Unit: Molecular Genetics of Bunyaviridae
Director: Bouloy Michèle
The main projects of the Unit deal with Rift Valley fever virus, which represents a model to study an hemorrhagic fever. The aim is to identify determinants of virulence, to decipher their mode of action and to produce an attenuated virus which could be utilized as a vaccin candidate.
Role of Interferon and NSs in pathogenesis
Rift Valley fever virus is an arbovirus of the Bunyaviridae family (Phlebovirus genus) transmitted by mosquitoes. It is endemic in sub-saharian Africa where it provokes many epizootics /epidemics. Recently it spread to Middle East. Ruminants are severely affected with a high mortality rate, abortions and teratogenesis. In humans, infection can lead to a fatal hemorrhagic fever with an acute hepatitis. The role of the non structural protein NSs has been determined only very recently. Studies on this phosphoprotein which forms filamentous structures in the nucleus whereas every step of the viral cycle occurs in the cytoplasm, represent our main axis of research. Genetic analysis of Clone 13, a naturally avirulent mutant, which possesses a large internal deletion in the NSs gene helped to show that NSs plays a major role in virulence and to demonstrate that NSs prevents the host antiviral response by blocking type I interferon production. Analysis of the mechanism utilized by NSs to act as antagonist of interferon production indicates that NSs is in fact a general inhibitor of cell transcription which interacts with p44 a sub-unit of the general transcription factor TFII H. By sequestering p44 and XPB, another sub-unit of TFII H in the nuclear filament of NSs, the cellular concnetration of TFII H is drastically reduced, making the cell unable to transcribe its RNAs.
Studies on transcription and replication with minigenomes.
To develop a system for reverse genetics, we established a basic transcription system in which viral proteins and RNAs were expressed from cDNA sequences cloned in plasmids. In a first step, the system was tested with a minigenome consisting in a viral like-RNA carrying the non coding sequences of the L, M or S genomic segments and the Chloramphenicol acetyl transferase gene in the antisense orientation. These RNA molecules were synthesized from plasmids under the control of the cellular Pol I promoter. Plasmids carrying the S-CAT, M-CAT and L-CAT sequences were constructed and these genome-like RNAs were efficiently recognized and transcribed by the viral complex composed of the L polymerase and the N nucleoprotein.
Recombinant antigen for serological diagnosis
Several Bunyaviruses replicate poorly in cell cultures (Hantavirus) or they must be handled in a high security level laboratory (Crimean-Congo hemorrhagic fever virus). During infection with Bunyaviruses, the nucleorprotein N is the major antigen which induces a strong humoral response. We developped an ELISA to detect antibodies using a recombinant form of Puumala hantavirus nucleoprotein. This antigen allows the diagnosis of this non fatal hemorrhagic fever which circulates in the North and Est of France as well as the detect viral infections in bank voles, the rodents carrying the virus.
Photo: Fig 1. The RVFV NSs protein forming a nuclear filament in infected Vero cells
Keywords: hemorrhagic fever, arbovirus, zoonosis, hantavirus, pathogenesis