Identification of Lysine Acetylation Sites on MERS-CoV Replicase pp1ab

MERS is a life-threatening disease and MERS-CoV has the potential to cause the next pandemic. Protein acetylation is known to play a crucial role in host response to viral infection. Acetylation of viral proteins encoded by other RNA viruses have been reported to affect viral replication. It is therefore of interest to see whether MERS-CoV proteins are also acetylated. Viral proteins obtained from infected cells were trypsin-digested into peptides. Acetylated peptides were enriched by immunoprecipitation and subject to nano-LC-Orbitrap analysis. Bioinformatic analysis was performed to assess the conservation level of identified acetylation sites and to predict the upstream regulatory factors. A total of 12 acetylation sites were identified from 7 peptides, which all belong to the replicase polyprotein pp1ab. All identified acetylation sites were found to be highly conserved across MERS-CoV sequences in NCBI database. Upstream factors, including deacetylases of the SIRT1 and HDAC families as well as acetyltransferases of the TIP60 family, were predicted to be responsible for regulating the acetylation events identified. Western blotting confirms that acetylation events indeed occur on pp1ab protein by expressing NSP4 in HEK293 cells. Acetylation events on MERS-CoV viral protein pp1ab were identified for the first time, which indicate that MERS-CoV might use the host acetylation machinery to regulate its enzyme activity and to achieve optimal replication. Upstream factors were predicted, which might facilitate further analysis of the regulatory mechanism of MERS-CoV replication. MERS is a life-threatening disease and MERS-CoV has the potential to cause the next pandemic. Protein acetylation is known to play a crucial role in host response to viral infection. Acetylation of viral proteins encoded by other RNA viruses have been reported to affect viral replication. It is therefore of interest to see whether MERS-CoV proteins are also acetylated. Viral proteins obtained from infected cells were trypsin-digested into peptides. Acetylated peptides were enriched by immunoprecipitation and subject to nano-LC-Orbitrap analysis. Bioinformatic analysis was performed to assess the conservation level of identified acetylation sites and to predict the upstream regulatory factors. A total of 12 acetylation sites were identified from 7 peptides, which all belong to the replicase polyprotein pp1ab. All identified acetylation sites were found to be highly conserved across MERS-CoV sequences in NCBI database. Upstream factors, including deacetylases of the SIRT1 and HDAC families as well as acetyltransferases of the TIP60 family, were predicted to be responsible for regulating the acetylation events identified. Western blotting confirms that acetylation events indeed occur on pp1ab protein by expressing NSP4 in HEK293 cells. Acetylation events on MERS-CoV viral protein pp1ab were identified for the first time, which indicate that MERS-CoV might use the host acetylation machinery to regulate its enzyme activity and to achieve optimal replication. Upstream factors were predicted, which might facilitate further analysis of the regulatory mechanism of MERS-CoV replication. First isolated in 2012 (1Zaki A.M. van Boheemen S. Bestebroer T.M. Osterhaus A.D. Fouchier R.A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.N. Engl. J. Med. 2012; 367: 1814-1820Crossref PubMed Scopus (3850) Google Scholar), Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus leading to severe and highly lethal respiratory diseases. According to WHO, MERS-CoV has a high case fatality of over 35% among more than 2500 cases reported from 27 countries worldwide (2Dawson P. Malik M.R. Parvez F. Morse S.S. What Have We Learned About Middle East Respiratory Syndrome Coronavirus Emergence in Humans? A Systematic Literature Review.Vector-Borne Zoonot. 2019; 19: 174-192Crossref PubMed Scopus (36) Google Scholar). MERS-CoV is a potential pandemic agent as person-to-person transmission has been observed, particularly within health care settings (3Oboho I.K. Tomczyk S.M. Al-Asmari A.M. Banjar A.A. Al-Mugti H. Aloraini M.S. Alkhaldi K.Z. Almohammadi E.L. Alraddadi B.M. Gerber S.I. Swerdlow D.L. Watson J.T. Madani T.A. 2014 MERS-CoV outbreak in Jeddah–a link to health care facilities.N. Engl. J. Med. 2015; 372: 846-854Crossref PubMed Scopus (347) Google Scholar). Therapeutic options are limited and unspecific (4Chan J.F.W. Lau S.K.P. To K.K.W. Cheng V.C.C. Woo P.C.Y. Yuen K.Y. Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease.Clin. Microbiol. Rev. 2015; 28: 465-522Crossref PubMed Scopus (610) Google Scholar). More importantly, unlike the epidemic of SARS-CoV that quickly faded off, the MERS epidemic has persisted over years with no signs of subsiding (5Woo P.C. Lau S.K. Yuen K.Y. Infectious diseases emerging from Chinese wet-markets: zoonotic origins of severe respiratory viral infections.Curr. Opin. Infect. Dis. 2006; 19: 401-407Crossref PubMed Scopus (128) Google Scholar). It is currently known that the clinical outcome might be dictated by the abilities of the virus to infect a wide range of DPP4-expressing cells, to induce dysregulation of cytokines and to evade host innate immune response (4Chan J.F.W. Lau S.K.P. To K.K.W. Cheng V.C.C. Woo P.C.Y. Yuen K.Y. Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease.Clin. Microbiol. Rev. 2015; 28: 465-522Crossref PubMed Scopus (610) Google Scholar). However, detailed molecular mechanisms for transmission and virus-host interaction remain poorly understood. Post-translational modifications (PTMs) generally refer to reversible addition of a functional group covalently to specific amino acid residue(s) on a protein (6Aebersold R. Agar J.N. Amster I.J. Baker M.S. Bertozzi C.R. Boja E.S. Costello C.E. Cravatt B.F. Fenselau C. Garcia B.A. Ge Y. Gunawardena J. Hendrickson R.C. Hergenrother P.J. Huber C.G. Ivanov A.R. Jensen O.N. Jewett M.C. Kelleher N.L. Kiessling L.L. Krogan N.J. Larsen M.R. Loo J.A. Loo R.R.O. Lundberg E. MacCoss M.J. Mallick P. Mootha V.K. Mrksich M. Muir T.W. Patrie S.M. Pesavento J.J. Pitteri S.J. Rodriguez H. Saghatelian A. Sandoval W. Schluter H. Sechi S. Slavoff S.A. Smith L.M. Snyder M.P. Thomas P.M. Uhlen M. Van Eyk J.E. Vidal M. Walt D.R. White F.M. Williams E.R. Wohlschlager T. Wysocki V.H. Yates N.A. Young N.L. Zhang B. How many human proteoforms are there?.Nat. Chem. Biol. 2018; 14: 206-214Crossref PubMed Scopus (379) Google Scholar). PTMs provide additional levels of regulation that could respond simultaneously to external stimuli. Among more than 400 different PTMs discovered so far, lysine acetylation is one of the most crucial types. Protein acetylation can control protein location, stability, and enzymatic activity (7Verdin E. Ott M. 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond.Nat. Rev. Mol. Cell Bio. 2015; 16: 258-264Crossref PubMed Scopus (523) Google Scholar). Particularly, accumulating evidence suggests that acetylation serves crucial roles in regulating both host response to viral infection and viral replication process (8Murray L.A. Sheng X. Cristea I.M. Orchestration of protein acetylation as a toggle for cellular defense and virus replication.Nat. Communications. 2018; 9: 4967Crossref PubMed Scopus (32) Google Scholar). Transcriptional activity of NF-κB complex is regulated by acetylation on its subunits (9Tummers B. Goedemans R. Pelascini L.P.L. Jordanova E.S. van Esch E.M.G. Meyers C. Melief C.J.M. Boer J.M. van der Burg S.H. The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NF kappa B activation.Nat. Communications. 2015; 6: 6537Crossref PubMed Scopus (50) Google Scholar). Viral DNA sensor IFI16 requires acetylation to translocate to the nucleus to initiate innate immune response (10Li T. Diner B.A. Chen J. Cristea I.M. Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 10558-10563Crossref PubMed Scopus (233) Google Scholar). Viruses can perturb host anti-viral response by inhibiting p53 acetylation (11Munoz-Fontela C. Gonzalez D. Marcos-Villar L. Campagna M. Gallego P. Gonzalez-Santamaria J. Herranz D. Gu W. Serrano M. Aaronson S.A. Rivas C. Acetylation is indispensable for p53 antiviral activity.Cell Cycle. 2011; 10: 3701-3705Crossref PubMed Scopus (39) Google Scholar). Emerging evidence shows that viral proteins are subject to acetylation by host factors. Acetylation of HIV-1 transactivator Tat is required for its transcriptional activity (12Kiernan R.E. Vanhulle C. Schiltz L. Adam E. Xiao H. Maudoux F. Calomme C. Burny A. Nakatani Y. Jeang K.T. Benkirane M. Van Lint C. HIV-1 Tat transcriptional activity is regulated by acetylation.EMBO J. 1999; 18: 6106-6118Crossref PubMed Scopus (363) Google Scholar); whereas acetylation of influenza A ribonucleoprotein NP was shown to be crucial for normal viral replication and packaging (13Giese S. Ciminski K. Bolte H. Moreira E.A. Lakdawala S. Hu Z. David Q. Kolesnikova L. Gotz V. Zhao Y. Dengjel J. Chin Y.E. Xu K. Schwemmle M. Role of influenza A virus NP acetylation on viral growth and replication.Nat. Commun. 2017; 8: 1259Crossref PubMed Scopus (35) Google Scholar). Considering that a viral protein might serve multiple functions during its replication cycle, acetylation provides a variety of protein isoforms that probably fit the needs. Particularly, acetylation occurs on different positions or functional domains to generate different protein isoforms with distinct functional activities. Moreover, to identify PTM sites on viral proteins provides crucial information for developing antiviral drugs (8Murray L.A. Sheng X. Cristea I.M. Orchestration of protein acetylation as a toggle for cellular defense and virus replication.Nat. Communications. 2018; 9: 4967Crossref PubMed Scopus (32) Google Scholar). However, no acetylation site has been reported for any MERS-CoV viral protein so far. In this study, we first reported multiple acetylation sites on MERS-CoV replicase pp1ab, suggesting a potential link of protein acetylation to the regulation of MERS-CoV replication. Three biological repeats of sample infected with MERS-CoV at 1 or 5 M.O.I. have been performed. As the focus of study is to search for potential acetylation sites on MERS-CoV proteins, no control or randomization was performed in the study. No quantitation analysis has been performed and therefore no statistic method was used in the study. All data and search result in msf format has been deposit to Harvard Dataverse and available for reviewing at following address: https://doi.org/10.7910/DVN/MTDOG7. MERS -CoV was a gift from Dr. Ron Fouchier (Erasmus Medical Center, Rotterdam, the Netherlands) and cultured in VeroE6 cells in serum free DMEM. Viruses were produced by transfection of the infectious clones into Vero-E6 cells according to previous described (14Siu K.L. Yuen K.S. Castano-Rodriguez C. Ye Z.W. Yeung M.L. Fung S.Y. Yuan S. Chan C.P. Yuen K.Y. Enjuanes L. Jin D.Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.FASEB J. 2019; 33: 8865-8877Crossref PubMed Scopus (322) Google Scholar) The experiment was carried out in a biosafety level 3 laboratory and strictly followed the disinfection protocol that boiling protein sample in 10% SDS. In solution digestion was performed as previously described (15Chan P.M.B. Zhu L. Wen C.Y. Chiu K.Y. Subchondral bone proteomics in osteoarthritis: Current status and perspectives.J. Orthop. Transl. 2015; 3: 71-77PubMed Google Scholar) with minor modifications. Briefly, the disinfected protein samples were firstly precipitated by 4 times volume of acetone, followed by resuspended with 8 m Urea, 50 mm Ammonium bicarbonate, 1% RapiGest SF Surfactant (Waters) to solubilize the sample. The protein sample was then reduced by 5 mm DTT and alkylated by 15 mm IAA. Sequencing grade trypsin (thermo scientific) was used at 1:50 enzyme/protein ratio to digest the sample at 37 °C overnight. The surfactant was removed by adding 200 mm HCl and centrifuged at 20,000 × g for 10 min. The resulting peptides were then used for acetylated peptide enrichment. Acetyl lysine antibody (Immunechem, Canada) conjugated on agarose beads was used to immunoprecipitated the acetylated peptides (16Guan K.L. Yu W. Lin Y. Xiong Y. Zhao S. Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides.Nat. Protoc. 2010; 5: 1583-1595Crossref PubMed Scopus (82) Google Scholar). Briefly, the tryptic digested peptide mixture was resuspend in NETNA buffer (50 mm HEPES, 100 mm NaCl, 1 mm EDTA, 0.5% NP-40 and 10% Acetonitrile) and incubated with the acetyl lysine antibody beads overnight. The beads was then washed with same buffer for three times, before eluted with 5% TFA in 10% Acetonitrile. The eluted acetylated peptides were freeze-dried in a freeze dryer and resuspend in 0.1% formic acid for LC-MS analysis. Peptides were loaded on a self-pack C18 analytical reverse phase column (ID 75 μm × 15 cm, 200Å, 3 μm particles) at flow rate of 300 nL/min and a 75 min LC gradient of 8% to 28% ACN in 0.1% FA was used. Orbitrap fusion tribrid (Thermo-Fisher) MS machine was used to analyze the peptide sample in a data-dependent acquisition mode, with 120,000 resolution at MS1 scan and 30,000 at MS2 (FWHM at m/z 400); cycle time 3 s, AGC target for MS2 is 50000, maximum injection time 60 ms, HCD collision energy set at 35%. The resulting Raw data file was then analyzed with Sequest-HT (2013) integrated in software Proteome Discoverer software (1.4.1.14, ThermoFisher) against Uniprot MERS-CoV database, which was composed of 10 reviewed sequences from Swiss-Prot on the Uniprot website released on Nov 18th, 2017. Trypsin is set as digestion enzyme and maximum allowed miss cleavage is 2. Precursor mass tolerance set at 20 ppm, fragment mass tolerance is 0.02 Da, carbamidomethyl on cysteine as fix modification and acetylation on lysine is variable modification. FDR is set at 0.01. The Protein FDR Validator node now bases its validation on protein scores calculated from the posterior error probability (PEP) values of the peptides if these values are available. A high confidence level (1% FDR) was set to filter out peptides identified with lower confidence based on the Proteome Discoverer software. For identified acetyl-lysine carrying peptides, manual validation has been carried out to confirm the acetylation site assignment as well as the identification of peptide. The validation is based on checking the fragmentation pattern of the peptides. A total of 540 sequences derived from MERS-CoV virus pp1ab were extracted from NCBI database, and sequence alignment was then performed by online tool Cluster Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) (17Sievers F. Wilm A. Dineen D. Gibson T.J. Karplus K. Li W.Z. Lopez R. McWilliam H. Remmert M. Soding J. Thompson J.D. Higgins D.G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.Mol. Syst. Biol. 2011; 7: 539Crossref PubMed Scopus (9214) Google Scholar). KAT-specific acetylation and SIRT1 site with pp1ab sequence was by a based on method L. Y. M. Li T. a for KAT-specific acetylation site 2012; PubMed Scopus Google T. B. Z. M. Zhu Systematic identification of HDAC PubMed Scopus Google Scholar) Briefly, the method on potential sites that have a sequence with known The was based on the amino acid peptides the first scores were obtained the potential peptide and known enrichment was then calculated as the of all scores for known peptides in the the of enrichment was against a peptide set including peptide acetyltransferases and and deacetylases HDAC and SIRT1 were by the one by one and sites were out at and To for lysine acetylation sites on MERS-CoV viral proteins more infected cells were used of cells a viral Briefly, Vero-E6 cells were infected with MERS-CoV at a of infection of 1 or 5 for before the viral proteins were followed by boiling in 10% SDS. The resulting and disinfected viral protein samples were and subject to digestion with modifications. As lysine acetylation was a acetylated peptides were enriched from trypsin-digested peptides by immunoprecipitation with and then analyzed by MS machine to a A total of peptides from 7 proteins were among which were reported to be acetylated The enrichment of peptides was therefore the to peptides with acetylation sites or poorly acetylated peptides with acetylation sites were reported 1 and All acetylation sites identified are on polyprotein pp1ab, a protein further into 15 of which different functions in MERS-CoV replication many acetylation sites were in the at the of pp1ab, acetylation sites were also found in and sequence alignment of 540 of MERS-CoV pp1ab sequence from NCBI that all lysine sites are highly conserved among MERS-CoV viruses suggesting a potential role of these amino acid in virus growth or replication. As other have that activity could be regulated by V. B. Young R. S. of activity and by and A of on protein and with a Google Scholar), and that acetylation on or could DNA L. J. P. R.A. Acetylation of and 1 by DNA by Biol. Chem. 2010; PubMed Scopus Google Scholar), MERS-CoV might also the host acetylation machinery to regulate its activity to achieve optimal acetyl-lysine carrying peptides in pp1ab in a of acetylation sites on of and of acetylation sites of from 540 replicase sequences from NCBI protein protein protein protein from 540 replicase sequences from NCBI in a To further the acetylation events might affect viral be of interest to the upstream factors that regulate lysine analysis of amino acid sequences of the identified acetylation sites a conserved which the of multiple acetyltransferases and deacetylases in the We therefore used a method to analyze which lysine on pp1ab could be acetylated in then the with acetyl-lysine acetyltransferases of and families as well as deacetylases HDAC and SIRT1 were to predict regulatory sites on pp1ab, based on known sequences identified acetylated lysine sites were indeed found to with the predicted was predicted to be a of SIRT1 whereas was predicted to be acetylated by TIP60 family, and by HDAC The analysis that these and deacetylases are by the MERS-CoV to its protein to achieve optimal upstream of acetylation in a To further confirm that the acetylation events identified was we the NSP4 which the SIRT1 site and in HEK293 cells. NSP4 protein is one of the 15 cleavage from pp1ab, which from to amino acid on pp1ab. the on the we were to the NSP4 proteins and its acetylation status with As shown in the NSP4 protein in HEK293 cells was indeed acetylated The data that pp1ab was acetylated in human cells. In three acetylated peptides were obtained and into Orbitrap MS machine to MS2 The for the peptides indeed fragment in previous identification We therefore that the acetylation sites identified were of acetylated peptides. of acetyl-lysine peptides. In this study, acetylation sites on MERS-CoV replicase polyprotein pp1ab have been identified for the first The lysine sites are all highly conserved and therefore might be of crucial in viral to host Bioinformatic analysis have potential upstream factors that acetylation including and TIP60 that SIRT1 was reported as a for MERS-CoV replication S. K.L. R. A. R. T. A used to identify SIRT1 as a for respiratory syndrome coronavirus 2019; PubMed Scopus Google Scholar) and with HDAC to regulate p53 activity R. L. Chen Y. D. J. B. P.C. Y. Zhao Y. SIRT1 with and to in 16: PubMed Scopus Google H. B. D. S. Y. Acetylation of and of SIRT1 a to regulate p53 acetylation during Dis. 2015; 6: PubMed Scopus (39) Google Scholar), which is known to suppress replication of coronavirus SARS-CoV Y. J. W. J. B. Y. B. D.R. S. C. H. H. M. R. A. p53 coronavirus replication and is by the and Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar); MERS-CoV replication is regulated by acetylation a complex As NSP4 and are known to with other to in RNA replication D. K. K. M. M. and cleavage of respiratory syndrome coronavirus polyprotein induce the of that with RNA 2017; 8: PubMed Scopus Google Scholar), acetylation might also be in the regulation of the MERS-CoV replication as The identification and analysis of acetylation sites on MERS-CoV viral proteins to further the detailed molecular mechanism by which virus to host to achieve optimal The information obtained in the study also the of a potential target against MERS-CoV as acetylation might be crucial in the host process of MERS-CoV on the acetylation site was regulated might to the role of NSP4 during MERS replication. Raw data and search result in format has been deposit to Harvard Dataverse and available for reviewing at address: https://doi.org/10.7910/DVN/MTDOG7. We to Dr. Ye of The of for the MERS-CoV infection with Middle East Respiratory Syndrome Coronavirus modification severe acute respiratory syndrome of infection.

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