Transmembrane Domains in Flavivirus Entry and Assembly

Enveloped viruses gain entry into host cells by fusing their membrane with a cellular membrane which leads to the release of the viral genome into the interior of the cell. Fusion is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving membrane merger (fusion proteins). Studies with all three structural classes of viral fusion proteins have shown that transmembrane domains (TMDs) can be involved in several steps of the viral life cycle including membrane fusion, but the underlying mechanisms remain poorly defined. The class II fusion protein E of flaviviruses is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane (TM) helices which execute important functions in the biosynthesis and processing of the viral polyprotein. During assembly of virus particles, the E protein interacts with a second viral membrane protein (prM) that possesses a similar double membrane anchor. In the course of this project, we want to study the interactions within and/or between the unique double membrane anchors of E and prM in the assembly and membrane fusion processes of one of the major human pathogenic flaviviruses, tick-borne-encephalitis virus (TBEV). This virus is closely related to the mosquito-borne yellow fever, dengue, Japanese encephalitis, and West Nile viruses. The atomic structures of soluble truncated pre- and post- fusion forms of the E proteins of several flaviviruses have been determined by X-ray crystallography, but these structures lack the membrane anchors targeted in this proposal. Using the infectious clone of TBEV we will replace the E and prM TM regions by the homologous elements of the related Japanese encephalitis virus in different combinations. This approach should allow the investigation of TMD interactions required for fusion and assembly of flaviviruses without affecting their functions necessary for polyprotein processing. Further information will be acquired by serial passaging of selected viruses and the identification of compensatory mutations. The results obtained in this project should provide new insights into the complex molecular mechanisms of flavivirus assembly and membrane fusion and could be important for the specific design of antiviral agents against flaviviruses which are not yet available.

Flaviviruses comprise over 70 viruses many of which are human pathogens with a significant public health impact in different parts of the world. The most important disease agents are the mosquito?borne dengue, Zika, yellow fever, West Nile and Japanese encephalitis viruses as well as tick?borne encephalitis virus. In this project, we have investigated how flavivirus particles are assembled in the host cell and how these viruses become infectious (virus maturation) using tick?borne encephalitis virus.Flaviviruses possess a lipid membrane that is derived from the host cell. Two proteins are embedded in this membrane which surrounds a protein shell (capsid) that contains the viral genome. It is still unclear how the capsid is packaged into an infectious virus particle during assembly. Our study provides evidence that interactions of the domains anchoring the envelope proteins in the membrane (transmembrane domains) are essential for virus assembly. Disturbances of these interactions lead to the preferential formation of non?infectious, capsid?less subviral particles at the expense of whole infectious virions. We were also able to show that the involvement of the transmembrane domains is not only restricted to the assembly process but extends to the maturation of virus particles.Our work has thus generated novel insights into key steps of the infection of a host cell by flaviviruses. A better understanding of the viral life cycle can help in the development of as yet unavailable antiviral agents against these viruses.