Epstein-Barr virus nuclear antigen-1 binds to nuclear transporter karyopherin alpha 1/NPI-1 in addition to karyopherin alpha 2/Rch1

We searched for cellular proteins that interact with Epstein-Barr (EBV) vir us nuclear antigen-1, which is a latent EBV origin-binding protein detected in all EBV latently infected cells and essential for maintenance of the la tent EBV genome, by a yeast two-hybrid screening of a B lymphocyte cDNA lib rary in this study. Interaction of polypeptides synthesized from three sele cted cDNA clones with EBNA-1 proteins was confirmed in vitro using their gl utathione-S-transferase-fusion polypeptides and by coimmunoprecipitation an alyses of B cell extracts with anti-EBNA-1 monoclonal antibodies and monosp ecific antibodies against cellular proteins of interest. We report the foll owing: (i) Karyopherin alpha (karyopherin alpha 1, hSRP1, and NPI-1), an ad aptor subunit of nuclear localization signal receptors, which direct protei ns to the nuclear pore, interacted with EBNA-1. (ii) EBNA-1 proteins endoge nous in the B cell line Raji of Burkitt lymphoma origin bound to another ad aptor protein, karyopherin alpha 2 (hSRP1 alpha, hRch1), interactions of wh ich to recombinant EBNA-1 polypeptides were previously reported. (iii) Near ly 90% of all the cDNA clones examined was p32 (SF2-associated P32, p32/TAP , and gC1q-R), and endogenous EBNA-1 proteins in the Raji cells bound to p3 2, a potential of which to affect localization of EBNA-1 in transfected Ver o cells has been recently suggested. These results suggest that EBNA-1, whi ch has the unique NLS containing Lys-Arg and overlapping with one of the ph osphorylation domains, is recognized and transported to the nuclei by these two distinct karyopherin alpha proteins, which are differentially expresse d in different cell types, implying a regulatory localization system for EB NA-1. (C) 2000 Academic Press.