Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

Abstract

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-Å crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA species of ∼100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous ∼100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.ribonucleoprotein | viral protein | segmented genome | negative-sense RNA virus

Expression, purification and crystallization of the Crimean–Congo haemorrhagic fever virus nucleocapsid protein

“…The percentage of each type of secondary structure was predicted to be 66% -helix, 0.005% -sheet and 29% random coil by the k2d neuralnetwork program. The high proportion of -helices in the CCHFV N protein is common to many negative-sense RNA virus nucleocapsid proteins and is consistent with the crystal structure of the RVFV N protein (Ferron et al, 2011;Raymond et al, 2010). Furthermore, the distinctive -helical spectrum suggests that very little -sheet secondary structure was present and suggests that the CCHFV N protein is unlikely to possess domains homologous to the Lassa fever virus N protein exonuclease C-terminal domain, which is predominantly composed of -sheet.…”

“…Attempts to determine initial phase information by molecular-replacement approaches using a monomer and a hexamer of the recently solved RVFV N protein (Raymond et al, 2010;Ferron et al, 2011) as a search model failed. This may reflect significant differences in secondary, tertiary and quaternary structure between these two nucleocapsids.…”

“…These data showed that the RVFV N protein forms a ring-shaped hexamer with sixfold symmetry. These hexamers reveal the structural basis for multimerization, as well as providing evidence for an RNA-binding groove populated with many positively charged amino acids postulated to bind the viral RNA within the RNP (Ferron et al, 2011;Raymond et al, 2010).…”

Crimean–Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses

“…Because the Bunyaviridae family includes hundreds of different genuses and the nucleoproteins of each genus show little homology or other features in common, the addressing of the exact function and mechanism of each group of nucleoproteins case by case is necessary for our understanding on bunyaviruses replication and assembly. To our knowledge, the only NP structure reported to date in the Bunyaviridae family is the Rift Valley fever virus nucleoprotein (9,10), which shows weak binding affinity with RNA and displays a conformational change before oligomerization into a ribonucleoprotein (RNP) complex (9). Previous structures of NPs from other families, such as the influenza virus (Orthomyxovidae) (11,12), rabies virus (Rhabdoviridae) (13), vesicular stomatitis virus (VSV) (Rhabdoviridae) (14), and borna disease virus (BDV) (Bornaviridae) (15) have shown how NPs assemble with RNA to form RNPs.…”

Helical capsids of plant viruses: architecture with structural lability

“…These observations suggest that in flexible viruses, the shape of RNA-protein assemblies, instead of being determined by axial contacts, more probably depends on the degree of freedom exerted by the flexible hinge interconnecting the protein core and the arm interacting with a neighbouring protein subunit. Indeed, the interacting arm of monomeric phlebovirus NP protein can fold back and interact with the core domain of the same protein molecule, therefore showing a wide range of hinge bending (Raymond et al, 2010), whereas the potexvirus CP molecule lacking an interacting counterpart has a structured N-terminal arm extending outward from the protein core (Yang et al, 2012). Therefore, we assume that the filamentous shape of potexvirus virions is determined by a limited flexibility of the hinge between the IA and core domains of CP.…”