PROJECT SUMMARY The herpesviruses have co-evolved with their hosts for hundreds of millions of years. As our adaptive immune systems evolved, so did the mechanisms of immune evasion encoded by herpesviruses. Because herpesviruses remain latent or persistent within their hosts throughout life, the herpesviruses have evolved a unique set of strategies to help them contend with the lifelong host immune response. This application?s central premise is that the herpesviruses, in their evolution, have performed a 100 million-year-long genetic screen to undermine host defense mechanisms, and in so doing, can illuminate cell biological processes that we may not yet understand; we seek to take advantage of their evolution to discover new concepts in cell biology. The long term goal of this project is to understand the underlying cell biological mechanisms by which human herpesviruses-6 and -7 (HHV-6/7) escape host defense mechanisms. The focus of this proposal is to search for the cellular proteins that assist HHV-7 U21 in rerouting class I MHC molecules to the lysosomal compartment and to explore the function of U21 in the context of virus infection. In Aim I, we will perform a CRISPR/Cas9 sgRNA library screen for cellular proteins that influence U21's ability to reroute class I MHC molecules to lysosomes. We will also attempt to identify cellular proteins in the vicinity of the U21/class I MHC complex using a promiscuous biotin ligase. In Aim II, to explore the physiological role of U21 in the context of infection, we will examine HHV-6A U21's role in host evasion, and determine whether HHV-6A U21 functions to downregulate cellular proteins from the cell surface by surveying the effects of HHV-6A U21 expression on the cell surface proteome. We will then widen our scope to examine the how the surface proteome changes in the context of HHV-6A infection. These exploratory experiments should extend our previous work on HHV-7 U21 in new directions; we aim to discover multiple cellular proteins that illuminate either novel lysosomal trafficking pathways or novel host evasion strategies for HHV-6A. In so doing, we work toward a more complete understanding of how these viruses have demonstrated such great success in evading the host immune response. RELEVANCE OF THIS RESEARCH TO PUBLIC HEALTH The high fevers resulting from HHV-6 infection are perhaps the most common cause of febrile seizures in young children, with a peak incidence in the second year of life. Childhood seizures have long been known to be associated with a substantially increased risk of temporal epilepsy (3). A recent study has shown that HHV- 6, and HHV-7 infection accounts for ~40% of all cases of severe seizures in young children (4). HHV6 DNA was found in 70% of brain tissue resections from mesial temporal lobe epilepsy (5). Together, these studies suggest a link between childhood infection with HHV-6 and -7, and the development of epilepsy. HHV-6 also reactivates in hematopoietic cell transplant recipients, resulting in poor neurocognitive outcome and can result in acute limbic encephalitis (3,4). The function of lysosomes is of critical importance to a cell's ability to break down both extracellular ingested material, as well as intracellular substrates. To maintain a functioning lysosome, proper targeting of lysosomal enzymes to lysosomes is critical, a point underscored by the growing number of devastating genetic deficiencies ascribed to lysosomal storage disorders. The U21 protein encoded by HHV-7 uses the cellular lysosomal sorting machinery to reroute class I MHC molecules for destruction in lysosomes. Discovering how U21 does this may illuminate novel cellular lysosomal sorting pathways. A clearer understanding of how lysosomal proteins reach their destination will inform our ability to treat lysosomal storage diseases.