Mechanisms of Conformational Dynamics and Inhibition of the HSV-1 Nuclear Egress Complex

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Project Summary/Abstract Herpesviruses are double-stranded DNA viruses that infect almost all mammals, including humans, making them highly effective pathogens. A hallmark event in the herpesvirus replication cycle requires immature capsids to bud through the inner nuclear membrane (INM) to the perinuclear space in a process termed nuclear egress. This essential first step in viral exit is mediated by the conserved viral nuclear egress complex (NEC) and is the focus of this proposal. The ability of the NEC to oligomerize on membranes is important for capsid budding, yet the conformational changes undergone to perform this are unknown. The long-term goal of this research is to determine how disruption of NEC oligomerization perturbs conformational changes that drive budding (Aim 1) and develop peptide-based screening platforms for identifying novel NEC inhibitors (Aim 2). This work stems from a central hypothesis, formulated from substantial preliminary data, that the NEC can be inhibited, specifically by perturbing NEC oligomerization. The scientific premise of this work is to formulate a detailed mechanism of herpesvirus nuclear egress to inform the design of innovative therapeutic compounds. A combination of cutting-edge biophysical techniques, including cryoelectron microscopy/tomography (cryoEM/T), along with mutational and functional approaches will be used to identify the molecular interactions undergone by Herpes Simplex NEC during budding and to identify routes for inhibiting this process. The K99 phase of this proposal is structured to not only answer these fundamental questions surrounding herpesvirus nuclear egress but also provide a platform for transitioning to an independent research career centered around identifying small molecule and peptide inhibitors targeting specific protein-protein interactions occurring at various stages of herpesviral replication. This type of protein targeting will provide a novel means for determining specific protein function within the virus and expand the repertoire of therapeutic targets available for the treatment of this disease. Focus during this phase will be on fine-tuning expertise in cryoEM/T by investigating NEC mutants designed to perturb oligomerization (in collaboration with cryoEM/T expert Dr. Zhiheng Yu at the HHMI Janelia Research Campus), gain instruction in virological techniques to perform functional studies on NEC mutants (in collaboration with herpes biology experts Dr. Richard Roller at the University of Iowa and Dr. David Knipe at Harvard Medical School) and establish peptide screening platforms (under advisement from Dr. Joshua Kritzer at Tufts University – an expert in therapeutic peptide design). Under the guidance of an extremely qualified mentor (Dr. Katya Heldwein – an expert in structural virology) and co-mentor (Dr. Ralph Isberg – an expert in postdoctoral training for successful academic careers), within the supportive institutional environment of Tufts University, this proposal ensures a timely transition to leading a successful independent academic career.