Morphology of influenza B/Lee/40 determined by cryo-electron microscopy

Abstract

Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (∼83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ∼460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.

Figure 1. Projection image of frozen-hydrated influenza B virions.

Surface spike are visible in all particles with intact or partially intact matrix. A defective virion which lacks surface proteins is identified in lower right and enlarged in inset.

Figure 2. Virion Size Distribution.

A) Histogram of virion diameter. B) Scatter plot of virion diameter; horizontal lines are mean and mean ±1 standard deviation.

Figure 3. Projection images of three virions.

A) Average-size spherical virion; B) large spherical virion; and C) irregular virion. The irregular virion exhibits considerable disruption in the RNPs indicated by low density regions.

Figure 4. Morphological classes of B/Lee/40 virions.

Central tomogram slices created with IMOD using R-weighted backprojections: A) spherical virion with well-ordered RNPs; B) oblong virion with some RNP disorder; and C) Irregular-shaped virion with little RNP order and a large region showing a very low density of RNP (lower left of virion).

Figure 5. ART Reconstruction of B/Lee/40.

A–E) Tomogram slices at 9 nm z-axis spacing. Docked atomic models of HA (yellow) and NA (red) are shown in panel B. RNPs are outlined in blue.

Figure 6. Amira Rendering of the RNPs by segment selection.

A) Complete virion. B) RNP elements exclusively presented showing the twisted confirmation.

Figure 7. RNP, HA, and NA connections into envelope.

A) Central tomogram slice; B) Polar plot of central slice shown in panel A, black arrows show glycoprotein extensions into the matrix and white arrows show contact points between RNPs and matrix. 0° in panel B corresponds to –x direction in panel A and angle increases in counter-clockwise direction. Images are inverted, i.e. higher density regions are lighter. C) Correlation of densities of the outer envelope spikes with their projections into the matrix.