Project Summary Arthropod-borne flaviviruses such as dengue virus, West Nile virus, yellow fever virus (YFV), and Zika virus, are a major cause of human disease. We are studying the early, post-fusion and pre-replication events in the flavivirus lifecycle by using the prototype flavivirus, YFV, as our model. Our overarching hypothesis is that flaviviruses, which alternately replicate in vertebrate and invertebrate hosts, have evolved to use i) highly conserved factors shared between hosts; and ii) multiple, redundant factors that may not be well conserved between hosts. Therefore, discoveries made with YFV will be validated and studied by comparison to other flaviviruses and between human and mosquito cells. Aim 1 focuses on the post-fusion process of nucleocapsid uncoating, dissecting the mechanisms by which cells unlock this fateful cargo. Aim 1 is a logical extension of our Preliminary Data showing that the delivery of a translatable YFV genome requires cellular ubiquitylation and VCP/p97, a cellular ATPase that extracts ubiquitylated proteins from large macromolecular complexes. Aim 2 focuses on identifying the cellular protease(s) that cleave the viral NS1-2A polyprotein intermediate. Cleavage is essential for flavivirus replication (shown here), yet the identity of this protease has remained elusive for over two decades. We have identified a small family of related candidate NS1-2A proteases and are validating their activity by rigorous, combinatorial genetic ablation. These efforts will solve long- standing puzzles in flavivirus gene expression and replication and identify targets for future development of broadly acting antivirals.