Project Abstract Herpesviruses are double-stranded-DNA viruses that infect most of the human population. These complex viruses establish lifelong, dormant infections, periodically reactivating under certain conditions. Reactivation is particularly detrimental to the immunocompromised, resulting in a variety of disease states, including blindness, encephalitis, cancers, and death, yet there is no cure. There are nine types of human herpesviruses, classified into three subfamilies, yet a vaccine is only available targeting one type. Furthermore, available antivirals are suboptimal due to viral mutation. The lack of pan-herpesvirus therapeutics likely stems from the variations in viral replication between subfamilies, yet certain aspects, such as the need for properly assembled capsids containing genetic content, are conserved. Therefore, the long-term goal of this research is to formulate a detailed mechanism as to how herpesviral capsids assemble and package DNA, both of which are essential for all herpesviruses to replicate. Although great strides have been made over the years to understand these processes, we still do not know how these dynamic and transient processes occur at the molecular level. Therefore, the scientific premise of this work is to develop biophysical methodologies to monitor capsid assembly and genome packaging in real-time. The work in this proposal capitalizes on an existing in-vitro capsid assembly platform that we will use in conjunction with new technologies in light scattering and mass spectrometry to understand how individual capsid proteins come together to form the capsid shell. Not only will this provide missing information regarding this essential process but it will also create new methodologies for other researchers studying large viruses. Additionally, work in this proposal will create a novel in-vitro herpesviral capsid packaging assay, something that has yet to be done in the field. We will subject this assay to various single-molecular approaches to understand how proteins involved in genome packaging, some with unknown or incompletely defined roles, coordinate this process to achieve successful encapsidation. Together, these innovative studies will not only provide fundamental knowledge regarding these essential processes but also challenge existing paradigms, resulting in a more complete understanding of herpesviral replication that can be exploited for preventative and therapeutic approaches.