Epstein-Barr Virus Nuclear Antigen 1 Forms a Complex with the Nuclear Transporter Karyopherin α2

Abstract

The Epstein-Barr virus (EBV) is implicated in the induction of several malignancies. The nuclear antigen 1 (EBNA1) is the only viral protein that is expressed consistently in all EBV-associated tumors. EBNA1 is involved in the replication and maintenance of the viral episome in the infected cell and exhibits oncogenic activity in transgenic mice. Here we report the identification of the nuclear transporter karyopherin ␣2 as a cellular partner of EBNA1 using the yeast "two-hybrid system." Karyopherin ␣2 is also called importin ␣ or Rch1. The binding to karyopherin ␣2 was mediated through a C-terminal region of EBNA1 encompassing the nuclear localization signal, whereas clones of EBNA1 devoid of the nuclear localization signal failed to bind to karyopherin ␣2. The interaction was biochemically confirmed by far-Western analysis using bacterially expressed karyopherin ␣2 and karyopherin ␣2-specific monoclonal antibodies. The nuclear transport of EBNA1 was impaired by expression of N-terminally truncated karyopherin ␣2. Zone velocity sedimentation in a sucrose gradient indicated that: (i) EBNA1 and Rch1 colocalize; and (ii) the association of karyopherin ␣2 with high molecular weight protein complexes might be impeded by the presence of EBNA1.The Epstein-Barr virus (EBV) 1 is the etiological agent of infectious mononucleosis and is implicated in the induction of several malignancies including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, T-cell lymphoma, and polyclonal B-cell lymphoma that arise under immunosuppression (reviewed in Ref. 1). The expression of EBV-encoded proteins is most stringently restricted in primary Burkitt's lymphoma in that only EBNA1 and the nontranslated EpsteinBarr virus-encoded small nuclear RNAs are transcribed. EBNA1 binds to the viral origin of replication (oriP)(2, 3) and is the only virus protein necessary and sufficient to replicate and maintain the EBV genome in the infected cell (4). In addition, experiments using transgenic animals show that EBNA1 by itself is able to induce tumors in vivo (5, 6). EBNA1 is a multifunctional phosphoprotein (7) with a variety of properties, including sequence-specific binding to DNA (8, 9), formation of homodimers (10), nuclear localization (11), and stimulation of cellular gene expression (12).Import of proteins into the nucleus is an active process that can be divided into at least two steps: 1) binding of proteins with a nuclear localization signal (NLS) to a cytosolic NLSreceptor and translocation to the nuclear pore complex; and 2) transport of NLS-bearing proteins into the nucleus. The NLSreceptor consists of two subunits, a 60-kDa protein that is called karypherin ␣2 (alternatively, it is called importin ␣ in vertebrates and Srp1 in yeast) and a 95-kDa protein, called karyopherin ␤ (also known as importin ␤ in vertebrates and Kap95p in yeast). Karyopherin ␣2 serves as the NLS-receptor, whereas karyopherin ␤ functions as an adapter that mediates binding to nucleoporins. Two additional proteins, GTPase Ran/ TC4 and p15 (NTF2...

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