Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.
Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.
Forsman
Alma
A
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.
Rüetschi
Ulla
U
Ekholm
Josefine
J
Rymo
Lars
L
D000345:Affinity Labels; D002460:Cell Line; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D050883:HSC70 Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M / Q000032:analysis / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D018832:Molecular Chaperones; D010455:Peptides / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D015203:Reproducibility of Results; D013205:Staphylococcal Protein A / Q000235:genetics; D047992:TNF Receptor-Associated Factor 2; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014162:Transfection; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000032:analysis / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; C492980:BAG2 protein, human; C421621:EBNA-5 protein, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M; D018832:Molecular Chaperones; C101338:PSMD2 protein, human; D010455:Peptides; D020033:Protein Isoforms; D013205:Staphylococcal Protein A; D047992:TNF Receptor-Associated Factor 2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014764:Viral Proteins