Reconstitution of Epstein-Barr virus-based plasmid partitioning in budding yeast

Abstract

The EBNA1 protein of Epstein-Barr virus (EBV) mediates the partitioning of EBV episomes and EBV-based plasmids during cell division by a mechanism that appears to involve binding to the cellular EBP2 protein on human chromosomes. We have investigated the ability of EBNA1 and the EBV segregation element (FR) to mediate plasmid partitioning in Saccharomyces cerevisiae. EBNA1 expression alone did not enable the stable segregation of FR-containing plasmids in yeast, but segregation was rescued by human EBP2. The reconstituted segregation system required EBNA1, human EBP2 and the FR element, and functionally replaced a CEN element. An EBP2 binding mutant of EBNA1 and an EBNA1 binding mutant of EBP2 each failed to support FR-plasmid partitioning, indicating that an EBNA1-EBP2 interaction is required. The results provide direct evidence of the role of hEBP2 in EBNA1-mediated segregation and demonstrate that heterologous segregation systems can be reconstituted in yeast.

Fig. 1. Plasmids used in budding yeast assay for plasmid loss. The stabilities of three different plasmids, all of which contain an ARS element and a TRP1 selectable marker, were examined. (A) Positive control plasmid for stable segregation containing a yeast CEN element (Sikorski and Hieter, 1989). (B) Negative control segregation plasmid lacking a segregation element (Stinchcomb et al., 1979). (C) Experimental plasmid containing the EBV segregation element (FR).

Fig. 2. Loss of YRp7FR in the presence and absence of EBNA1. Yeast (KY320) were transformed with one of the segregation plasmids shown in Figure 1 and a URA3 expression plasmid that did (+) or did not (–) contain the EBNA1 gene. After 48 h of growth (approximately eight doublings) without selection for the segregation plasmids, 10-fold dilutions of the cultures were plated on SC-Ura (no selection for test plasmid) or SC-Ura,Trp (selection for test plasmid) plates.

Fig. 3. Stable segregation of YRp7FR in the presence of EBNA1 and hEBP2. Plasmid loss assays were conducted over 11 generations in KY320 (left panel) or W303-1A (right panel) yeast strains containing a segregation plasmid, a URA3 expression plasmid that contains or lacks the EBNA1 gene, and a LEU2 expression plasmid that contains or lacks the hEBP2 gene. Dilutions of the cultures were then grown on SC-Ura,Leu (no selection for test plasmid) or SC-Ura,Leu,Trp (selection for test plasmid) plates.

Fig. 4. EBNA1-mediated segregation in KY320.hEBP2. KY320.hEBP2, which contains an integrated hEBP2 gene under control of a constitutively active promoter, was used in plasmid loss assays over 11 generations as described in Figure 2. The assays were conducted in the presence of either wild-type EBNA1 (+) or the EBNAΔ325–376 mutant (Δ325–376), or in the absence of EBNA1 (–).

Fig. 5. The hEBP2 binding region of EBNA1 is required for EBNA1-mediated segregation in yeast. The loss of YRp7FR was monitored in KY320 (left panel) and W303-1A (right panel) yeast strains in the presence of either wild-type EBNA1 (+) or the EBNAΔ325–376 mutant (Δ325–376), or in the absence of EBNA1 (–). All cultures expressed hEBP2 from p425PGK.hEBP2. Assays were conducted as in Figure 3.

Fig. 6. The EBNA1 binding region of hEBP2 is required for EBNA1-mediated segregation. (A) Schematic representation of hEBP2 showing the most conserved region, which corresponds to a predicted coiled-coil domain. Amino acids numbers are indicated. (B) EBP2 from yeast (yEBP2) and humans (hEBP2), and hEBP2 fragments 1–220, 220–306, 1–100, 95–220 and 95–306 were tested for binding to EBNA1 in a yeast two-hybrid assay, as determined by activation of the HIS3 reporter gene. As a negative control, hEBP2 was tested for binding to EBNA452–641, which lacks the hEBP2 binding sequence. Ten-fold serial dilutions of log-phase cultures were grown on plates containing histidine (left panel) or lacking histidine and containing 50 mM AT (right panel). (C) Plasmid loss assay comparing the ability of hEBP2 (+) and the hEBP2 1–220 mutant (1–220) to support the segregation of YRp7FR in the presence of EBNA1. Assay results in the absence of hEBP2 expression (–) are also shown. Assays were conducted as in Figure 3.