show Abstracthide AbstractPrimary infection with Human cytomegalovirus (HCMV) results in a persistent lifelong infection due to its ability to establish latent infection. During productive HCMV infection, viral genes are expressed in a coordinated cascade that is characteristic of all herpesviruses and traditionally relies on the dependencies of viral genes on protein synthesis and viral DNA replication. In contrast, the transcriptional landscape associated with HCMV latency is still disputed and poorly understood. Here, we examine viral transcriptomic dynamics during the establishment of both productive and latent HCMV infections. These temporal measurements reveal that viral gene expression dynamics along productive infection and their dependencies on protein synthesis and viral DNA replication, do not fully align. This illustrates that the regulation of herpesvirus genes does not represent a simple sequential transcriptional cascade and surprisingly many viral genes are regulated by multiple independent modules. Using our improved classification of viral gene expression kinetics in conjunction with transcriptome-wide measurements of the effects of a wide array of chromatin modifiers, we unbiasedly show that a defining characteristic of latent cells is the unique repression of immediate early (IE) genes. In particular, we demonstrate that IE1 (a central IE protein) expression is the principal barrier for achieving a full productive cycle. Altogether, our findings provide an unbiased and elaborate definition of HCMV gene expression in lytic and latent infection states. Overall design: For the time course analysis fibroblasts and CD14+ monocytes were infected with the same stock of HCMV strain TB40E-GFP at a multiplicity of infection (MOI) of 1 and 10, respectively. For all other experiments CD14+ monocytes or THP1 cells were infected with HCMV strain TB40E-GFP at an MOI of 5. Cells were incubated with the virus for 2h, washed, and supplemented with fresh medium. CHX was added to infected fibroblasts and CD14+ monocytes immediately after infection at a final concentration of 100 ug/ml and 200 ug/ml or at 100 ug/ml, respectively. PFA was added to fibroblasts and CD14+ monocytes immediately after infection at a final concentration of 400 ug/ml. for epigenetic drug treatments - 48 hpi, TB40-infected CD14+ monocytes were divided into 1.2 ml tubes containing ~100,000 cells per tube and inhibitors or DMSO for negative control were added to a final concentration of 1uM for 24 hours