Over 90% of the world’s population is seropositive for three or more of the nine human herpesviruses (HHVs), which possess the ability to establish lifelong latent infections that can be reactivated. Reactivation of HHV infections is particularly serious in immunocompromised patients leading to disseminated life-threatening infections. Although many of the anti HHV drug discovery efforts to date have focused on nucleoside/tide HHV polymerase inhibitors, the large number of essential replication proteins encoded by the herpesviruses provide excellent novel targets for antiviral therapy. New agents are needed to better prevent pathological sequelae of reactivation as well as viral shedding and transmission to new hosts. The need for new modalities of therapeutics to treat HHV infections underscores the importance of a more thorough understanding of HHV DNA replication. In addition to the seven essential herpes simplex virus (HSV) replication proteins identified by us and others, we have shown that the viral alkaline nuclease UL12 is also essential for the production of viral DNA that can be packaged into infectious virus. UL12 interacts with the HSV ssDNA binding protein ICP8 to form a two-component recombinase (exo/SSAP) that can promote single strand annealing (SSA). We have suggested that HSV uses an unusual mechanism of DNA replication that has more in common with bacteriophage than eukaryotic cells or other eukaryotic viruses. In fact, all DNA viruses of bacteria, protozoa, plants, insects and mammals that replicate through concatemer formation encode a similar exo/SSAP complex. We hypothesize that ICP8 and UL12 promote a series of reactions in which UL12 resects dsDNA leaving a 3’ ssDNA overhang that is recognized by ICP8. ICP8 then promotes annealing of the ssDNA to an active replication fork to promote DNA synthesis by the viral DNA polymerase (UL30). UL30 is comprised of two functional domains: a 3’ to 5’ exonuclease, PolExo, that plays a role in proofreading and the catalytic polymerase domain required for extending primers during viral DNA replication. While the SSA model for DNA replication is consistent with available evidence, questions remain about how UL12 and the two activities of the polymerase function during DNA synthesis. In parallel studies, the Wright and Weller labs have been interested in both AN and PolExo and their othologs from other HHVs as targets for novel antiviral therapeutics and have generated focused libraries designed for their ability to engage a two-metal binding motif found in both AN and PolExo active sites. We have identified several lead compounds that are potent antivirals and inhibit one or more of the exonuclease activities and in some cases additional activity against polymerase activity itself. In this proposal we will use our lead compounds as molecular probes to study mechanisms of viral DNA replication and to continue our efforts to develop broad spectrum antiviral agents that inhibit AN, PolExo an...