Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation

Abstract

Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus, is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP-RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ringshaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization was observed for the recombinant BUNV NP-RNA complex, which was similar to the Rift Valley fever virus NP-RNA complex. This result suggested that the flexibility of one NP protomer with adjacent protomers underlies the BUNV ribonucleoprotein complex (RNP) formation. Electron microscopy revealed that the monomer-sized NP-RNA complex was the building block of the natural BUNV RNP. Combined with previous results indicating that mutagenesis of the interprotomer or protein-RNA interface affects BUNV replication, our structure provides a great potential for understanding the mechanism underlying negative-sense single-stranded RNA RNP formation and enables the development of antiviral therapies targeting BUNV RNP formation.assembly | packaging B unyaviruses constitute the largest segmented negative-sense single-stranded RNA (-ssRNA) virus family, which is subdivided into Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus genera (1). The genomes of Bunyaviridae members are featured by three segments, the large (L), middle (M), and small (S) segments (2, 3). The L segment encodes an RNA-dependent RNA polymerase (RdRp), the M segment encodes a precursor of glycoproteins (Gn and Gc), and the S segment encodes a nucleocapsid protein (NP). Moreover, a few Bunyaviridae members possess a nonstructural protein (NSs and/or NSm) using an ambisense coding strategy by the S and M segments (4).Similar to other -ssRNA viruses, all members of the Bunyaviridae family contain a ribonucleoprotein (RNP) complex composed of genomic RNA enwrapped by the NP (4). After entry into the cytoplasm through membrane fusion mediated by glycoproteins, the RNP is released from the virion and serves as the template with which the copackaged RdRp transcribes mRNAs from the viral genome in the RNP. In the later stage of virus replication, complementary positive-strand RNA (cRNA) is produced in the form of an RNP. The RNP serves as the template for replication that generates the viral genomic RNA in the form of an RNP ready to be packaged in the virion. Throughout...

Structure of the Leanyer orthobunyavirus nucleoprotein–RNA complex reveals unique architecture for RNA encapsidation

“…While the manuscript was under review, the structure of BUNV N belonging to the same Orthobunyavirus genus as LEAV N of the present study (the sequence identity between BUNV N and LEAV N is 39.8%) was solved by Li et al (39). The overall structure of BUNV N is similar to that of LEAV N. Superimposition studies reveal that the two structures can be superimposed with an rmsd of 1.4 Å over 204 matching Cα atoms.…”

Crystal structure of Schmallenberg orthobunyavirus nucleoprotein–RNA complex reveals a novel RNA sequestration mechanism

“…Note added in Revision: While responding to the reviewers' comments on this manuscript, lower resolution structures of N protein-ssRNA complexes from other viruses in the Orthobunyavirus genus, including La Crosse (3.4 Å resolution), Leanyer (2.78 Å resolution), and Bunyamwera viruses (3.2 Å resolution) were reported (Ariza et al 2013;Li et al 2013;Niu et al 2013;Reguera et al 2013). They all share similar structural features and proposed mode of RNA binding.…”

Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein ofTomato spotted wilt virus

“…Recently, the structures of N proteins from three animal virus genera of the Bunyaviridae family have been reported and several encapsidation mechanisms of viral genome RNAs have been suggested based on the formation of different oligomers of N proteins (Raymond et al, 2010(Raymond et al, , 2012Ferron et al, 2011;Guo et al, 2012;Carter et al, 2012;Wang et al, 2012;Dong et al, 2013;Niu et al, 2013;Li et al, 2013;Reguera et al, 2013). Interestingly, the sequence and structure of N proteins are highly conserved within a genus but differ between genera.…”