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

Abstract

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 replicationdefective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/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.

Valosin‐containing protein ATPase activity regulates the morphogenesis of Zika virus replication organelles and virus‐induced cell death

“…Although potential contributions of VCP in HCV or WNV vRF fate were never reported, it is tempting to speculate based on our results that VCP contribution to the biogenesis of vRFs overlaps with several Flaviviridae family members. While this study was under evaluation, Ramanathan et al (2020) reported a novel and nicely designed yellow fever virus (YFV) entry assay that they used to demonstrate that VCP inhibition impairs a post‐fusion pre‐translation step of the life cycle. Since NS4B is not expressed and vRFs are absent during flavivirus entry, this VCP function is not related to the one described here.…”

Beyond the Surface: Endocytosis of Mosquito-Borne Flaviviruses

“…Viral binding and internalization assays with WNV-Kun specifically implicated LY6E function in virus internalization [153]. Parallel studies confirmed the infection-enhancing and post-attachment, pre-replication function of LY6E during YFV infection [192,193]. Interestingly, LY6E was shown to be dispensable for cellular uptake of canonical CME cargo, such as transferrin (~6 nm diameter), but necessary for internalization of virions, which are larger (~50 nm diameter) [153].…”

How Viruses Use the VCP/p97 ATPase Molecular Machine

“…These same siRNAs did not affect replication of VSV [39], a negative-stranded RNA virus, which also uses receptor-mediated endocytosis for cell entry [45]. Using a replication-defective reporter virus system to study entry and pre-replication events of multiple flaviviruses, an inhibitor of the ubiquitin-activating enzyme E1 also blocked YFV, ZIKV, and WNV uncoating [40]. This study further showed that two small molecule inhibitors of p97, DBeQ and NMS-873, which have different modes of action [46,47], interfered with reporter virus expression.…”

“…Members of the positive-stranded RNA-containing Flaviviridae family of enveloped viruses, particularly the Flavivirus genus [38], use p97 for early stages of viral infection [39,40] (Figure 2). This genus includes multiple viruses causing disease in humans, e.g., West Nile virus (WNV), Zika virus (ZIKV), Dengue virus (DENV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) [38,41].…”