A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVASK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVASK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.