Structures and Mechanisms of Flavivirus Membrane Fusion

Membrane fusion plays a fundamental role in numerous biological processes such as intracellular transport and the entry of enveloped viruses into host cells. In the case of viruses, this process is controlled by the activity of specific but structurally different classes of viral membrane proteins (fusion proteins) primed to undergo conformational changes that drive fusion. A triggering event leads to the exposure of a hydrophobic sequence element of the fusion protein (fusion peptide) that inserts into the target membrane. Upon refolding of the protein into a hairpin-like structure with the fusion peptide and the membrane anchor on the same side of the molecule, the membranes are drawn together, lipid mixing occurs and finally a fusion pore opens through which the viral genetic information is released into the interior of the cell. In this project we will address as yet unknown aspects of the flavivirus fusion process. Flaviviruses are a genus of enveloped viruses - including important human pathogens like yellow fever, dengue, West Nile (WN), Japanese encephalitis, and tick-borne encephalitis (TBE) viruses - and enter host cells by receptor-mediated endocytosis and low-pH-induced fusion. The atomic structures of the pre- and postfusion conformations of the fusion proteins (envelope protein E) of dengue, WN and TBE virus have been determined by X-ray crystallography, but they lack important structural elements ( the so-called `stem` and membrane anchor regions) that have been hypothesized to be essential for the completion of fusion. The goal of this research proposal is to define these final stages of the flavivirus membrane fusion process by several approaches. For this purpose we will 1) attempt to determine the post-fusion structure of the E protein containing the stem-region by X-ray crystallography, in collaboration with specialists at the Institut Pasteur in France, and 2) investigate the role of the stem and the fusion peptide in the final stages of membrane fusion by site-directed mutagenesis in combination with biochemical and functional analyses related to the fusion process. The structural and mechanistic details revealed in this study should provide new insights into the regulation of the entry of flaviviruses into host cells, which - as shown for the human immunodeficiency virus (HIV) - can lead to the identification of specific targets for the development of antiviral agents.

In the context of this project, we investigated molecular details of how flaviviruses, especially tick-borne encephalitis virus (TBEV), infect host cells. TBEV causes more than 10,000 cases per year in large parts of Europe and Asia. It is closely related to the human pathogenic mosquito-borne dengue, yellow fever, Zika, West Nile and Japanese encephalitis viruses. Specific drugs are not available against flaviviruses and a detailed molecular understanding of virus specific-processes in host cells might therefore increase the probability of success of antivirals development. Our work focused on virus entry, i.e. virus host cell binding and membrane fusion in which the viral membrane fuses with a cellular membrane thus allowing infection of the cell. Both processes are mediated by a viral surface protein (fusion protein) that is triggered by cellular factors to adopt its fusion-active form. In this project, we established experimental set-ups for the investigation of the different steps of virus entry. We identified structural elements of the fusion protein that are involved in host-cell binding as well as different steps of membrane fusion and characterized their role in these processes. As shown for human immune deficiency virus (HIV), a detailed knowledge of virus entry offers excellent targets for antiviral drugs. The results generated in our studies provide new insights into the molecular understanding of key steps required for flavivirus infection and can thus facilitate the specific design of antiviral agents targeting specifically these stages of the viral life cycle.