We searched for cellular proteins that interact with Epstein-Barr (EBV) virus nuclear antigen-1, which is a latent EBV origin-binding protein detected in all EBV latently infected cells and essential for maintenance of the latent EBV genome, by a yeast two-hybrid screening of a B lymphocyte cDNA library in this study. Interaction of polypeptides synthesized from three selected cDNA clones with EBNA-1 proteins was confirmed in vitro using their glutathione-S-transferase-fusion polypeptides and by coimmunoprecipitation analyses of B cell extracts with anti-EBNA-1 monoclonal antibodies and monospecific antibodies against cellular proteins of interest. We report the following: (i) Karyopherin α (karyopherin α, hSRP1, and NPI-1), an adaptor subunit of nuclear localization signal receptors, which direct proteins to the nuclear pore, interacted with EBNA-1. (ii) EBNA-1 proteins endogenous in the B cell line Raji of Burkitt lymphoma origin bound to another adaptor protein, karyopherin α2 (hSRP 1α, hRch1), interactions of which to recombinant EBNA-1 polypeptides were previously reported. (iii) Nearly 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 p32, a potential of which to affect localization of EBNA-1 in transfected Vero cells has been recently suggested. These results suggest that EBNA-1, which has the unique NLS containing Lys-Arg and overlapping with one of the phosphorylation domains, is recognized and transported to the nuclei by these two distinct karyopherin α proteins, which are differentially expressed in different cell types, implying a regulatory localization system for EBNA-1.
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