The chromatin regulatory factors CTCF and cohesin have been implicated in the 
coordinated control of multiple gene loci in Epstein-Barr virus (EBV) latency. 
We have found that CTCF and cohesin are highly enriched at the convergent and 
partially overlapping transcripts for the LMP1 and LMP2A genes, but it is not 
yet known how CTCF and cohesin may coordinately regulate these transcripts. We 
now show that genetic disruption of this CTCF binding site (EBVΔCTCF166) leads 
to a deregulation of LMP1, LMP2A, and LMP2B transcription in EBV-immortalized B 
lymphocytes. EBVΔCTCF166 virus-immortalized primary B lymphocytes showed a 
decrease in LMP1 and LMP2A mRNA and a corresponding increase in LMP2B mRNA. The 
reduction of LMP1 and LMP2A correlated with a loss of euchromatic histone 
modification H3K9ac and a corresponding increase in heterochromatic histone 
modification H3K9me3 at the LMP2A promoter region in EBVΔCTCF166. Chromosome 
conformation capture (3C) revealed that DNA loop formation with the origin of 
plasmid replication (OriP) enhancer was eliminated in EBVΔCTCF166. We also 
observed that the EBV episome copy number was elevated in EBVΔCTCF166 and that 
this was not due to increased lytic cycle activity. These findings suggest that 
a single CTCF binding site controls LMP2A and LMP1 promoter selection, chromatin 
boundary function, DNA loop formation, and episome copy number control during 
EBV latency.