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• W4296520220 abstract "The human cytomegalovirus (HCMV) UL97 protein is a conserved herpesvirus protein kinase (CHPK) and a viral cyclin-dependent kinase (v-CDK). However, mechanisms regulating its activity in the context of infection are unknown. Here, we identified several cellular regulatory 14-3-3 proteins as UL97-interacting partners that promote UL97 stability. Humans are known to encode seven isoforms of 14-3-3 proteins (β, ε, η, γ, σ, θ, and ζ) that bind phosphoserines or phosphothreonines to impact protein structure, stability, activity, and localization. Our proteomic analysis of UL97 identified 49 interacting partners, including 14-3-3 isoforms β, η, and γ. Furthermore, coimmunoprecipitation with Western blotting assays demonstrated that UL97 interaction with 14-3-3 isoforms β, ε, η, γ, and θ occurs in a kinase activity-dependent manner. Using mutational analysis, we determined the serine residue at amino acid 13 of UL97 is crucial for 14-3-3 interaction. We demonstrate UL97 S13A (serine to alanine substitution at residue 13) retains kinase activity but the mutant protein accumulated at lower levels than WT UL97. Finally, we show both laboratory (AD169) and clinical (TB40/E) strains of HCMV encoding UL97 S13A replicated with WT kinetics in fibroblasts but showed decreased UL97 accumulation. Taken together, we conclude that 14-3-3 proteins interact with and stabilize UL97 during HCMV infection. The human cytomegalovirus (HCMV) UL97 protein is a conserved herpesvirus protein kinase (CHPK) and a viral cyclin-dependent kinase (v-CDK). However, mechanisms regulating its activity in the context of infection are unknown. Here, we identified several cellular regulatory 14-3-3 proteins as UL97-interacting partners that promote UL97 stability. Humans are known to encode seven isoforms of 14-3-3 proteins (β, ε, η, γ, σ, θ, and ζ) that bind phosphoserines or phosphothreonines to impact protein structure, stability, activity, and localization. Our proteomic analysis of UL97 identified 49 interacting partners, including 14-3-3 isoforms β, η, and γ. Furthermore, coimmunoprecipitation with Western blotting assays demonstrated that UL97 interaction with 14-3-3 isoforms β, ε, η, γ, and θ occurs in a kinase activity-dependent manner. Using mutational analysis, we determined the serine residue at amino acid 13 of UL97 is crucial for 14-3-3 interaction. We demonstrate UL97 S13A (serine to alanine substitution at residue 13) retains kinase activity but the mutant protein accumulated at lower levels than WT UL97. Finally, we show both laboratory (AD169) and clinical (TB40/E) strains of HCMV encoding UL97 S13A replicated with WT kinetics in fibroblasts but showed decreased UL97 accumulation. Taken together, we conclude that 14-3-3 proteins interact with and stabilize UL97 during HCMV infection. The human cytomegalovirus (HCMV) UL97 protein functions as a viral cyclin-dependent kinase (v-CDK) (1Hume A.J. Finkel J.S. Kamil J.P. Coen D.M. Culbertson M.R. Kalejta R.F. Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function.Science. 2008; 320: 797-799Crossref PubMed Scopus (180) Google Scholar, 2Kuny C.V. Chinchilla K. Culbertson M.R. Kalejta R.F. Cyclin-dependent kinase-like function is shared by the beta- and gamma- subset of the conserved herpesvirus protein kinases.PLoS Pathog. 2010; 6e1001092Crossref PubMed Scopus (82) Google Scholar, 3Steingruber M. Marschall M. The cytomegalovirus protein kinase pUL97: host interactions, regulatory mechanisms and antiviral drug targeting.Microorganisms. 2020; 8: 515Crossref PubMed Scopus (23) Google Scholar). UL97 complements the growth of CDK-deficient yeast and shares phosphorylation substrates with cellular CDKs such as the retinoblastoma (Rb) tumor suppressor and the Rb family members p107 and p130 (1Hume A.J. Finkel J.S. Kamil J.P. Coen D.M. Culbertson M.R. Kalejta R.F. Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function.Science. 2008; 320: 797-799Crossref PubMed Scopus (180) Google Scholar, 4Iwahori S. Hakki M. Chou S. Kalejta R.F. Molecular determinants for the inactivation of the retinoblastoma tumor suppressor by the viral cyclin-dependent kinase UL97.J. Biol. Chem. 2015; 290: 19666-19680Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 5Iwahori S. Umana A.C. VanDeusen H.R. Kalejta R.F. Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins.J. Biol. Chem. 2017; 292: 6583-6599Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Also, like the CDKs, UL97 phosphorylates and inactivates the deoxy-NTP (dNTP) hydrolase SAMHD1 (6Kim E.T. Roche K.L. Kulej K. Spruce L.A. Seeholzer S.H. Coen D.M. et al.SAMHD1 Modulates early steps during human cytomegalovirus infection by limiting NF-kappaB activation.Cell Rep. 2019; 28: 434-448.e436Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 7Businger R. Deutschmann J. Gruska I. Milbradt J. Wiebusch L. Gramberg T. et al.Human cytomegalovirus overcomes SAMHD1 restriction in macrophages via pUL97.Nat. Microbiol. 2019; 4: 2260-2272Crossref PubMed Scopus (27) Google Scholar, 8De Meo S. Dell'Oste V. Molfetta R. Tassinari V. Lotti L.V. Vespa S. et al.SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity.PLoS Pathog. 2020; 16e1008855Crossref PubMed Scopus (9) Google Scholar) and Lamin A/C (9Hamirally S. Kamil J.P. Ndassa-Colday Y.M. Lin A.J. Jahng W.J. Baek M.C. et al.Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress.PLoS Pathog. 2009; 5e1000275Crossref PubMed Scopus (167) Google Scholar) proteins. Furthermore, UL97 phosphorylates viral proteins to support capsid nuclear egress and virion assembly (10Sharma M. Bender B.J. Kamil J.P. Lye M.F. Pesola J.M. Reim N.I. et al.Human cytomegalovirus UL97 phosphorylates the viral nuclear egress complex.J. Virol. 2015; 89: 523-534Crossref PubMed Scopus (50) Google Scholar, 11Kamil J.P. Coen D.M. Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65.J. Virol. 2007; 81: 10659-10668Crossref PubMed Scopus (73) Google Scholar). Recombinant HCMVs lacking the UL97 gene or deficient for UL97 kinase activity grow poorly (12Prichard M.N. Gao N. Jairath S. Mulamba G. Krosky P. Coen D.M. et al.A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency.J. Virol. 1999; 73: 5663-5670Crossref PubMed Google Scholar, 13Kamil J.P. Hume A.J. Jurak I. Munger K. Kalejta R.F. Coen D.M. Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 16823-16828Crossref PubMed Scopus (38) Google Scholar). Mechanisms that regulate the activity of cellular CDKs are well understood and include the phosphorylation of specific CDK residues, the interaction with cyclins and CDK inhibitors (CKIs), and associations with chaperone complexes (14Malumbres M. Barbacid M. Mammalian cyclin-dependent kinases.Trends Biochem. Sci. 2005; 30: 630-641Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar). Despite the functional similarities between UL97 and cellular CDKs, our understanding of UL97 regulatory mechanisms is limited. UL97 is phosphorylated by CDK9/cyclin T1, but the significance of this event is unknown (15Graf L. Webel R. Wagner S. Hamilton S.T. Rawlinson W.D. Sticht H. et al.The cyclin-dependent kinase ortholog pUL97 of human cytomegalovirus interacts with cyclins.Viruses. 2013; 5: 3213-3230Crossref PubMed Scopus (18) Google Scholar). Furthermore, UL97 associates with the cellular cyclins A2, B1, H, and T1, but whether these associations affect kinase activity or substrate specificity has not been established (15Graf L. Webel R. Wagner S. Hamilton S.T. Rawlinson W.D. Sticht H. et al.The cyclin-dependent kinase ortholog pUL97 of human cytomegalovirus interacts with cyclins.Viruses. 2013; 5: 3213-3230Crossref PubMed Scopus (18) Google Scholar, 16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar, 17Steingruber M. Kraut A. Socher E. Sticht H. Reichel A. Stamminger T. et al.Proteomic interaction patterns between human cyclins, the cyclin-dependent kinase ortholog pUL97 and additional cytomegalovirus proteins.Viruses. 2016; 8: 219Crossref PubMed Scopus (18) Google Scholar). While the CDK inhibitor p21 does not efficiently inhibit UL97-mediated phosphorylation compared with cellular CDKs (1Hume A.J. Finkel J.S. Kamil J.P. Coen D.M. Culbertson M.R. Kalejta R.F. Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function.Science. 2008; 320: 797-799Crossref PubMed Scopus (180) Google Scholar), other CKIs have not been tested for their ability to regulate UL97 kinase activity. Similarly, if and how the other v-CDKs (EBV BGLF4; HHV-6a, HHV-6b, and HHV-7, U69; KSHV ORF36) (2Kuny C.V. Chinchilla K. Culbertson M.R. Kalejta R.F. Cyclin-dependent kinase-like function is shared by the beta- and gamma- subset of the conserved herpesvirus protein kinases.PLoS Pathog. 2010; 6e1001092Crossref PubMed Scopus (82) Google Scholar) are regulated is not well established. Compared to the other v-CDKs, UL97 contains an N-terminal extension predicted to be largely disordered (18Jones D.T. Cozzetto D. DISOPRED3: precise disordered region predictions with annotated protein-binding activity.Bioinformatics. 2015; 31: 857-863Crossref PubMed Scopus (533) Google Scholar). This N-terminal domain contains multiple autophosphorylation sites (19Baek M.C. Krosky P.M. Coen D.M. Relationship between autophosphorylation and phosphorylation of exogenous substrates by the human cytomegalovirus UL97 protein kinase.J. Virol. 2002; 76: 11943-11952Crossref PubMed Scopus (42) Google Scholar, 20Oberstein A. Perlman D.H. Shenk T. Terry L.J. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.Proteomics. 2015; 15: 2006-2022Crossref PubMed Scopus (34) Google Scholar, 21Steingruber M. Keller L. Socher E. Ferre S. Hesse A.M. Coute Y. et al.Cyclins B1, T1, and H differ in their molecular mode of interaction with cytomegalovirus protein kinase pUL97.J. Biol. Chem. 2019; 294: 6188-6203Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), including Ser-2, Ser-3, Ser-11, Ser-13, Thr-16, Thr-18, Ser-133, Thr-134, and Thr-177. While the N-terminal domain is dispensable for in vitro kinase activity (19Baek M.C. Krosky P.M. Coen D.M. Relationship between autophosphorylation and phosphorylation of exogenous substrates by the human cytomegalovirus UL97 protein kinase.J. Virol. 2002; 76: 11943-11952Crossref PubMed Scopus (42) Google Scholar), it promotes UL97 nuclear localization and overall viral fitness (22Webel R. Hakki M. Prichard M.N. Rawlinson W.D. Marschall M. Chou S. Differential properties of cytomegalovirus pUL97 kinase isoforms affect viral replication and maribavir susceptibility.J. Virol. 2014; 88: 4776-4785Crossref PubMed Scopus (23) Google Scholar). More insight into the functions of this unique N-terminal extension of UL97 is required to understand its role during infection. To understand the roles of UL97 and its N terminus, we, as well as others (3Steingruber M. Marschall M. The cytomegalovirus protein kinase pUL97: host interactions, regulatory mechanisms and antiviral drug targeting.Microorganisms. 2020; 8: 515Crossref PubMed Scopus (23) Google Scholar, 11Kamil J.P. Coen D.M. Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65.J. Virol. 2007; 81: 10659-10668Crossref PubMed Scopus (73) Google Scholar, 16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar), have used molecular proteomics to identify UL97 interacting proteins. Here, we report the capture and identification of 49 candidate UL97 binding proteins. Among the novel interactors we found were a subset of the 14-3-3 proteins. Humans encode 7 isoforms of 14-3-3 proteins (β, ε, η, γ, σ, θ, and ζ) that form either homodimers or heterodimers depending on the isoforms (23Reinhardt H.C. Yaffe M.B. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.Nat. Rev. Mol. Cell Biol. 2013; 14: 563-580Crossref PubMed Scopus (200) Google Scholar). 14-3-3 proteins bind to phosphorylated Ser and/or Thr residues on client proteins and can affect their structure, stability, activity, and localization (23Reinhardt H.C. Yaffe M.B. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.Nat. Rev. Mol. Cell Biol. 2013; 14: 563-580Crossref PubMed Scopus (200) Google Scholar, 24Dougherty M.K. Morrison D.K. Unlocking the code of 14-3-3.J. Cell Sci. 2004; 117: 1875-1884Crossref PubMed Scopus (401) Google Scholar). Proteins involved in cell cycle progression and the DNA damage response are prominent 14-3-3 clients (25Hermeking H. Benzinger A. 14-3-3 proteins in cell cycle regulation.Semin. Cancer Biol. 2006; 16: 183-192Crossref PubMed Scopus (248) Google Scholar, 26Gardino A.K. Yaffe M.B. 14-3-3 proteins as signaling integration points for cell cycle control and apoptosis.Semin. Cell Dev. Biol. 2011; 22: 688-695Crossref PubMed Scopus (206) Google Scholar, 27Kasahara K. Goto H. Izawa I. Kiyono T. Watanabe N. Elowe S. et al.PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3gamma and is required for metaphase-anaphase transition.Nat. Commun. 2013; 4: 1882Crossref PubMed Scopus (47) Google Scholar, 28Dar A. Wu D. Lee N. Shibata E. Dutta A. 14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation.Mol. Cell Biol. 2014; 34: 4049-4061Crossref PubMed Scopus (37) Google Scholar, 29Jiang K. Pereira E. Maxfield M. Russell B. Goudelock D.M. Sanchez Y. Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345.J. Biol. Chem. 2003; 278: 25207-25217Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). We show here that UL97 binds to five different 14-3-3 proteins (β, ε, η, γ, and θ) dependent upon the Ser-13 residue of UL97, an autophosphorylation site found in its unique N-terminal extension. Interaction with 14-3-3 proteins increases UL97 stability but did not substantially contribute to viral fitness in the in vitro productive replication assays commonly employed to study HCMV. Our work identifies a unique function for the N terminus of UL97 and highlights 14-3-3 proteins as additional cellular regulators of HCMV. To discover potential substrates and regulatory factors, we identified UL97-associated proteins from the HEK-293T cells commonly used for interactome studies (30Gordon D.E. Jang G.M. Bouhaddou M. Xu J. Obernier K. White K.M. et al.A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.Nature. 2020; 583: 459-468Crossref PubMed Scopus (2570) Google Scholar, 31Shah P.S. Link N. Jang G.M. Sharp P.P. Zhu T. Swaney D.L. et al.Comparative flavivirus-host protein interaction mapping reveals mechanisms of dengue and zika virus pathogenesis.Cell. 2018; 175: 1931-1945.e1918Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Carboxy-terminally Halo-tagged UL97 or an unanchored Halo peptide were expressed by transient transfection in HEK-293T cells, and Halo-containing or Halo-associated proteins were collected from lysates with HaloLink beads and identified by mass spectrometry (LC-MS/MS). Two independent biological replicates were analyzed. Forty-nine UL97-interacting proteins (Table 1 and Supporting information) were detected in each of the two biological replicates but not in either of the two Halo-only negative control pull downs. Of these 49, 16 (AIFM1, CCT3, CCT7, CDC20, CDC37, CDK1, CDK2, CCNA2, CCNB1, DNAJA1, DNAJA2, EIF4A1, HSPA6, NUMA1, TRIM28, and VCP) have been detected in previous UL97 interactome analysis (3Steingruber M. Marschall M. The cytomegalovirus protein kinase pUL97: host interactions, regulatory mechanisms and antiviral drug targeting.Microorganisms. 2020; 8: 515Crossref PubMed Scopus (23) Google Scholar, 11Kamil J.P. Coen D.M. Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65.J. Virol. 2007; 81: 10659-10668Crossref PubMed Scopus (73) Google Scholar, 15Graf L. Webel R. Wagner S. Hamilton S.T. Rawlinson W.D. Sticht H. et al.The cyclin-dependent kinase ortholog pUL97 of human cytomegalovirus interacts with cyclins.Viruses. 2013; 5: 3213-3230Crossref PubMed Scopus (18) Google Scholar, 16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar, 17Steingruber M. Kraut A. Socher E. Sticht H. Reichel A. Stamminger T. et al.Proteomic interaction patterns between human cyclins, the cyclin-dependent kinase ortholog pUL97 and additional cytomegalovirus proteins.Viruses. 2016; 8: 219Crossref PubMed Scopus (18) Google Scholar) and 5 (CCNB1, MARK2, PGRMC1, STUB1, and TRIM28) have been previously identified as UL97 substrates (20Oberstein A. Perlman D.H. Shenk T. Terry L.J. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.Proteomics. 2015; 15: 2006-2022Crossref PubMed Scopus (34) Google Scholar, 32Li R. Zhu J. Xie Z. Liao G. Liu J. Chen M.R. et al.Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication.Cell Host Microbe. 2011; 10: 390-400Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Although not all known interacting proteins and substrates were detected, our ability to identify multiple known UL97-interacting proteins and substrates suggests that the novel proteins we identified are promising candidates for true UL97-binding partners or targets.Table 1List of UL97 interacting proteinsAccession #Protein name (49)Ave. CtrlaAverage of quantitative value normalized total spectra for control (HALO only) or UL97 in duplicates.Ave. UL97aAverage of quantitative value normalized total spectra for control (HALO only) or UL97 in duplicates.Previously identified as UL97 binding partner or kinase substrateP17066HSP76_Heat shock 70 kDa protein 6 GN = HSPA6021.8795Binding partner (11Kamil J.P. Coen D.M. Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65.J. Virol. 2007; 81: 10659-10668Crossref PubMed Scopus (73) Google Scholar)bReference number.P619811433G_14-3-3 protein gamma GN = YWHAG012.164P319461433B_14-3-3 protein beta/alpha GN = YWHAB011.104Q16543CDC37_Hsp90 co-chaperone Cdc37 GN = CDC37011.104Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)Q13263TIF1B_Transcription intermediary factor 1-beta GN = TRIM28010.8147Kinase substrate (20Oberstein A. Perlman D.H. Shenk T. Terry L.J. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.Proteomics. 2015; 15: 2006-2022Crossref PubMed Scopus (34) Google Scholar); Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)Q049171433F_14-3-3 protein eta GN = YWHAH08.6553Q92598HS105_Heat shock protein 105 kDa GN = HSPH105.319P31689DNJA1_DnaJ homolog subfamily A member 1 GN = DNAJA105.18545Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)P06493CDK1_Cell division protein kinase 1 GN = CDK104.85785Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)P14635CCNB1_G2/mitotic-specific cyclin-B1 GN=CCNB104.5691Binding partner (3Steingruber M. Marschall M. The cytomegalovirus protein kinase pUL97: host interactions, regulatory mechanisms and antiviral drug targeting.Microorganisms. 2020; 8: 515Crossref PubMed Scopus (23) Google Scholar, 15Graf L. Webel R. Wagner S. Hamilton S.T. Rawlinson W.D. Sticht H. et al.The cyclin-dependent kinase ortholog pUL97 of human cytomegalovirus interacts with cyclins.Viruses. 2013; 5: 3213-3230Crossref PubMed Scopus (18) Google Scholar, 16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar, 17Steingruber M. Kraut A. Socher E. Sticht H. Reichel A. Stamminger T. et al.Proteomic interaction patterns between human cyclins, the cyclin-dependent kinase ortholog pUL97 and additional cytomegalovirus proteins.Viruses. 2016; 8: 219Crossref PubMed Scopus (18) Google Scholar); Kinase substrate (32Li R. Zhu J. Xie Z. Liao G. Liu J. Chen M.R. et al.Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication.Cell Host Microbe. 2011; 10: 390-400Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar)Q99832TCPH_T-complex protein 1 subunit eta GN = CCT704.04745Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)P49368TCPG_T-complex protein 1 subunit gamma GN = CCT304.04745Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)P27448MARK3_MAP/microtubule affinity-regulating kinase 3 GN=MARK303.43105P49411EFTU_Elongation factor Tu, mitochondrial GN=TUFM03.1311P24941CDK2_Cell division protein kinase 2 GN = CDK202.6983Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)P26641EF1G_Elongation factor 1-gamma GN = EEF1G02.4095Q12931TRAP1_Heat shock protein 75 kDa, mitochondrial GN = TRAP102.1207O60884DNJA2_DnaJ homolog subfamily A member 2 GN = DNAJA202.0819Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)Q7KZI7MARK2_Serine/threonine-protein kinase MARK2 GN = MARK202.0819Kinase substrate (20Oberstein A. Perlman D.H. Shenk T. Terry L.J. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.Proteomics. 2015; 15: 2006-2022Crossref PubMed Scopus (34) Google Scholar)Q92841DDX17_Probable ATP-dependent RNA helicase DDX17 GN = DDX1702.04315Q12834CDC20_Cell division cycle protein 20 homolog GN = CDC2002.00434Binding partner (3Steingruber M. Marschall M. The cytomegalovirus protein kinase pUL97: host interactions, regulatory mechanisms and antiviral drug targeting.Microorganisms. 2020; 8: 515Crossref PubMed Scopus (23) Google Scholar, 16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)P55072TERA_Transitional endoplasmic reticulum ATPase GN = VCP01.71555Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)Q15084PDIA6_Protein disulfide-isomerase A6 GN = PDIA601.67674O95831AIFM1_Apoptosis-inducing factor 1, mitochondrial GN = AIFM101.67674Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)Q14697GANAB_Neutral alpha-glucosidase AB GN = GANAB01.426735P62306RUXF_Small nuclear ribonucleoprotein F GN = SNRPF01.38795P07737PROF1_Profilin-1 GN = PFN101.38795P18669PGAM1_Phosphoglycerate mutase 1 GN = PGAM101.38795Q9UNE7CHIP_E3 ubiquitin-protein ligase CHIP GN = STUB101.38795Kinase substrate (32Li R. Zhu J. Xie Z. Liao G. Liu J. Chen M.R. et al.Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication.Cell Host Microbe. 2011; 10: 390-400Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar)Q14980NUMA1_Nuclear mitotic apparatus protein 1 GN = NUMA101.34914Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)P78406RAE1L_mRNA export factor GN = RAE101.34914Q9HCN8SDF2L_Stromal cell-derived factor 2-like protein 1 GN = SDF2L101.060385P05141ADT2_ADP/ATP translocase 2 GN = SLC25A501.060385P35613BASI_Basigin GN = BSG01.060385P60842IF4A1_Eukaryotic initiation factor 4A-I GN = EIF4A101.060385Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar)Q15233NONO_Non-POU domain-containing octamer-binding protein GN = NONO01.060385O43175SERA_D-3-phosphoglycerate dehydrogenase GN = PHGDH01.060385P31040DHSA_Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial GN = SDHA01.060385P60660MYL6_Myosin light polypeptide 6 GN = MYL601.02154P30086PEBP1_Phosphatidylethanolamine-binding protein 1 GN = PEBP101.02154O00264PGRC1_Membrane-associated progesterone receptor component 1 GN = PGRMC101.02154Kinase substrate (20Oberstein A. Perlman D.H. Shenk T. Terry L.J. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.Proteomics. 2015; 15: 2006-2022Crossref PubMed Scopus (34) Google Scholar)Q93008USP9X_Probable ubiquitin carboxyl-terminal hydrolase FAF-X GN = USP9X01.02154P31942HNRH3_Heterogeneous nuclear ribonucleoprotein H3 GN=HNRNPH301.02154P00558PGK1_Phosphoglycerate kinase 1 GN = PGK101.02154P20248CCNA2_Cyclin-A2 GN = CCNA201.02154Binding partner (16Bogdanow B. Schmidt M. Weisbach H. Gruska I. Vetter B. Imami K. et al.Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.Nat. Commun. 2020; 11: 4845Crossref PubMed Scopus (11) Google Scholar, 17Steingruber M. Kraut A. Socher E. Sticht H. Reichel A. Stamminger T. et al.Proteomic interaction patterns between human cyclins, the cyclin-dependent kinase ortholog pUL97 and additional cytomegalovirus proteins.Viruses. 2016; 8: 219Crossref PubMed Scopus (18) Google Scholar)P30101PDIA3_Protein disulfide-isomerase A3 GN = PDIA301.02154P301532AAA_Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform GN = PPP2R1A01.02154Q9UBS4DJB11_DnaJ homolog subfamily B member 11 GN = DNAJB1100.693975Q99615DNJC7_DnaJ homolog subfamily C member 7 GN = DNAJC700.693975a Average of quantitative value normalized total spectra for control (HALO only) or UL97 in duplicates.b Reference number. Open table in a new tab Our interactome contained three 14-3-3 proteins, β, η, and γ. Because UL97 interacted with multiple 14-3-3 proteins and because very little is known about 14-3-3 regulation of HCMV infection (33Gupta S. Yla-Anttila P. Sandalova T. Achour A. Masucci M.G. Interaction with 14-3-3 correlates with inactivation of the RIG-I signalosome by herpesvirus ubiquitin deconjugases.Front. Immunol. 2020; 11: 437Crossref PubMed Scopus (16) Google Scholar), we further analyzed the physical and functional interaction between UL97 and the cellular 14-3-3 proteins. We screened all seven human 14-3-3 isoforms (β, ε, η, γ, σ, θ, and ζ) with transient transfection coimmunoprecipitation (IP) assays for their ability to bind HA-tagged UL97. We found that the three isoforms detected in the Halo-UL97 pulldown (β, η, and γ), as well as two others (ε and θ) interacted with HA-UL97 (Fig. 1A). Any putative binding of UL97 to 14-3-3 σ or ζ was below the limit of detection of this assay. The steady state levels of the UL97-interacting 14-3-3 proteins did not change during HCMV infection (Fig. 1B). Of the five isoforms that interact with UL97, we found antibodies for two (ε and θ) that worked for IP and used them to examine the interaction between UL97 and 14-3-3 protein in HCMV-infected cells. Antibodies for 14-3-3 ε or θ coimmunoprecipitated UL97 at both 48 hpi (Fig. 1C) and 72 hpi (Fig. 1D). Recip" @default.
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• W4296520220 date "2022-11-01" @default.
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• W4296520220 title "Serine 13 of the human cytomegalovirus viral cyclin-dependent kinase UL97 is required for regulatory protein 14-3-3 binding and UL97 stability" @default.
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• W4296520220 doi "https://doi.org/10.1016/j.jbc.2022.102513" @default.
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