Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization

Abstract

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N-RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

Figure 1.

Crystal structures of the apo SBV N protein and BUNV N protein in complex with RNA. (A) Sequence alignment of SBV N and BUNV N with secondary structure assignments for SBV shown above in cartoon form (β-strands as arrows; α-helices as cylinders; 3₁₀ helices as coils). Conserved residues are highlighted. The N-terminal arm is underscored in blue, globular domain in yellow and C-terminal domain in red. (B) SBV N tetramer in ribbons representation. (C) BUNV N tetramer (ribbons) bound to RNA (cyan, sticks). In both B and C, the N- and C- termini of the yellow monomer are marked with a blue and red sphere, respectively (note that a small part of the N-terminal arm in both SBV and BUNV N is not visible in electron density; therefore, it is not built as part of the models). (D) Protein monomers of SBV N (left), BUNV N (right) and the superposition of both (centre). Figure 1A has been made using A-line (41). Figure 1 B–D and all other figures were made using PyMol (Version 1.5.0.4 Schrödinger).

Figure 2.

RNA binding by orthobunyavirus N proteins. (A) Electrostatic surface of a SBV N protomer reveals the electropositive RNA-binding groove. (B) Two molecules of BUNV N bound to RNA (as seen from inside the tetramer; RNA is yellow sticks representation). (C) BUNV N bound to RNA (red) with solvent accessible surface shown (yellow/greens, transparent).

Figure 3.

Structure–function analysis. (A) Structure of the BUNV N protein monomer bound to RNA shown with residues highlighted in cyan that have been mutated and tested for RNA-binding affinity, and mutant residues that generate temperature-sensitive viruses in yellow tested in the replicon assay. (B) Fluorescence anisotropy RNA-binding assay. Wild-type (WT) BUNV N, SBV N and mutant BUNV N proteins were tested for binding to a fluorescein-labelled 48mer. (C) Table of affinities of BUNV N mutants and SBV N wild-type relative to the BUNV N WT. (D) The replicon assay tests relative activity of the N protein in the replication and transcription of a model BUNV segment; ts19 shows considerably diminished transcription activity, whereas ts63 shows dramatically reduced replication. See text for details of mutations.

Figure 4.

EM of purified tetramers and native RNPs. (A) Micrograph of negative-stained RNP purified from BUNV virus particles in cell culture. (B) Single particle average of purified SBV tetramers bound to synthetic 48mer RNA (inset shows the crystal structure of the BUNV N tetramer bound to RNA). (C) Zoomed in view of a section of native RNP at the same scale as B (the 10-nm scale bar applies to both B and C).