Metaphase Chromosome Tethering Is Necessary for the DNA Synthesis and Maintenance of oriP Plasmids but Is Insufficient for Transcription Activation by Epstein-Barr Nuclear Antigen 1

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Abstract
Epstein-Barr Virus (EBV) infects resting B cells, within which it establishes latency as a stable, circular episome with only two EBV components, the cis element oriP and the latently expressed protein EBNA1. It is believed that EBNA1's ability to tether oriP episomes to metaphase chromosomes is required for its stable replication. We created fusions between the DNA-binding domain (DBD) of EBNA1 and the cellular chromatin-binding proteins HMGA1a and HMG1 to determine the minimal requirements for stable maintenance of an oriP -based episome. These two proteins differ in that HMGA1a can associate with metaphase chromosomes but HMG1 cannot. Interestingly, coinciding with metaphase chromosome association, HMGA1a-DBD but not HMG1-DBD supported both the transient replication and stable maintenance of oriP plasmids, with efficiencies quantitatively similar to that of EBNA1. However, HMGA1a-DBD activated transcription from EBNA1-dependent episomal reporter to only 20% of the level of EBNA1. Furthermore, EBNA1 but not HMGA1a-DBD activated transcription from a chromosomally integrated EBNA1-dependent transcription reporter. This indicates that EBNA1 possesses functional domains that support transcription activation independent of its ability to tether episomal oriP plasmids to cellular chromosomes. We provide evidence that metaphase chromosome tethering is a fundamental requirement for maintenance of an oriP plasmid but is insufficient for EBNA1 to activate transcription.