Structural basis for the regulation of nuclear import of Epstein-Barr virus nuclear antigen 1 (EBNA1) by phosphorylation of the nuclear localization signal

https://doi.org/10.1016/j.bbrc.2017.01.063Get rights and content

Highlights

  • Nuclear import of EBNA1 can be regulated by phosphorylation of NLS.

  • Crystal structures of importin-α1 bound to the NLS peptides of EBNA1 are solved.

  • Structures provide insights into how phosphorylation can regulate nuclear import.

Abstract

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is expressed in every EBV-positive tumor and is essential for the maintenance, replication, and transcription of the EBV genome in the nucleus of host cells. EBNA1 is a serine phosphoprotein, and it has been shown that phosphorylation of S385 in the nuclear localization signal (NLS) of EBNA1 increases the binding affinity to the nuclear import adaptor importin-α1 as well as importin-α5, and stimulates nuclear import of EBNA1. To gain insights into how phosphorylation of the EBNA1 NLS regulates nuclear import, we have determined the crystal structures of two peptide complexes of importin-α1: one with S385-phosphorylated EBNA1 NLS peptide, determined at 2.0 Å resolution, and one with non-phosphorylated EBNA1 NLS peptide, determined at 2.2 Å resolution. The structures show that EBNA1 NLS binds to the major and minor NLS-binding sites of importin-α1, and indicate that the binding affinity of the EBNA1 NLS to the minor NLS-binding site could be enhanced by phosphorylation of S385 through electrostatic interaction between the phosphate group of phospho-S385 and K392 of importin-α1 (corresponding to R395 of importin-α5) on armadillo repeat 8.

Introduction

Epstein-Barr virus (EBV) is a ubiquitous human herpes virus associated with a diverse range of tumors of both lymphoid and epithelial origin [1]. EBV nuclear antigen 1 (EBNA1) is a DNA-binding protein expressed in every EBV-positive tumor. EBNA1 plays an essential role in the maintenance and replication of the episomal EBV genome through its direct interaction with sequences in the EBV latent origin of replication (oriP) [2], [3], [4], [5], and also acts as a transcriptional regulator [6], [7]. It has been shown that EBNA1 induces B cell lymphomas in transgenic mice [8], enhances cell survival [9], and induces genetic instability [10], indicating that EBNA1 might contribute directly to oncogenesis. EBNA1 is phosphorylated at multiple serine residues when expressed in human and insect cells [11], [12], [13], [14]. Although the physiological significance of EBNA1 phosphorylation remains incompletely understood, it has been suggested that phosphorylation of EBNA1 serine residues contributes to segregation and maintenance of the EBV genome, transcriptional activation, and nuclear import of EBNA1 [15], [16], [17], [18].

Transport of macromolecules between the cytoplasm and nucleus occurs through nuclear pore complexes (NPCs) [19]. Eukaryotic cells control and finely tune many biological processes by regulating nuclear transport [20]. Phosphorylation of cargoes has emerged as one of the important mechanisms to regulate a multitude of nuclear transport pathways [20], including the importin (Imp) α:β-dependent nuclear import pathway [21], [22]. The Impα adaptor proteins bind cargo proteins possessing the nuclear localization signal (NLS), and heterodimerize with Impβ through the N-terminal Impβ-binding (IBB) domain, forming the heterotrimeric Impα:β:NLS-cargo complexes that permeate through NPCs and deliver NLS-cargoes into the nucleus [19], [20], [21], [22]. Mammalian cells have at least seven Impα isoforms, whose expression is tightly regulated depending on cell type and developmental stage [23], [24]. Each isoform of Impα has different substrate specificity, and many cellular and viral cargoes have been shown to associate preferentially with specific isoforms of Impα [23], [24].

Previous cell biological and biochemical studies identified the NLS of EBNA1 (379KRPRSPSS386) [25] and demonstrated that EBNA1 binds to the nuclear import adaptor Impα1 [26], [27], [28] as well as Impα5 [28]. The amino-terminal K379 and R380 of the EBNA1 NLS are essential for nuclear translocation [18]. Although the serine residues (S383, S385, and S386) are not essential for the EBNA1 NLS, both S383 and S385 are important for nuclear translocation [18]. Phosphorylation of S385 increases nuclear import efficiency [18] and also increases the binding affinity of the EBNA1 NLS to Impα1 as well as Impα5 [18], [29]. In this study, we used X-ray crystallography to elucidate how phosphorylation of EBNA1 NLS can regulate its interaction with Impα.

Section snippets

Preparation of protein-peptide complexes for crystallization

N-terminally His6-and S-tagged ΔIBB Impα1 (mouse, residues 70–529) was expressed from pET30a (Novagen) [30] in the E. coli host strain BL21-CodonPlus(DE3)RIL (Stratagene), and was purified over Ni-NTA (Novagen) and gel filtration over Superdex200 (GE Healthcare). S385-phosphorylated EBNA1 NLS peptide 378EKRPRPRSP-pS-S386 (pS stands for phosphoserine) and non-phosphorylated EBNA1 NLS peptide 378EKRPRPRSPSS386 were synthesized by GenScript. Prior to crystallization, ΔIBB Impα1 and the NLS peptide

Crystal structure of Impα1 bound to S385-phosphorylated EBNA1 NLS peptide

We obtained crystals of the NLS-binding armadillo (ARM) repeat domain of Impα1 bound to the S385-phosphorylated NLS peptide of EBNA1, and determined the structure at 2.0 Å resolution by molecular replacement (Table 1). The structure was refined to free and working R-factor values of 21.2% and 18.6%, respectively. Residues 378–385 and 378–383 of the NLS peptide bound to the minor and major NLS-binding sites, respectively, were identified unambiguously in the electron density map (Fig. 1A and B).

Acknowledgements

We thank the staff of Photon Factory for assistance during X-ray diffraction data collection.

References (41)

  • J.L. Yates et al.

    Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells

    Nature

    (1985)
  • S. Lupton et al.

    Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells

    Mol. Cell Biol.

    (1985)
  • D. Reisman et al.

    A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components

    Mol. Cell Biol.

    (1985)
  • D. Reisman et al.

    Trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1

    Mol. Cell Biol.

    (1986)
  • T.A. Gahn et al.

    An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene

    J. Virol.

    (1995)
  • J.B. Wilson et al.

    Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice

    EMBO J.

    (1996)
  • G. Kennedy et al.

    Epstein-Barr virus provides a survival factor to Burkitt's lymphomas

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • B. Gruhne et al.

    The Epstein-Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species

    Proc. Natl. Acad. Sci. U. S. A.

    (2009)
  • J.C. Hearing et al.

    The Epstein-Barr virus nuclear antigen (BamHI K antigen) is a single-stranded DNA binding phosphoprotein

    Virology

    (1985)
  • M. Polvino-Bodnar et al.

    Mutational analysis of Epstein-Barr virus nuclear antigen 1 (EBNA 1)

    Nucleic Acids Res.

    (1988)
  • Cited by (11)

    View all citing articles on Scopus
    View full text