Kaposi Sarcoma-associated herpesvirus (KSHV) is a medically important virus that causes two types of human cancer. Infection with KSHV can result in viral persistence for the lifetime of the host. Viral persistence, commonly referred to as latency, is characterized by the absence of gene expression. Reactivation from latency results in entry into the lytic cycle which is characterized by the full repertoire of viral gene expression. A key pathogenic step in KSHV-mediated disease is viral reactivation and viral lytic gene expression. Immunosuppression, regardless of the source (HIV-mediated or iatrogenic), results in log-fold increases in the risk of developing KSHV-mediated diseases.We adopted a tractable system to study a KSHV genome derived from a clinical isolate. This KSHV genome has been engineered for stable propagation in E. coli. The strain of E. coli harboring the KSHV genome has been engineered for the use of a scarless, site-directed mutagenesis procedure. Parental and mutagenized virus can be introduced into a human cell line via transfection. Transfection of parental virus into this cell line results in stable latency. This cell line has also been engineered to express the major viral-transactivator under an inducible promoter. Expression of the viral-transactivator is sufficient to reactivate KSHV from latency, drive the full repertoire of viral gene expression, and results in significant cell-free progeny. We implemented this experimental workflow to mutagenize all known canonical Open Reading Frames (ORFs) and several other uncanonical ORFs. Transfection and cell-free progeny analyses resulted in the identification of ORFs essential for completion of the viral lifecycle. Growth analyses of non-essential mutants resulted in the identification of ORFs important for induced and spontaneous viral reactivation. Phenotypic screening of essential mutants using light and electron microscopy resulted in the identification of ORFs playing vital roles in viral lytic gene expression and capsid formation and maturation. The results of my thesis reveal the identification of novel antiviral targets and live-attenuated vaccine candidates. Moreover, these data suggest a new model for KSHV latency and reactivation. This model proposes that KSHV actively communicates with and responds to the host’s immune system in a concerted effort to fine-tune viral latency and reactivation. Supplementary analyses, not provided herein, are underway to elucidate the molecular details for discrete steps in viral egress.