The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein–Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance

Abstract

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

Identifying Sites Bound by Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) in the Human Genome: Defining a Position-Weighted Matrix To Predict Sites Bound by EBNA1 in Viral Genomes

“…S2 in the supplemental material) (48). It was determined previously that EBNA1 of EBV can maintain CeHV-12 plasmid replicons (containing CeHV's oriP) and, conversely, that the EBNA1 homologue of CeHV-12 can maintain EBV plasmid replicons, with preference for their homologous oriP sequences (21,39,56). In CeHV-15, site III was predicted, consistent with a previous report (44).…”

Optimal Transactivation by Epstein-Barr Nuclear Antigen 1 Requires the UR1 and ATH1 Domains

“…All plasmids were constructed by site-directed mutagenesis using overlap extension PCR (2), and the desired alteration was confirmed by automated sequencing. Plasmid 53 was used as the FR-TKp-luciferase (where TKp is the thymidine kinase promoter) reporter (20), and plasmid 1033 was used as the oriP-BamHI-Cp-luciferase reporter (25). Plasmid 2145 expresses enhanced green fluorescent protein (EGFP) and was used to normalize for transfection efficiency.…”

“…Experiments were performed in C33a (herpesvirus [HPV] negative) cervical cancer cells (44), BJAB (EBV-negative) Burkitt's lymphoma cells (39), and BJAB/FR-TK-luciferase cells (23). Cells were propagated as described earlier in serum and antibiotic-containing medium (20,23). C33a cells were transfected using calcium phosphate precipitates.…”

Identification of Properties of the Kaposi's Sarcoma-Associated Herpesvirus Latent Origin of Replication That Are Essential for the Efficient Establishment and Maintenance of Intact Plasmids

“…EBV OriP deletion mutants containing 6 or few EBNA1-binding sites in the FR region support establishment with low efficiencies, and the established plasmids are maintained as rearranged plasmids containing head-to-tail multimers of the initial plasmids (32). The presence of at least 7 copies of EBNA1-binding sites in the FR, however, completely rescues the plasmids from their inability to be established by supporting efficient partitioning (32)(33)(34).…”

“…Early after introduction into cells, a majority of the plasmids are lost precipitously within the first 15 to 20 generations, such that only a small fraction of the initially infected cells go on to harbor the plasmids stably in the long term (29,30). The process of establishment is not fully understood, but it appears to depend on the efficiency with which a plasmid can be synthesized and partitioned (31)(32)(33)(34) and may involve epigenetic modifications to the viral genomes (30,35).…”