Establishment of persistent Epstein-Barr virus (EBV) infection requires 
transition from a program of full viral latency gene expression (latency III) to 
one that is highly restricted (latency I and 0) within memory B lymphocytes. It 
is well established that DNA methylation plays a critical role in EBV gene 
silencing, and recently the chromatin boundary protein CTCF has been implicated 
as a pivotal regulator of latency via its binding to several loci within the EBV 
genome. One notable site is upstream of the common EBNA gene promoter Cp, at 
which CTCF may act as an enhancer-blocking factor to initiate and maintain 
silencing of EBNA gene transcription. It was previously suggested that increased 
expression of CTCF may underlie its potential to promote restricted latency, and 
here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B 
associated with latency I. Within B-cell lines that maintain latency I, however, 
stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination 
did not result in activation of latency III protein expression or EBNA gene 
transcription, nor did knockdown of DNMTs significantly alter CpG methylation 
within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not 
critical for maintenance of restricted latency. Finally, mutant EBV lacking the 
Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV 
in a recently developed B-cell superinfection model but ultimately was able to 
transition to latency I, suggesting that CTCF contributes to but is not 
necessarily essential for the establishment of restricted latency.