This proposal focuses on regulation of y-herpesvirus latency-associated gene expression in the long-term latency reservoir(s). We have recently shown that murine gammaherpesvirus 68 (yHV68), like EBV, persists within memory B cells. In EBV latently infected memory B cells, there is very limited viral gene expression. In contrast, EBV infection of naive and geminal center B cells leads to expression of a panel of latency-associated genes whose products play a pivotal role in driving differentiation of these virus infected B cells into the memory B cell reservoir. We hypothesize that yHV68 infection of naive B cells also leads to their differentiation into geminal center and memory B cells. We further hypothesize that this differentiation is accompanied by changes in the viral latency-associated gene expression. Importantly, we and others have consistently shown a tight correlation between methylation of the EBV genome and establishment of restricted viral latency programs. The under-representation of CpG dinucleotides in the EBV and yHV68 genomes is evidence that methylation of these viral genomes is a normal part of their life cycle in the infected host. The parallels between EBV and yHV68 latency in vivo justify the proposed characterization of the role of DNA methylation in regulating chronic yHV68 infection in mice. Importantly, EBV genome methylation is a hallmark of all characterized EBV-associated tumors that arise in immunocompetent hosts. The latter provides further impetus to understand the underlying mechanisms involved in, and the relationship between, methylation of y-herpesvirus genomes and chronic infection. Aim 1. Analysis of EBV restricted latency analyze EBNA1 gene transcription from Qp; assess specificity/targeting of Cp/Wp methylation; Aim 2. Characterization of yHV68 latency programs characterize latency-associated gene transcription in distinct latency reservoirs; develop yHV68 latency models; map latency-associated viral promoters; Aim 3. Investigate role of methylation in regulating yHV68 latency-associated gene expression analyze viral genome methylation in naive and memory B cell reservoirs; characterize expression of de novo methyltransferases in B cells; develop recombinant yHV68 that inhibit Dnmt3a/3b expression.