Nucleocapsid-like cryoEM structure of influenza B virus

Fig 1. Recombinant B/NPWT, B/NP∆64 and B/NP∆68 RNA affinity and oligomeric state. A. Schematic representation of B/NPWT, B/NP∆64 and B/NP∆68 in light blue, pink and purple, respectively. The rectangle represents the folded core of the protein. B. Fluorescence anisotropy measurements to quantify B/NPWT, B/NP∆64 and B/NP∆68 affinity for the 12-mer or 24-mer synthetic RNAs, in 50 or 150 mM NaCl. Color coding consistent with A. C. Visualization by electron microscopy with negative staining of B/NPWT, B/NP∆64 and B/NP∆68 purified in 50 mM NaCl buffer. Arrows points at monomeric protomers. Scale bars represent 100 nm. D. Size exclusion chromatography profiles of B/NPWT, B/NP∆64 and B/NP∆68 eluted in the same buffer with 50 mM NaCl.

Fig 2. Visualization of RNP-like particles and vRNP. A. Observation by electron microscopy with negative staining of B/NPWT, B/NP∆64 and B/NP∆68 incubated with the 12-mer or 24-mer synthetic RNA. The formation of RNP-like filaments is indicated with green arrows. B. B/vRNP extracted from viruses observed by electron microscopy with negative staining. Bottom right, close up view of one B/vRNP, with white arrows pointing to the major groove. Scale bars represent 100 nm.

Fig 3. Cryo-EM structure of B/NP∆68 RNP-like particles. A. Left, cryo-EM 3D reconstruction of B/NP∆68 RNP-like particle at 6 Å resolution. Right, fitting of B/NP∆68 atomic model in the helically symmetrized cryo-EM structure. The antiparallel helical structure displays two turns. Each strand is colored with a sky-blue-to-blue or light-yellow-to-orange color gradient to indicate the positive polarity of each strand (light toward dark color). Red asterisks indicate the major grooves. B. Atomic model built from the focused refinement cryo-EM map encompassing 2-2 NPs. Consecutive NPs from the Strand-1 are colored in gradient of blue, while NPs on the Strand-2 appear in gradient of orange. The RNA is shown as a pink ribbon. C-F. Close-ups on residues involved in NP-NP and NP-RNA interactions. NP protomers are displayed in a cartoon representation, with colors identical to B. Dotted lines represent putative hydrogen bonds.

Fig 4. Conformational changes induced by RNA binding. A, C, E. Cartoon representations, with cylindric α -helices, of crystal structures of a single protomer of B/NP, A/NP and D/NP from trimeric or tetrameric oligomers in the absence of RNA. The N-terminal is marked with a red asterisk, unfolded loops 127-146 in B/NP, 72-90 in A/NP and 66-77, 199-217 in D/NP are highlighted in orange, purple and green, respectively. B, D, F. Cartoon representations, with cylindric α-helices, of single protomers of B/NP, A/NP and D/NP from RNP-like or vRNP cryo-EM structures. RNA is shown as a dark grey line, and N-termini are marked with a red asterisk. Newly folded α-helices and ß-strands are highlighted with darker color and a dotted line. The green stars (B, D) indicate the short loops in A/NP and B/NP while in D/NP the corresponding longer loop folds into the 210 hairpin (F).

Fig 5. A. Comparison of D/vRNP (EMDB: 44980), A/RNP-like (EMDB: 51188) and B/RNP-like structures. Each double stranded antiparallel helical map is displayed with one strand colored in white (NP) and blue (RNA), and the other strand in grey (NP) and yellow (RNA). The applied helical parameters and resolutions are indicated below each structure. B. Proposed model of the location of the B/NP disordered N-terminal tail in the helical RNP architecture. Models were generated with the RANCH program and fitted into our experimental cryo-EM structure. Views are from the top (left) or side (right) of the helical filament shown as a surface. NPs on Strand-1 are colored in a blue gradient and NPs on Strand-2 are colored in an orange gradient. RNA is colored in grey. The modeled disordered tails are shown as cartoons and residues of the NLS are colored in red.