Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures

Abstract

Ribonucleoprotein (RNP) complexes of influenza viruses are composed of multiple copies of the viral nucleoprotein (NP) that can form filamentous supra-structures. RNPs package distinct viral genomic RNA segments of different lengths into pleomorphic influenza virions. RNPs also function in viral RNA transcription and replication. Different RNP segments have varying lengths, but all must be incorporated into virions during assembly and then released during viral entry for productive infection cycles. RNP structures serve varied functions in the viral replication cycle, therefore understanding their molecular organization and flexibility is essential to understanding these functions. Here, we show using electron tomography and image analyses that isolated RNP filaments are not rigid helical structures, but instead display variations in lengths, curvatures, and even tolerated kinks and local unwinding. Additionally, we observed NP rings within RNP preparations, which were commonly composed of 5, 6, or 7 NP molecules and were of similar widths to filaments, suggesting plasticity in NP-NP interactions mediate RNP structural polymorphism. To demonstrate that NP alone could generate rings of variable oligomeric state, we performed 2D single particle image analysis on recombinant NP and found that rings of 4 and 5 protomers dominated, but rings of all compositions up to 7 were directly observed with variable frequency. This structural flexibility may be needed as RNPs carry out the interactions and conformational changes required for RNP assembly and genome packaging as well as virus uncoating.

Keywords: Electron microscopy; Flexibility; Influenza virus; RNPs; Structure; Tomography.

Fig. 1. Characterization of purified influenza RNPs

(A) SDS-PAGE analysis of purified ribonucleoprotein complexes (RNPs). Lane 1 contains a molecular weight standard and lane 2 contains purified RNPs. (B) Image of a field of RNP complexes by negative-staining electron microscopy. Black arrows denote the longer filamentous complexes while white arrows denote the smaller complexes. Scale bar, 100 nm. (C) Zoomed–in view of RNP complexes from the area in brackets in panel B. (D) A lateral section through a denoised tomographic reconstruction of RNPs. Examples of RNP ring-like structures (white arrows) and RNP filaments (black arrows) are indicated. (E) Gallery of examples of RNPs with their long filament axis approximately vertically oriented. RNP filaments are of varying lengths arranged from longest to shortest (I, II, III, IV). Images were not rotated. Light-green arrows denote apparent subunits that appear as white dots within the structures. Three vertical black arrows in panel III indicate three interpretable distinct structural units. (F) Filament with three units indicated by brackets. (G) Filament with each unit outlined with a blue schematic. The ends of two striations within each unit are indicated by asterisks. Image contrast is shown with protein represented by white contrast. Scale bars (C–G) 20 nm.

Fig. 2. Characterization of RNP size, curvature and organization

(A) Gallery of lateral sections through subtomograms of filamentous structures (I–VI), illustrating RNPs with straight and bent appearances. Scale bar, 10 nm. (B) Distribution of RNP lengths as a histogram of RNP lengths measured directly from the RNP tomograms, which indicates the percentage of filaments within that length range. Overlayed on the same plot is a histogram of the anticipated RNA segment lengths in kilobases (blue dashed line, blue upper axis). The secondary axis was aligned using a scaling factor to indicate correspondence between these two distributions. These two different axes were related by a best fit of 167 nucleotides to 10 nm of RNP. (C) Image of an RNP with high curvature. (D) Image of an RNP that is approximately linear. Traces along the center of the RNPs were fit to the surface of a sphere to quantitate curvature, illustrated as a cyan circle in panel C, or merely the visible arc of that circle in panel D. (E) Distribution of the curvature of RNPs as a histogram of curvature values for all RNPs. As curvature approaches zero RNPs are approximately straight. (F) Class averages of projection images of RNP filaments with stacked array (1), double-helical (2), globular array (3), and loose ring patterned structures that represented a collection of particles largely defined as not being members of classes 1–3 (4). (G) Spacing between turns in a putatively resolved double-helical filament was 17 nm (left). In comparison, a previously reported NP filament reconstruction, EMD 2209, indicates a closely matching distance of 16 nm (right).

Fig. 3. Analysis of RNP configurations by cryo-electron tomography

(A) Gallery (I–XII) of x–y sections through subtomograms of individual RNP filaments indicating RNPs with twisted, straight and curved appearances. Scale bar is 50 nm. (B) Column scatter plot of RNP lengths measured from cryo-tomography. (C) Histogram of curvature values from 3D fitting of the RNP traces to the surface of spheres, illustrating both linear and curved RNPs were present. (D, E) Molecular envelopes of two RNPs were fit with individual NP proteins (PDB ID 2IQH) to illustrate the relative size and NP occupancy in individual RNPs. Individual NP molecules are indicated by different colors.

Fig. 4. Analysis of subunit number and size within ring structures

(A) Gallery of lateral slices through representative subvolumes of individual ring complexes (I–XII). Protein density is white (positive contrast). Each panel is an individual non-averaged subvolume. (B) 3D map from tomography of a RNP ring structure (panel A, II) with six subunits. No symmetry was imposed. The map is show as a wire mesh. (C) A near-central slice through the 3D map illustrates the subunits, arbitrarily numbered. (D) Top view of the nucleoprotein (NP) monomer coordinates (PDB ID 2IQH) on the same scale as the map. The C-terminal loop of the NP molecule is indicated. Scale bar 10 nm.

Fig. 5. Classification and subtomogram averaging for molecular models of NP rings

(A) Computational sorting of 3D ring volumes into different classes. Corresponding numbers of NP molecules in each class are indicated, based upon rotational correlation analysis. Scale bar is 10 nm. (B) A histogram of class occupancy indicated that 6-mer rings were most populated (class 3), followed by 7-mers (class 2) and 5-mers (class 1). Classes with open rings (class 4) and skewed rings (class 5) were less commonly observed. (C) Radially averaged density for each class average of rings was plotted as a function of radius of the ring, such that the peak density describes the average radius of the particle class. (D) Symmetrized subtomogram averages were calculated representing classes of the most prevalent oligomeric states. Electron density is indicated as the gray surface rendering, and corresponding docked NP coordinates (PDB ID 2IQH) are shown as ribbon models with different colored NP monomers. (E) An oblique view of the 6-mer coordinate model illustrates how the NP tail loop (black arrows) could facilitate an interaction with a neighboring molecule.

Figure 6. 2D single particle image analysis of the oligomeric state of purified recombinant NP

(A) 2D Class averages of nucleoprotein (NP) molecules purified from a baculovirus expression system. Micrographs of negatively stained NP were exhaustively picked and 2D classification parameters were optimized to delineate oligomeric states of NP. Classes are order by occupancy. (B) Histogram depicting the occupancy for each class out of 11,752 picked particles. (C) Histogram depicting the oligomeric state of NP by aggregating classes according to the following: (3-mer, class 4), (4-mer, classes 2, 3, 6, 15), (5-mer, classes 1, 9), (6-mer, classes 5, 13) and (7-mer, class 7).

Fig. 7. Schematic of RNP flexibility mediated by transitions between helical and ring conformations

(A) Schematic model of straight double-helical RNP. Helical repeats are represented as a double-striated coil. (B) Segments of the NP proteins within the RNP may adopt a local ring conformation, disrupting helical symmetry. (C) The local ring conformation may gain flexibility after breaking contacts above and below in the filament. (D) Helical segments above and below the local ring conformation may undergo independent motion, creating bends and kinks in the RNP structure. (E) RNPs may arrive at stable state with an induced kink in the RNP structure, where the ring structure has found an equilibrium conformation. The ring is blurred slightly in panels C and D to denote dynamics in transition between states.