There is fundamental gap in our understanding of KSHV genome replication during primary infection. Infected cells go on to establish latent infection with multiple copies of viral episomes even after being infected with single multiplicity of infection (moi). Interestingly, latent virus replicates only once per cell cycle thus suggests tat viral genome amplifies during primary infection before establishing latency. However, the mechanism of genome replication during primary infection is not known. Understanding the mechanism of viral genome replication including the involvement of proteins will be important in identifying targets, which can be exploited to block KSHV primary infection and thus suppress latency establishment. The long-term goal of this project is to use interventional strategies to target primary infection for thwarting the virus from infected cells and thus treat KSHV associated malignancies, which are a major health problem for HIV infected and organ transplant patients undergoing immunosuppressive therapies. The objective of this application is to identify the roles of viral origins utilized for viral genome replication during primary infectin and the involvement of viral and cellular factors assisting DNA replication. Our next generation sequencing data of chromatin immunoprecipitated (ChIP-seq) with a lytic replication protein, RTA suggested the usage of lytic origin during primary infection. Additionally, next-generation sequencing of chromatin bound by a long non- coding RNA, PAN identified its binding to oriLyt during primary infection suggesting its role as scaffold to recruit replication complex at the origin. Our central hypothesis is that lytic replication origins are primarily activated during intial infection, which helps in synthesizing multiple copies of the viral genome through rolling circle replication. This hypothesis has been formulated based on our preliminary data produced by using Single Molecule Analysis of the Replicated DNA (SMARD), and next generation sequencing of Chromatin Immunoprecipitation (ChIP-seq) and Chromatin Isolation by RNA Purification (ChIRP-seq) approaches. The rationale for the proposed research is that, once it is known how KSHV replicates by using specific viral and cellular proteins, expression of those targets can be altered, which may open new and innovative avenues to prevent KSHV primary infection. This proposal will provide a better understanding of molecular events of genome replication during primary infection, thus will determine the critical targets which can be exploited for therapeutic benefits. Guided by strong preliminary data, this hypothesis will be tested by three specific aims: 1) Determining the roles of latent and lytic origins during primary infection using single molecule analysis of the replicated DNA (SMARD), 2) Determining the accumulation of replication complexes at the origins will identify the replication mechanism used for genome amplification and 3) Determining the role of PAN RNA in recruiting replication proteins at origins in regulating DNA replication during primary infection. The preliminary data of SMARD and next generation sequencing strongly suggests that viral genes are synthesized during primary infection which accumulate at viral origins most likely with the help of PAN RNA to initiate DNA replication. The approach is innovative, because we are utilizing a powerful single molecule analysis of the replicated DNA approach and next generation sequencing, which analyzes DNA replication and gene expression, respectively in an unbiased approach at single copy levels. The proposed research is significant because it is expected to vertically advance and expand the understanding of viral genome replication and the establishment of latent infection after primary infection. Ultimately, such knowledge has the potential to determine critical targets, which can be exploited for suppressing KSHV infection and associated malignancies.