Essential early events in the flavivirus lifecycle

Illustrated here are the early, pre-replication events in the flavivirus life cycle. Virus particles bind to cells through specific receptors, internalized via clathrin-mediated endocytosis, and trafficked to endosomes, where the low pH induces viral Envelope (E) glycoprotein-mediated fusion of the viral envelope bounding endosomal membranes. Our work indicates that the cellular AAA+-ATPase VCP/p97, which recognizes ubiquitylated proteins and extracts them from larger macromolecular complexes, has an important role after fusion and preceding viral genome translation, most likely in uncoating the nucleocapsid. Following uncoating, the viral genomes is translated to express a large polyprotein, which is processed by viral and cellular proteases. We are working to identify the cellular protease that cleaves NS1-2A, a cleavage event that is essential for replication complex assembly, as well as the role of uncleaved NS1-2A in this process.

Arthropod-borne flaviviruses — such as dengue, West Nile, yellow fever, and Zika viruses — exact a huge toll on human health, yet only a few flavivirus vaccines have been developed and specific antiviral therapies are not yet available. A key to unlocking new antiviral approaches is to identify the molecular basis of flavivirus replication, including druggable virus-host interactions. We are focusing on early steps in the flavivirus life cycle: events that take place between viral entry and RNA replication. Little is known about these early post-fusion and pre-replication events, in large part because the tools to dissect them were previously unavailable. To address this deficit, we have developed tools and techniques to examine the initial round of gene expression and a key polyprotein cleavage for the prototype flavivirus, yellow fever virus (YFV).

The central hypothesis of this Project 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 (e.g., members of a diverse protein family) that may not be well conserved between hosts. A key strength of our approach is that we will compare essential, early events across a well curated panel of flaviviruses and between human and mosquito host cells. Thus, we aspire to address the larger questions, “What features are shared by different flaviviruses?” and “How do flaviviruses solve the same problem in different environments?”

Identify essential determinants of genome uncoating.

By using a YFV entry reporter to quantitively monitor the translation of incoming virus particle-delivered genomes, we discovered that viral gene expression requires VCP/p97, a cellular enzyme that extracts ubiquitylated proteins from macromolecular complexes. VCP/p97 is needed at a post-fusion step of viral entry, suggesting that it functions during uncoating; these requirements were conserved among flaviviruses and between human and mosquito host cells. We are currently: i) evaluating the hypothesis that incoming capsid protein is ubiquitylated prior to genome uncoating; ii) applying complementary genetic and chemical-genetic approaches to identify determinants within the incoming nucleocapsid required for flavivirus uncoating; and iii) identifying components of the cellular ubiquitylation machinery required for flavivirus entry and uncoating.

Dissect the mechanisms of NS1-2A cleavage in flavivirus gene expression and replication.

Once uncoated, flavivirus genomes are directly translated, producing a large polyprotein that is proteolytically cleaved by viral and cellular proteases into mature viral proteins. However, the identity of the cellular protease(s) that cleaves the flavivirus NS1-2A polyprotein intermediate has remained elusive for decades. We have identified several candidate NS1-2A proteases and have shown that NS1-2A cleavage is essential for YFV replication. Intriguingly, YFV mutants designed to express NS1 and NS2A independent of NS1-2A cleavage were unable to replicate and could not be complemented in trans, indicating that the NS1-2A intermediate plays an important, if transient, cis-acting role at an early step in flavivirus replication. We are currently working to: i) definitively identify the cellular protease that cleaves the flavivirus NS1-2A polyprotein intermediate; and ii) examining the role of NS1-2A cleavage in the flavivirus lifecycle.

Funding: R01 AI154844