The replicator of the Epstein-Barr virus latent cycle origin of DNA replication, oriP, is composed of multiple functional elements.
Replication of the Epstein-Barr virus genome initiates at one of several sites in latently infected, dividing cells. One of these replication origins is close to the viral DNA maintenance element, and, together, this replication origin and the maintenance element are referred to as oriP. The replicator of oriP contains four binding sites for Epstein-Barr virus nuclear antigen 1 (EBNA-1), the sole viral protein required for the replication and maintenance of oriP plasmids. We showed previously that these EBNA-1 sites function in pairs and that mutational inactivation of one pair does not eliminate replicator function. In this study we characterized the contribution of each EBNA-1 site within the replicator and flanking sequences through the use of an internally controlled replication assay. We present evidence that shows that all four EBNA-1 sites are required for an oriP plasmid to be replicated in every cell cycle. Results from these experiments also show that the paired EBNA-1 binding sites are not functionally equivalent and that the low affinity of sites 2 and 3 compared to that of sites 1 and 4 is not essential for replicator function. Our results suggest that a host cell protein(s) binds sequences flanking the EBNA-1 sites and that interactions between EBNA-1 and this protein(s) are critical for replicator function. Finally, we present evidence that shows that the minimal replicator of oriP consists of EBNA-1 sites 3 and 4 and two copies of a 14-bp repeat that is present in inverse orientation flanking these EBNA-1 sites. EBNA-1 sites 1 and 2, together with an element(s) within nucleotides 9138 to 9516, are ancillary elements required for full replicator activity.
Koons
M D
MD
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222, USA.
Van Scoy
S
S
Hearing
J
J
D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000235:genetics*; D008969:Molecular Sequence Data; D018741:Replication Origin / Q000502:physiology*; D014779:Virus Replication
D019309:Epstein-Barr Virus Nuclear Antigens; C417029:EBV-encoded nuclear antigen 1