Host Regulation of Flavivirus Infection - PROJECT SUMMARY Viruses of the family Flaviviridae, including dengue virus (DENV), Zika virus (ZIKV) and West Nile virus (WNV), are important pathogens with regard to human health across the globe. These flaviviruses, which are transmitted by mosquito vectors, either usurp or antagonize a myriad of host cell factors for their optimal replication in the human host. Whereas most efforts in the field have focused on characterizing flaviviral interactions with components of innate and adaptive immunity, little is still known about how host factors regulate key enzymatic activities of flaviviral proteins. Specifically, how the activity of NS3, which is an essential viral enzyme that encodes both a protease and a helicase, is regulated by host factors remains elusive. The proposed study builds on a recent discovery by the Gack laboratory that showed that dynamic acetylation of ZIKV NS3’s helicase domain critically regulates its RNA binding and unwinding activities, and thereby is crucial for ZIKV replication. An unbiased RNAi screen targeting all human acetyltransferases, combined with molecular and biochemical studies, identified that an as-of-yet uncharacterized isoform of the acetyltransferase KAT5 (KAT5) is responsible for NS3 acetylation. Cell biological and virological studies showed that KAT5 translocates from the nucleus to viral replication complexes (RCs) at the endoplasmic reticulum, where it binds to and acetylates NS3, facilitating ZIKV replication. Importantly, our preliminary data indicated that NS3 acetylation by KAT5, as well as its proviral role, are also conserved for WNV and DENV. Molecular, biochemical, and cell biological approaches combined with gene-targeting screens will focus on defining in precise detail how acetylation/deacetylation regulates the enzymatic activities of flavivirus NS3. This study will also give detailed insight into the mechanisms by which flavivirus infection induces the relocalization of KAT5 from the nucleus to viral RCs, and will define the molecular determinants of KAT5’s binding to NS3. Moreover, we will determine the physiological relevance of NS3 acetylation by KAT5 for effective virus replication and flavivirus-associated pathology. Our studies will provide a molecular understanding of how the host acetylation machinery controls flaviviral helicase function and thereby flavivirus replication, which may provide the foundation for new, potentially broadly effective therapeutic approaches.
