The DNA segregation mechanism of Epstein-Barr virus nuclear antigen 1

Abstract

Latent Epstein-Barr virus (EBV) genomes are maintained in human cells as low copy number episomes that are thought to be partitioned by attachment to the cellular mitotic chromosomes through the viral EBNA1 protein. We have identified a human protein, EBP2, which interacts with the EBNA1 sequences that govern EBV partitioning. Here we show that, in mitosis, EBP2 localizes to the condensed cellular chromosomes producing a staining pattern that is indistinguishable from that of EBNA1. The localization of EBNA1 proteins with mutations in the EBP2 binding region was also examined. An EBNA1 mutant (delta325-376) disrupted for EBP2 binding and segregation function was nuclear but failed to attach to the cellular chromosomes in mitosis. Our results indicate that amino acids 325-376 mediate the binding of EBNA1 to mitotic chromosomes and strongly suggest that EBNA1 mediates EBV segregation by attaching to EBP2 on the cellular mitotic chromosomes.

Fig. 1. Localization of EBP2 and EBNA1 on mitotic chromosomes. Mitotic chromosome spreads are shown from Raji (right panels) and BL41 (left panels) Burkitt’s lymphoma cells that do and do not express EBNA1, respectively. (A) and (B) EBNA1 staining with monoclonal antibody. (C) and (D) EBP2 staining with rabbit polyclonal antibody. (E) and (F) DAPI staining. (G) Overlay of images (B) and (D) showing co-localization (yellow). Images were captured at 630-fold magnification.

Fig. 2. Summary of the functionalities and interactions of the EBNA1 mutants. EBNA1–EBP2 interactions and DNA replication and oriP plasmid maintenance (requiring DNA replication and segregation) activities are from Shire et al. (1999). nd, not determined.

Fig. 3. Cellular localization of wild-type and mutant EBNA1 proteins in C33A cells. C33A cells that are EBNA1-negative (A and B) or that express EBNA1 (C and D), Δ325–376 (E and F), Δ356–362 (G and H) or Δ367–376 (I and J) were grown on slides then fixed for microscopy. EBNA1 proteins were detected with mouse monoclonal antibody (left panels), and DNA was detected with DAPI (right panels). Images in the left panels were captured using similar exposure times and 400-fold magnification.

Fig. 4. Mitotic localization of EBNA1 mutants. C33A cells that are EBNA1-negative (A and B) or that express EBNA1 (C and D), Δ325–376 (E–H), Δ356–362 (I and J) or Δ367–376 (K and L) were blocked in mitosis by colcemid treatment, and chromosomes were spread for microscopy. EBNA1 proteins were detected with monoclonal antibody (left panels), and DNA was detected with DAPI (right panels). Images in left panels were captured using similar exposure times and 630-fold magnification.