E protein interactions in flavivirus membrane fusion

Flaviviruses enter cells by endosomal uptake followed by fusion of viral and endosomal membranes. The main player in these essential entry events is the envelope protein E, a class II viral fusion protein. Important structural information was obtained on E and its role in membrane fusion including the crystal structures of the E ectodomain in its pre- and post-fusion form. E is composed of three domains (DI-III) that make an extended rod in the pre- fusion conformation with DI at its center. DII has a "fusion loop" (FL) at its distal end, and DIII connects to the C- terminal transmembrane domains (TMDs) via a region termed "stem". Exposure to the acidic endosomal environment leads to E dimer dissociation with concomitant FL exposure, which can insert into the endosomal membrane. The E monomers then trimerize and adopt a hairpin conformation, with the C-terminus of DIII directed towards the FLs. The current models postulate that the stem displays key interactions with the body of the trimer, such that the TMDs become juxtaposed to the FLs. However, a crystal structure of the complete ectodomain of flavivirus E containing the stem in its post-fusion conformation is not available today. Recent data on dengue virus E suggest that the upstream region of the stem is disordered. The stem of the class II fusion proteins of togaviruses is much shorter and structural details of the interactions of the stem with DII have been elucidated recently. In addition, the stem of the EFF-1 fusion protein of C. elegans a distant homolog of flavivirus E has about the same number of amino acids as in flavivirus E. The upstream half of the EFF-1 stem is disordered in the post-fusion structure and the downstream region shows similar interactions as in togaviruses. Because stem interactions are essential in providing the energy to bring together the FLs and the TMDs during fusion, the structure of this region could provide an important target for antiviral strategies. In this project, we therefore propose to bring structural information on the stem to gain novel insights into the flavivirus fusion mechanism. We will employ two strategies for producing E proteins for crystallography and functional studies. The first is to introduce deletions based on the structural data mentioned above, eliminating the upstream part of the stem. The second is to study E from flaviruses that infect exclusively insects. The C-terminal part of their E protein appears to be more similar to that of togaviruses, containing a much shorter stem. Structural information on the interactions of the stem with the body of E will be a major leap forward in understanding the mechanism of flavivirus membrane fusion, and open the way for new antiviral strategies targeting this region.

Flaviviruses comprise a number of important human pathogens such as the dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and tick-borne encephalitis (TBE) viruses. Since they are enveloped viruses, infection of host cells includes a step in which the viral membrane fuses with a cellular membrane. This fusion process results in the delivery of the viral genome into the cell and the initiation of virus replication. Flavivirus fusion is controlled by the major envelope protein E (a class II viral fusion protein) that is primed to undergo triggered structural changes, thereby driving the merger of the two membranes. In this project, we gained novel insights into the flavivirus fusion mechanism by investigating interactions of different parts of E that are formed during the conversion into the fusogenic state. We obtained new atomic details of the TBE virus E protein and identified important elements of E that are involved in this crucial step. Our data thus extend the existing models of the flavivirus fusion process and can open the way for new antiviral strategies targeting these regions.