EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance

Abstract

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient replication of oriP plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.

FIG. 1

Activation of the HIS3 reporter by EBNA1 and EBP2 in the two-hybrid system. Tenfold serial dilutions of log-phase cultures of Y190 strains expressing SNF1, EBNA1 or lamin as GAL4 DNA binding domain fusions and SNF4, EBP2, or nothing (pACT2) fused to the GAL4 activation domain were plated on SC-Trp,Leu (left panel) or SC-Trp,Leu,His plus 50 mM AT (right panel). The SNF1/SNF4 culture is a positive control for the two-hybrid interaction.

FIG. 2

Interaction of EBNA1 and EBP2 in the one-hybrid system. Triplicate cultures of LF100 expressing EBNA1 (from pEBNA1) and EBP2 fused to the GAL4 activation domain (from pACT63) were grown in liquid SC-Trp,Leu,His plus 5 mM AT, and the growth was monitored by measuring the OD₆₀₀ (triangles). The growth of negative control cultures containing pEBNA1 with pACT2 (circles) or pACT63 with pAS2 (squares) is also shown.

FIG. 3

The EBP2 cDNA. The cDNA isolated from the 5′-Stretch Plus library is shown. The Kozak consensus sequence is in boldface and the polyadenylation sequence is underlined.

FIG. 4

Alignment of human EBP2 with the S. cerevisiae (YKL172w), Schizosaccharomyces pombe (SPAC17H9), and C. elegans (C18A3.3) homologues. The alignment was performed by using the Clustal method in DNASTAR. Identical and highly conserved amino acids are shaded.

FIG. 5

Predicted structure of human EBP2. The predicted secondary structure (2°) of EBP2 was determined by using PHDsec (EMBL, Heidelberg, Germany). L, loop; H, α-helix (no β-sheets were predicted). Predicted structures are only shown for residues with secondary structure reliability indices of 5 or more. The position of coiled coils (C-C) was determined by using PairCoil, and residues with a 40% or greater probability of forming coils are shown (C).

FIG. 6

Coimmunoprecipitation of EBNA1 and EBP2. Insect cells were infected with baculoviruses expressing EBNA1 or EBP2 alone or were coinfected with both baculoviruses. Lysates from metabolically labeled cells were prepared 3 days (A and C) or 2 days (B) postinfection and immunoprecipitated with anti-EBNA1 antibody (IP). (C) Immunoprecipitates from lysates coexpressing EBNA1 and EBP2 are shown before and after treatment with RNase A and DNase I.

FIG. 7

Coimmunoprecipitation of EBP2 with EBNA1 mutants. (A) Lysates from insect cells expressing EBNA1 proteins alone (−) or EBNA1 proteins with EBP2 (+) were prepared 2 days postinfection and immunoprecipitated with anti-EBNA1 antibody as in Fig. 6. Immunoprecipitated proteins are shown. (B) Insect cell lysates coexpressing EBNA1 proteins and EBP2 (1/140 the amount used in panel A) were analyzed by Western blotting with EBNA1 antisera. The position of the band corresponding to the full-length EBNA1 protein in question is marked by the bracket.

FIG. 8

Interaction of EBNA1 mutants with EBP2 in the two-hybrid system. (A) Activation of the HIS3 reporter was determined as in Fig. 1. Tenfold serial dilutions of log-phase cultures of Y190 strains expressing EBNA1 or EBNA1 mutants as GAL4 DNA binding domain fusions and EBP2 or nothing (pACT2) fused to the GAL4 activation domain were plated on SC-Trp,Leu (left panel) or SC-Trp,Leu,His plus 50 mM AT (right panel). (B) Equal numbers of Y190 cells expressing the EBNA1 fusion proteins indicated were lysed and subjected to Western blot analysis with antibodies against the HA tag.

FIG. 9

Summary of EBP2 interactions with EBNA1 fragments and mutants. A schematic representation of the EBNA1 polypeptides tested for EBP2 binding in co-immunoprecipitation (IP) and two-hybrid (2H) assays.

FIG. 10

Long-term plasmid maintenance ability of EBNA1 mutants. C33A cells were transfected with a plasmid containing oriP and expressing EBNA1, EBNAΔ325–376 (duplicate samples are shown), EBNAΔ41–376 (duplicate samples are shown), EBNAΔ367–376, EBNAΔ356–362, or no EBNA1 (pc3oriP) and maintained under selection for 14 days. Plasmid DNA from 5 × 10⁶ cells was collected, digested with XhoI and DpnI, and analyzed by Southern blotting. A 100-pg linearized pc3oriPE marker is also shown.

FIG. 11

Transient-replication assays of EBNA1 mutants. C33A cells were transfected with a plasmid containing oriP and expressing EBNA1, EBNAΔ325–376, EBNAΔ41–376, or no EBNA1 (pc3oriP) and grown without selection for 3 days. The plasmid DNA from each plate of cells was harvested; 1/10 was linearized with XhoI, and 9/10 was digested with both XhoI and DpnI prior to Southern blotting. The results from two experiments are shown.