Project Summary The standard treatments against other human herpesviruses have limited efficacy against Kaposi’s sarcoma- associated herpesvirus (KSHV). A mechanistic understanding of KSHV infection and virion production will help us develop new therapies to limit spread of the virus and thereby prevent the associated diseases, such as Kaposi’s sarcoma. Viral packaging is an essential, conserved process in herpesviruses and is therefore a promising therapeutic target. Although the essential proteins and necessary molecular events of packaging are known, we lack a detailed mechanistic understanding of the process. These knowledge gaps must be addressed to enable rational drug design. ORF68 is an essential packaging protein of unknown function whose role in this multi-step packaging process is unknown. Recombinant ORF68 forms a pentamer with a positively central channel that binds dsDNA in vitro. This work will bridge a critical knowledge gap between these in vitro observations and ORF68 molecular function during infection in vivo. In Aim 1, I will determine the oligomeric state of ORF68 during KSHV infection in vivo. In Aim 2, I will determine if the ORF68 oligomerization interface is required during KSHV infection in vivo. In Aim 3, I will directly assess whether DNA binds inside the ORF68 pentamer channel. This work will unveil the molecular function of ORF68 in the context of infection, bringing us closer to a mechanistic model of KSHV packaging.