PROJECT SUMMARY/ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Like all herpesviruses, the KSHV life cycle consists of latent and lytic phases, and the virus relies on a sophisticated cascade of gene expression during lytic reactivation from latency. KSHV uses the host cell gene expression machinery to transcriptionally and posttranscriptionally control the timing and levels of gene expression. For host genes, proper transcription involves regulation of RNA polymerase II (pol II) elongation illustrated by pol II pausing at the 5´ and 3´ ends of genes. The near ubiquitous regulation of pol II elongation on host genes coupled with KSHV’s use of the host machinery suggest that the virus employs similar mechanisms of regulation. Despite this potential importance of pol II control by elongation, regulation of KSHV transcription elongation has been largely unexplored. Using an unbiased genome-wide CRISPR screen, host factors involved in pol II elongation and mRNA 3´ end formation were identified as negative regulators of KSHV gene expression. Depletion of these factors in cells latently infected with a KSHV infectious bacmid clone (BAC16) robustly increases the speed and overall production of infectious virions upon lytic reactivation. In the proposed work, the mechanisms linking elongation and 3´-end formation factors to KSHV transcription will be defined. Aim 1 will explore the importance of these elongation factors in PEL cells and during lytic gammaherpesvirus infection. Aim 2 uses viral gene reporter constructs and reductionist molecular biology to test whether pol II pausing is induced by cellular factors on viral genes. Moreover, the cis-acting and trans-acting requirements for elongation control of viral genes will be determined. In Aim 3, a combination of high-throughput methods will be performed to examine the roles of host elongation factors on viral gene expression. These will include RNA-seq to test gene expression levels, PAC-seq to address mRNA 3´-end formation, and PRO-seq to determine pol II occupancy on the viral genome. Finally, preliminary and published data suggest that reversible phosphorylation of elongation factors may play essential roles in the control of pol II pausing. In Aim 4, the targets and role of dephosphorylation of elongation factors on KSHV genes will be defined. Successful completion of the proposed studies will have considerable impact on the field by defining a novel host factor that negatively regulates viral gene expression by a previously undescribed mechanism(s). Moreover, it is likely that the mechanisms described will apply to additional DNA viruses and inform human gene expression as well. These studies will generate a deeper understanding of the mechanisms of a pathogenic virus which may lead to insights into how to combat KSHV related diseases.