Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.
The Rift Valley fever virus (RVFV), a negative strand RNA virus spread by infected mosquitoes, affects livestock and humans who can develop a severe disease. We studied the structure of its nucleoprotein (N), which forms a filamentous coat that protects the viral RNA genome and is also required for RNA replication and transcription by the polymerase of the virus. We report the structure of the RVFV N protein at 1.6 Å resolution, which reveals hexameric rings with an external diameter of 100 Å that are formed by exchanges of N-terminal arms between the nearest neighbors. Electron microscopy of recombinant protein in complex with RNA shows that N also forms rings in solution. A reconstruction of the hexameric ring at 25 Å resolution is consistent with the hexamer structure determined by crystallography. We propose that slight structural variations would suffice to convert a ring-shaped oligomer into subunits with a super-helical arrangement and that this mode of protein-protein association forms the basis for the formation of filamentous ribonucleocapsids by this virus family. Both the RNA binding cleft and the multimerization site of the N protein can be targeted for the development of drugs against RVFV.