Abstract
The Epstein-Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors. EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters. The mechanisms by which EBNA-1 performs these functions are not known. Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1. We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa. It has been previously reported that P32/TAP is expressed on the cell surface. Our transient expression assays detected full-length P32/TAP (1-282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus. Three lines of evidence support P32/TAP interaction with EBNA-1. First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40-60 and aa 325-376, each of which contains arginine-glycine repeats. These regions interact with the C-terminal half of P32/TAP. Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm. Third, P32/TAP co-immunoprecipitated with EBNA-1. We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244-282) transactivates expression from a reporter containing upstream Gal4-binding sites. Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays. Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation.
Original language | English |
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Pages (from-to) | 18-29 |
Number of pages | 12 |
Journal | Virology |
Volume | 236 |
Issue number | 1 |
DOIs | |
Publication status | Published - 15 Sept 1997 |
Keywords
- Amino Acid Sequence
- Animals
- Arginine
- Binding Sites
- Carrier Proteins
- Cell Line
- Cercopithecus aethiops
- Chloramphenicol O-Acetyltransferase
- Cloning, Molecular
- Consensus Sequence
- DNA Primers
- DNA, Complementary
- Epstein-Barr Virus Nuclear Antigens
- Gene Library
- Genome, Viral
- Glycine
- Herpesvirus 4, Human
- Humans
- Hyaluronan Receptors
- Journal Article
- Lymphocytes
- Membrane Glycoproteins
- Mice
- Mitochondrial Proteins
- Molecular Sequence Data
- Polymerase Chain Reaction
- Receptors, Complement
- Recombinant Fusion Proteins
- Research Support, Non-U.S. Gov't
- Research Support, U.S. Gov't, P.H.S.
- Saccharomyces cerevisiae
- Sequence Alignment
- Transfection