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.

Subject headings

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)

Keyword

Affinity Labels

Cell Line

Cell Line

Tumor

Chromatography

Affinity

methods

Chromatography

Gel

Epstein-Barr Virus Nuclear Antigens

genetics

metabolism

Genetic Vectors

genetics

HSC70 Heat-Shock Proteins

analysis

metabolism

HSP70 Heat-Shock Proteins

analysis

metabolism

Heterogeneous-Nuclear Ribonucleoprotein Group M

analysis

metabolism

Humans

Immunoprecipitation

Peptides

genetics

Protein Binding

Protein Interaction Mapping

methods

Protein Isoforms

analysis

metabolism

Reproducibility of Results

Staphylococcal Protein A

genetics

Tandem Mass Spectrometry

methods

Transfection

Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

analysis

metabolism

Viral Proteins

genetics

metabolism

Publication and Content Type

ref (subject category)

art (subject category)