCoV, SARS Coronavirus 2, β-CoVs, 2019 Novel, betacoronavirus, Viruses, Coronavirus Disease 2019 Virus, Wuhan Seafood Market Pneumonia Virus, 2019 Novel Coronaviruses., severe acute respiratory syndrome coronavirus 2, Wuhan, beta-CoVs, SARS-coronavirus 2, Protein Gene Products, Gene Proteins, COVID-19 Virus, 2019, COVID 19 Virus, Wuhan Coronavirusliquid chromatography tandem mass spectroscopy, cluster, Viridae, AIPC, viral infectious cycle, Host Microbial Interface, nucleocytoplasm, syndrome associated with disease or disorder, Microbe-Host Interactions, determination, Calf Serum, Host Virus Interactions, DELTA AND GAMMA, 2019 novel coronavirus, protein, Stage, betaTub1, Serum, Life History, Microbiota-Host Interaction, phosphorylation, Host-Virus, F6D5_2, 2-Amino-3-hydroxypropionic acid, Viral Host Interactions, Techniques, Background, B1t, Fetal Bovine Serum, Microbiota-Host Interactions, Virus Replications, "syndrome, PDZK3, Method, Coronaviruses, Bacterial Host Interactions, Cultural, F6D5.2, Novel Coronavirus, Virus, Rabbit Coronavirus, Life Cycle, symptom, Microbe Host Interaction, protein aggregate, Virus-Host Interaction, Host-Bacteria Interactions, A, ethnicity, Bacterial-Host Interactions, Cell Culture Technique., LCMSMS, kus, beta1tub, CG9277, Determination, entire life cycle, Host-Bacteria, Sequence Homology Determination, T, beta-particle, Host Microbial Interaction, Microbiota Host Interactions, beta-CoVs, Viral Gene Products, Virus-Host Interactions, Viral-Host Interactions, Bacterial-Host Interaction, 1t, e, Viral Replications, Methodological Studies, 2-amino-3-hydroxypropanoic acid, e-, beta1Tub, Vira, Fetal, syndromic disease, Virus Host Interaction, Symptom Clusters, Host Microbe Interaction, Host-Microbe Interaction, Microbe Host Interactions, Alignments, accepteur, LC-MS2, Sequence Homology Determinations, ARABIDOPSIS THALIANA PIN-FORMED 1, SARS-CoV-2 Viruses, BETA 56D, Viral, β-CoV, Host Microbe Interactions, COVID-19 Viruses, Arts, LC-MS/MS, UBL5, SARS-CoV-2, Bovine Albumin, Coronavirus 2, Clusters, Host Fungal Interactions, Procedure, not genetically inherited, results, Determinations, ubl5, sigma virus replication, COVID19 Viruses, Cultural Background, Interaction, and release, symptom cluster, l(1)G0108, Cultures, 3-Hydroxyalanine, SARS-CoV-2 Virus, Fetal Calf Serum, Coronavirus, FLOWERING TIME CONTROL PROTEIN FCA ALPHA, internal to cell, region, Host-Virus Interaction, Microbial-Host Interactions, Culture Technique, Fetal Calf, Hydrogen-acceptor, Viral Replication, beta-tub, Industrial, Industrial Arts, Beta <eudicots>, Host-Fungal Interaction, Cultural Beliefs, Coronavirus Disease 2019 Virus, life, Life Cycle Stage, Sequence Homology, History Stage, FCAALL.331, modulation by virus of host anatomical structure or process, Host-Bacteria Interaction, lytic viral life cycle, Methodological, beta56D, DmelCG9277, Methodological Study, Bovine Serum, clusters, beta1-tub, beta(-), DTB2, Life Cycles, Acceptor, beta-Tub, Host-Microbial Interface, Replications, Syndromes, Cluster, liquid chromatography-tandem mass spectroscopy, i6, DL4180C, 2019, Cell Cultures, Viral Protein, Animal Viruses, L Serine, Microbial Host Interaction, beta-Tub56D, L-Serine, betaTub, Specbeta, beta1-Tubulin, pin1a, bis-bisPMAP, PIN-FORMED 1, Host Microbiota Interaction, COVID19, lifespan, Akzeptor, pin1, Procedures, Host-Microbial Interactions, nucleocapsid, COVID-19, e(-), Alignment, Gene, electron, DOD, SARS CoV 2 Virus, protein-containing complex, syndromic disease or disorder, COVID19 Virus, LC-MS-MS, protein amino acid phosphorylation, method, Bacterium-Host Interactions, Host-Microbe, SARS, B-spec, betaspec, method used in an experiment, Cycle, "syndrome" EXACT [MTH:NOCODE], LC-MSMS, Host-Fungal Interactions, Gene Products, Studies, symptom clusters, viral replication, Bacterium Host Interactions, Animal, β-coronavirus, CG17051, Life, dod, Microbe-Host, Cell Culture, Technique, 2019-nCoV, Backgrounds, 2019 Novel Coronaviruses, protoplasm, Cycles, study, Microbial-Host Interaction, Animal Virus, Host Bacteria Interactions, SARS Coronavirus 2, β-CoVs, betatub(56D), Rabbit, protoplast, Viruses, entire lifespan, ligand, Elektron, egress, Wuhan Seafood Market Pneumonia Virus, beta[[1]] tubulin, modulation by virus of host morphology or physiology, beta1t, D9Bwg1161e, syndrome, severe acute respiratory syndrome coronavirus 2, Cultural Relativisms, Microbial-Host, Bacterium-Host Interaction, syndromes, Oxidized donor, Life History Stages, Symptom, Host-Virus Interactions, Cell Culture Technique, anon-EST:fe1B3, Microbiota Host Interaction, Study, PAPIN, COVID-19 Virus, Bovine, NOS" EXACT [SNOMEDCT_2005_07_31:64572001], Stages, Life History Stage, SPEC8, Host Bacteria Interaction, Bacteria Host Interactions, Customs, site, Albumin Bovine, culture, CG5870, Pin1, PIN1, Wuhan Coronavirus, beta-tubulin56D, Spec-beta, Dmbeta1, Culture Techniques, 2019 Novel Coronavirus, beta-CoV, PTF1, Rabbit Coronaviruses, protein complex, Host Microbiota Interactions, Proteins, Phosphorylations, maturation, Tubulin, 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Gene Products, Gene Proteins, COVID-19 Virus, 2019, COVID 19 Virus, Wuhan Coronavirus, bis-bisPMAPCOVID19, SARS-CoV-2 Viruses, 2019 Novel Coronavirus, β-CoV, beta-CoV, COVID-19 Viruses, Arts, COVID-19, Phosphorylations, SARS-CoV-2, 2019 novel coronavirus, Coronavirus 2, SARS CoV 2 Virus, Phosphopeptide., phosphorylation, COVID19 Virus, COVID19 Viruses, Severe Acute Respiratory Syndrome Coronavirus 2, SARS, SARS-CoV-2 Virus, Novel Coronavirus, Virus, Coronavirus, β-coronavirus, Animal, Zoophaginae, 2019-nCoV, 2019 Novel Coronaviruses, Animal Virus, SARS Coronavirus 2, Industrial, β-CoVs, 2019 Novel, betacoronavirus, Industrial Arts, Viruses, Nucleoprotein, phos-tag, Coronavirus Disease 2019 Virus, Wuhan Seafood Market Pneumonia Virus, severe acute respiratory syndrome coronavirus 2, Wuhan, beta-CoVs, SARS-coronavirus 2, COVID-19 Virus, 2019, Animal Viruses, COVID 19 Virus, Wuhan Coronavirus, bis-bisPMAPfalsePhosphoproteomics of nucleocapsid protein of SARS-CoV-2We aimed to identify the functional phosphorylation of SARS-CoV-2 nucleoprotein. For this purpose, we used the Phos-tag technology to purify and enrich virus-derived phosphopeptides. Fri Jan 28 00:00:00 GMT 2022PXD02793913156735093569