Recombination and packaging of flaviviruses

The genus Flavivirus comprises several arthropod-transmitted human pathogens of global importance, three of which will be investigated in this project: Tick-borne encephalitis virus (TBEV), West Nile virus (WNV) and Japanese encephalitis virus (JEV). Flaviviruses have a single-stranded, positive sense RNA genome. It associates with the viral capsid protein C to form the nucleocapsid and is engulfed by a lipid envelope carrying two further viral proteins. This project addresses two fundamental issues that underlie the biology of these disease agents, namely packaging and recombination of the flavivirus genome. Packaging of the viral genome into nucleocapsids is a crucial step during assembly of infectious virions. However, the determinants which provide specificity and efficiency to this process are mostly unknown. A better understanding of packaging will not only elucidate evolutionary constraints of these viruses, but potentially open new avenues for therapeutic interventions. Many RNA viruses exhibit a large degree of evolutionary flexibility which can also be caused by recombination events between different RNA molecules. In the case of flaviviruses, the rate, role and molecular premise of recombination are controversial. Systems to study recombination in the laboratory do not yet exist but are badly needed in face of the continuing emergence of flaviviruses leading to frequent co-circulation of different flaviviruses, the use of attenuated live vaccines in endemic regions, and the development of new multivalent flavivirus vaccines. Both packaging and recombination are closely coupled to the process of RNA replication. We have developed a new system which allows the investigation of both of these processes when two flavivirus RNA molecules simultaneously replicate in the same host cell. Each replicon lacks part of the structural protein genes and is therefore incapable of forming infectious progeny. However, upon introduction of two different replicons into the same host cell, they can form an "infectious alliance" by trans-complementing each other. This approach for the first time allows the analysis of the determinants of packaging in a context which closely mimics the natural situation. At the same time, the experimental setup represents a "recombination trap" which provides a sensitive tool to detect recombination events between the two replicons potentially leading to the emergence of infectious virus progeny. The new system will be used to study packaging and recombination of TBEV in detail. In order to obtain a more comprehensive view, it will also be applied to two other flaviviruses (WNV and JEV). Furthermore, it will provide the tools to investigate packaging and recombination between heterologous flavivirus genomes, an issue of increasing importance due to the growing risk of simultaneous infection by different flaviviruses and live vaccines. The results of this study will be instrumental towards understanding flavivirus evolution, predicting new health risks, and guiding future antiviral strategies.

In this project, we have investigated fundamental aspects of the molecular biology of flaviviruses that are also of practical relevance. These viruses are transmitted by mosquitoes or ticks and comprise important human pathogens such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis viruses. The results obtained have an impact on the development and use of flavivirus vaccines and can provide new leads for finding targets of antiviral agents. Specifically, we have addressed the question whether during mixed infections with different flaviviruses and/or vaccination with live vaccines new and potentially harmful viruses can develop by a process termed RNA recombination. Thereby two different but related viruses replicate their genetic information in one and the same cell and theoretically allow for the generation of mixed genomes that can give rise to a new virus with unpredictable properties. For resolving this important question in the case of flaviviruses, we have established a highly sensitive experimental system termed recombination trap - that allows the detection of newly formed viruses even when recombination would be a very rare event. Our data are reassuring for those developing live flavivirus vaccines, because we demonstrated that the risk of generating new viruses through recombination is apparently very low with flaviviruses and not expected to be a serious problem for vaccine usage. The results of this part of our project thus resolved a controversial discussion among researchers, regulators, and vaccine manufacturers that was of special importance in the context of new tetravalent live dengue vaccines requiring the simultaneous application and replication of four different apathogenic viruses in the vaccine recipient.In further parts of our project, we investigated the role of specific structural elements contained in the viral RNA (which provides the genetic information for this kind of viruses) and in proteins at the surface of the virus particle that control the entry into cells, i.e. the initial phase of infection. In both instances, our findings provide new insights into the mechanisms of the flavivirus infection process and this knowledge can provide new leads for identifying vulnerable targets in the virus that could be used for developing specific antiviral agents. This is especially true for the structures involved in virus entry, because they are accessible when the virus is still outside the cell and drugs targeting such sites would therefore not have to penetrate into infected cells where the virus already replicates.