Dissection of the flavivirus fusion mechanism

Membrane fusion is an essential part of the life cycle of enveloped viruses, allowing virus entry and the release of the genetic information into the cell. This process is mediated by specific but structurally different classes of viral membrane proteins (fusion proteins) primed to undergo triggered conformational changes that drive the fusion reaction. In this proposal as yet undefined aspects of the fusion machinery of flaviviruses will be addressed using tick-borne encephalitis (TBE) virus as a model. Flaviviruses are small icosahedral enveloped viruses and include major human pathogens such as yellow fever, dengue, West Nile, Japanese encephalitis, and TBE viruses. These viruses enter cells by receptor-mediated endocytosis and fusion is triggered by the acidic pH in endosomes. Atomic details of soluble forms of the flavivirus fusion protein E have been determined by X-ray crystallography, but they lack important structural elements (the so-called `stem` and transmembrane anchor regions) that are essential for interactions during specific steps of the fusion process. Two important facets of the flavivirus fusion mechanism will be studied in this project: 1) Which amino acids in E are involved in the protonation-induced conformational changes that drive the fusion reaction (the low-pH trigger), and 2) what is the role of the `stem` in late stages of fusion. Both objectives will be investigated by the use of recombinant virus-like particles and their site specific mutagenesis in combination with biochemical and functional analyses related to the fusion process. These studies should allow to trap as yet elusive fusion intermediates and to reveal important mechanistic details of the flavivirus fusion process. They have also the potential to provide leads for the development of antiviral agents.

Membrane fusion is an essential part of the life cycle of enveloped viruses, allowing virus entry and the release of the genetic information into the cell. This process is mediated by specific but structurally different classes of viral membrane proteins (fusion proteins) primed to undergo triggered conformational changes that drive the fusion reaction. In this proposal as yet undefined aspects of the fusion machinery of flaviviruses will be addressed using tick-borne encephalitis (TBE) virus as a model. Flaviviruses are small icosahedral enveloped viruses and include major human pathogens such as yellow fever, dengue, West Nile, Japanese encephalitis, and TBE viruses. These viruses enter cells by receptor-mediated endocytosis and fusion is triggered by the acidic pH in endosomes. Atomic details of soluble forms of the flavivirus fusion protein E have been determined by X-ray crystallography, but they lack important structural elements (the so-called "stem" and transmembrane anchor regions) that are essential for interactions during specific steps of the fusion process. Two important facets of the flavivirus fusion mechanism will be studied in this project: 1) Which amino acids in E are involved in the protonation-induced conformational changes that drive the fusion reaction (the low-pH trigger), and 2) what is the role of the "stem" in late stages of fusion. Both objectives will be investigated by the use of recombinant virus-like particles and their site specific mutagenesis in combination with biochemical and functional analyses related to the fusion process. These studies should allow to trap as yet elusive fusion intermediates and to reveal important mechanistic details of the flavivirus fusion process. They have also the potential to provide leads for the development of antiviral agents.