ABSTRACT This project aims to elucidate the interactions of the herpes simplex virus portal vertex with replicated viral DNA and the HSV packaging machinery. The packaging and cleavage of replicated viral DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus type 1 (HSV-1), this process requires the products of seven viral genes: UL6, UL15, UL17, UL25, UL28, UL32, and UL33. Most of the proteins involved in capsid assembly and DNA packaging are conserved suggesting that these mechanisms will also be similar for all herpesviruses. The proposed research by the Homa and[CJF1] Conway laboratories combines expertise in virology and structural biology for determining the interaction of the cleavage/packaging proteins with the capsid portal vertex leading to stable packaging of a unit length genome. Understanding the herpesvirus portal structure and its interaction with the packaging machinery has been hampered due to the subtle morphological differences between the portal and penton vertices. However, recent advances in cryoEM techniques have allowed portals to be localized in images of intact capsids yielding important structural information about the portal and associated proteins. The pUL6 portal dodecamer appears to be anchored to the capsid by interactions with the peri-pentonal triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the capsid vertex specific component (CVSC). Here we propose combined biochemical and structural studies focusing on the unique portal vertex of wild type HSV and packaging mutants that will provide insights into the mechanism of HSV genome packaging. We aim to understand the functions of the CVSC components, their interactions with the portal vertex and with the terminase complex in cleavage and retention of the packaged viral genome. Based on published and preliminary studies, we believe the pUL17 is emerging as a key player in capsid assembly and maturation. We hypothesize that pUL17 anchors the terminase complex to the portal and, after packaging is complete, recruits pUL25 to the portal to retain the viral genome. We will test the hypothesis in two specific Aims that together investigate the structure and function of the portal vertex. Aim 1 focuses on the viral proteins required for the portal complex to interact with the replicated viral genome and initiate the cleavage packaging reaction. Aim 2 follows a complementary structural approach to understand the architecture of the portal vertex, include the components required for different stages of assembly and packaging. Our results will inform the development of innovative and specific therapeutics designed to prevent herpesvirus replication by blocking DNA packaging.