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The ribonucleoprotein from Rift-Valley Fever Virus

People involved : Dr Julien Lescar, Dr Fran├žois Ferron, Joelle Boretto

Bunyaviridae is a family of negative-stranded RNA viruses. Bunyaviridae are vector-borne viruses responsible for hemorrhagic fever, with a high mortality level, for which no efficient vaccines or antiviral drugs are available. Though generally found in arthropods or rodents, certain viruses that belong to this family occasionally infect humans. This family encompasses Orthobunyavirus, Hantavirus, Nairovirus, Tospovirus, and Phlebovirus, gathering more than 250 viruses. The Phlebovirus includes Rift Valley fever virus (RVFV), Toscana virus (TOSV), sandfly fever Sicilian virus, Punta Toro virus (PTV), and Uukuniemi virus (UUKV). RVFV causes a disease endemic to sub-Saharan Africa that has emerged in explosive, mosquito-borne epidemics that resulted in massive economic loss of sheep and cattle, while also causing hemorrhagic fever, encephalitis, retinal vasculitis, and lesser diseases in humans. Since the late 1970s, several large outbreaks of Rift Valley fever have occurred outside of sub-Saharan Africa, e.g., in Egypt, Madagascar, Saudi Arabia, and Yemen. RVFV have a segmented genome composed of three single negative stranded linear RNA named L, M, S. The L segment encodes the RNA Dependent RNA-polymerase, necessary for viral RNA replication and mRNA synthesis. The M segment encodes the viral glycoproteins, which project from the viral surface and aid the virus in attaching to and entering the host cell. The S segment encodes the nucleoprotein (N). N protects the RNA and forms tracks for the polymerase forming the ribonucleoprotein complex (RNP). N is the major component of this complex. N is a 27 kDa protein. At the start of the project, little was known about the structure of this protein and the way it assembles. Different strategies to assemble N are used by various Bunyaviridae. For Hantavirus, N has the ability to pre-form trimeric complexes through a coiled-coil domain prior to RNA encapsidation. For Phlebovirus (Toscana virus, Rift Valley Fever Virus), N forms dimers, while for Orthobunyavirus N seems to remain in its monomeric form.

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The work performed by Fran├žois Ferron [1] has been to characterize the N protein from Bunyaviridae (starting with RVFV) using both X-ray crystallography and Electron Microscopy (EM), in order to understand the mechanisms for self-assembly and characterize the mechanisms for RNA encapsidation. We successfully cloned, expressed and purified N in quantities suitable for crystallography. We already obtained crystals that diffract up to 2 A˚ that belong to the space group P6 and with unit cell dimensions: a=b=175.24 A˚ , c=47.32 A˚. We solved the structure (Fig. 1) using crystals of the seleniated protein. Thanks to a collaboration with the group of Dr Thomas Walz at Harvard Medical School, we also used cryo-electron microscopy to characterize the structure of the ribonucleoprotein.

The analysis of negatively stain samples shows that the Nucleoprotein forms several assemblies ranging from pentamers to octamers with a majority of hexameric rings. We carried out reconstruction by preparing cryocooled and negatively stained samples. We have obtained the reconstruction of the pentameric complex at a resolution of 20 A˚. We could fit the X-ray crystal structure into the cryo-EM reconstruction (Fig. 2).

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We solved the crystallographic structure of N from RVFV [2], the major component of the ribonucleoprotein complex at 1.6 A˚ resolution. The results reveal a helical bundle whose folding is stabilized by two N-terminal helices contributed by another monomer. Hexameric rings about 45 A˚ thick and with an external diameter of 100 A˚ are formed via directional exchanges of N-terminal arms between nearest neighbors around the crystallographic 6-fold axis. Likewise, using recombinant N proteins expressed in E. coli and purified also under native conditions, Cryo-EM single-particle reconstructions show rings into which the crystallographic hexamer can be fitted. Both the RNA binding groove and the multimerization site form potential targets for antiviral drug development. We intend to carry on this work by characterizing the interactions between N and RNA both functionally and using X-ray crystallographic methods. In collaboration with the screening platform AD2P (JC Guillemot), we are also planning to screen for compounds able to interfere with multimerization of the N protein or RNA binding. Once identified, these compounds will be cocrystallized with the N protein and their efficacy improved using medicinal chemistry methods (Karine Alvarez) informed by 3D structural information.


[1] during his Post-doctoral fellowship supported by an ATIP grant from the CNRS
[2] Ferron F, Li Z, Danek EI, Luo D, Wong Y, Coutard B, Lantez V, Charrel R, Canard B, Walz T, Lescar J (2011) PLoS Pathog 7 e1002030
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