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

Abstract

Negative-stranded RNA viruses cover their genome with nucleoprotein (N) to protect it from the human innate immune system. Abrogation of the function of N offers a unique opportunity to combat the spread of the viruses. Here, we describe a unique fold of N from Leanyer virus (LEAV, Orthobunyavirus genus, Bunyaviridae family) in complex with single-stranded RNA refined to 2.78 Å resolution as well as a 2.68 Å resolution structure of LEAV N-ssDNA complex. LEAV N is made up of an N-and a C-terminal lobe, with the RNA binding site located at the junction of these lobes. The LEAV N tetramer binds a 44-nucleotide-long single-stranded RNA chain. Hence, oligomerization of N is essential for encapsidation of the entire genome and is accomplished by using extensions at the N and C terminus. Molecular details of the oligomerization of N are illustrated in the structure where a circular ring-like tertiary assembly of a tetramer of LEAV N is observed tethering the RNA in a positively charged cavity running along the inner edge. Hydrogen bonds between N and the C2 hydroxyl group of ribose sugar explain the specificity of LEAV N for RNA over DNA. In addition, base-specific hydrogen bonds suggest that some regions of RNA bind N more tightly than others. Hinge movements around F20 and V125 assist in the reversal of capsidation during transcription and replication of the virus. Electron microscopic images of the ribonucleoprotein complexes of LEAV N reveal a filamentous assembly similar to those found in phleboviruses.crystal structure | nucleoprotein complex with ssRNA | RNP

Genome encapsidation by orthobunyavirus nucleoproteins

“…Each NP subunit binds 11 nucleotides of RNA according to Niu et al, Reguera et al, and Ariza et al (4,5,7). Bound RNA is largely inaccessible, consistent with the observation that Orthobunyavirus NP-RNA complexes are generally resistant to RNase treatment (3-5, 7).…”

“…Like in other −ssRNA viruses, the genomic RNAs of bunyaviruses are extensively coated by NP and associated with the viral RNA polymerase or L protein to form RNPs, which are responsible for viral RNA synthesis as well as viral genome packaging (1,8,9). Electron microscopy (EM) images show that bunyavirus RNPs form closed circular structures that are often loosely coiled without apparent symmetry (3,4,8).…”

“…Each Orthobunyavirus NP folds into a compact structure that can be divided into four parts: the N-arm, N-terminal domain, C-terminal domain, and C-arm. The N-/C-terminal domains, formed by the N-/C-terminal halves of the polypeptide chain, comprise the NP structure core (3)(4)(5)(6)(7). The N-/C-arms are short sequences from the N/C termini, and they play important roles in mediating NP-NP interaction.…”

Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage

“…Human cystatin C and the activated cathepsins were mixed in the assay buffer for 1 min, 5 min, 30 min, 1 h and 4 h at 16 °C or 25 °C and analyzed using SDS-PAGE analysis. npg NaCl) were collected on the SIBYLS beamline at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, and the data were treated as previously described [44,45]. Briefly, each sample was measured at three exposures (0.5, 1.0, and 6.0 sec) and three concentrations (2.5, 5.0, and 10.0 mg/ml) at 10 °C to exclude aggregation and radiation-damage effects.…”

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.…”