Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human uridine kinase/uracil phosphoribosyltransferase.
BACKGROUND
Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts.


RESULTS
In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A), using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538). The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538) was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human uridine kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain uridine kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3.


CONCLUSIONS
We suggest that EBNA-3 by direct protein-protein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation.
Microbiology and Tumor Biology Centre, Karolinska Institute, S-171 77, Stockholm, Sweden. elena.kashuba@mtc.ki.se
Elena
E
Kashuba
Vladimir
V
Kashuba
Tatjana
T
Sandalova
George
G
Klein
Laszlo
L
Szekely
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D016785:Cryptosporidium parvum; D019281:Dimerization; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D015800:Protozoan Proteins; D016415:Sequence Alignment / Q000379:methods; D014541:Uridine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; C422762:EBNA-3B antigen; D019309:Epstein-Barr Virus Nuclear Antigens; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D014541:Uridine Kinase