Tick-borne flavivirus structures and maturation triggers

Arthropod-transmitted flaviviruses encompass several human pathogens which pose significant threats to public health in different parts of the world and have the potential to emerge in previously unaffected regions. Most of these especially those found in tropical and subtropical regions - are transmitted to their vertebrate hosts by mosquitoes including yellow fever, dengue, Japanese encephalitis and West Nile viruses. Tick-borne encephalitis virus, on the other hand, is found in many parts of Europe as well as Central and Eastern Asia where it circulates in natural foci between ticks and small rodents. Despite being an RNA virus with an intrinsically high mutation frequency, TBE virus is very stable under natural ecological conditions, indicating strong forces imposed by adaptation to its specific ecological niche that counteract divergent evolution. Structural information on flavivirus particles in immature, mature and intermediate forms and the mechanisms of transitions between these stages has so far been generated only for mosquito-borne viruses, primarily for dengue virus serotype 2. There are several lines of evidence, however, that tick-borne flaviviruses differ in specific aspects of viral particle structure and the control of acidic-pH-induced conformational changes required for virus maturation and/or cell entry. It is therefore a major objective of this proposal to determine the protein organization of the viral envelope in immature and mature TBE virus particles as well as in intermediate stages of the maturation process and to obtain a high resolution structure of the complex between two different envelope proteins found in immature particles only. This part of the project will rely on an established collaboration with excellent structural biologists (Felix Rey, Institut Pasteur, Paris for X-ray crystallography and Jean Lepault, CNRS, in Gif-sur-Yvette for cryo electron microscopy) which has already proven to be extremely effective and successful in the past. In addition, the project will specifically investigate potential sensors of acidic pH that differ between mosquito-borne and tick- borne flaviviruses and control the triggers of virus maturation and/or cell entry. Together, the proposed work will yield a combination of novel structural and functional information of an important human pathogen and provide insights into host-specific adaptations. The data generated will also increase our understanding of the molecular antigenic structure of TBE virus which is highly relevant for the structure-based design of novel vaccines and knowledge of molecular details of the viral maturation process can provide new targets for the development of antiviral agents.

Viruses are small non-living molecular entities that have the capacity to enter living cells and parasite cellular processes for their own reproduction. The infection process is completed by the release of newly produced virus particles, competent to infect new cells. Important stages of the viral replication cycle are dependent on structural changes in viral proteins that are induced by cellular triggers, requiring control mechanisms to ensure that viral particles disassemble during virus entry but are stable during assembly and release from infected cells. In our project, we addressed these crucial aspects of virus biology in the area of flaviviruses, which comprise a number of important human pathogens transmitted either by mosquitoes (yellow fever, dengue, Zika, West Nile viruses) or by ticks (tick-borne encephalitis - TBE-virus). In these cases, the structural changes in the viral life cycle are controlled by the acidic pH in intracellular compartments encountered by the virus during entry and egress. By combining X-ray crystallography and mutational analyses of TBE virus envelope proteins we provide novel insights into the atomic details of how these viruses protect their structural integrity in the processes of virus assembly, maturation and release and at the same time allow the generation of molecular machines capable of disintegration when infecting new cells. The results of this project fill an important gap in our understanding of flavivirus biology, including differences between mosquito-borne and tick-borne viruses, and provide new molecular targets for the development of antiviral substances.