title: P32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus.
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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205-2185, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: J E
authors.initials: JE
authors.lastname: Finan
authors.forename: J M
authors.initials: JM
authors.lastname: Middeldorp
authors.forename: S D
authors.initials: SD
authors.lastname: Hayward
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002352:Carrier Proteins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D016679:Genome, Viral; D005998:Glycine; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D018960:Hyaluronan Receptors; D008214:Lymphocytes / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011951:Receptors, Complement / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D014162:Transfection
chemical_list: C087299:C1QBP protein, human; C490193:C1qbp protein, mouse; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D019309:Epstein-Barr Virus Nuclear Antigens; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; C032402:complement 1q receptor; D001120:Arginine; D015500:Chloramphenicol O-Acetyltransferase; D005998:Glycine