Structural variability and complexity of the giant Pithovirus sibericum particle revealed by high-voltage electron cryo-tomography and energy-filtered electron cryo-microscopy

Abstract

The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM. We detected a previously undescribed low-density outer layer covering the tegument and a periodical structuring of the fibres in the striated apical cork. Energy-filtered Zernike phase-contrast cryo-EM images show distinct substructures inside the particles, implicating an internal compartmentalisation. The density of the interior volume of Pithovirus particles is three quarters lower than that of the Mimivirus. However, it is remarkably high given that the 600 kbp Pithovirus genome is only half the size of the Mimivirus genome and is packaged in a volume up to 100 times larger. These observations suggest that the interior is densely packed with macromolecules in addition to the genomic nucleic acid.

Figure 1

Image gallery of ice-embedded Pithovirus particles using 1 MV cryo-HVEM. The small, medium, and large sized viruses are indicated in (A1–10). The arrows indicate the positions of apical pores in the particles. (B) Size distribution of the Pithovirus particles, showing histograms of the length and width on each axis. The dotted lines mark the mean values for the length and the width of Pithovirus particles. Dimensions of particles shown in Figures 1–10 are marked with red circles in the histogram in B. The particles with dual pores are marked with yellow stars. (C) The size distribution in volume of the Pithovirus particles with single pore or dual pores.

Figure 2

Pithovirus particles with dual corks in energy-filtered regular defocus phase contrast (DPC) (A) and Zernike phase contrast (ZPC) cryo-EM images (B) at 200 kV. Black arrows indicate the positions of the apical corks. Dark spots in A show fiducial markers of gold colloids.

Figure 3

Peripheral structures of the Pithovirus particle imaged using energy-filtered cryo-EM at 200 kV. (A) The entire structure of the Pithovirus particle. Arrows indicate the low-density outer layer identified in this study. (B) Close-up view of the viral surface. The surface of the Pithovirus particle consists of four layers, those are, (a) nucleoid, (b) interior gap, (c) tegument and (d) low-density outer layer. (C) Close-up view of the apical cork. Arrows indicate vertical stripes that are formed at intersections of the horizontal parallel lines. Dark dots show fiducial markers of gold colloids.

Figure 4

Periodic Moiré pattern of the parallelly aligned fibre structures of the apical cork in a side view of the Pithovirus particle using energy-filtered cryo-EM at 200 kV. (A) Close-up view of the Moiré pattern from the fibre structures of the apical cork. Red arrows indicate two sets of parallel lines in the apical cork. (B) Fourier transform of (A).

Figure 5

Tomographic 3D reconstructions of the Pithovirus particles using cryo-HVEM at 1 MV. (A) Non-tilted image of ice-embedded Pithovirus particles. Arrows indicate the positions of the apical pores. (B) A Z-slice of the tomographic 3D reconstruction. (C–F) Structural segmentations of the entire 3D structures of the individual Pithovirus particles. Pithovirus particles with small length (C–D, particle 1), and medium length (E–F, particle 2). (C and E) Outer surface image of the segmentations. (D and F) Cross-section images of the segmentations. In the segmentation images, yellow, green, red, blue and pink surfaces indicate nucleoid, tegument, a root of the apical cork, apical cork, and low-density outer layer, respectively.

Figure 6

Empty Pithovirus particles imaged by cryo-HVEM at 1 MV. Membranous and other densely organized structures are seen inside the empty particles.

Figure 7

Energy-filtered Zernike phase contrast cryo-EM images of the dsDNA-full Pithovirus particles at 200 kV. (A,C) Original raw images and (B,D) raw images with higher contrast level. White arrows indicate putative interior substructures of the Pithovirus particles.

Figure 8

Measurement of nucleoid densities in Pithovirus and Mimivirus particles. (A) A bright field image without energy-filtering (cryo-EM at 200 kV). The small hexagonal and large oval shapes correspond to Mimivirus and Pithovirus particles, respectively. (B) Calculated EELS log-ratio image (t/λ, see Methods). Red and blue rectangles highlight regions for estimating nucleoid density profiles in Pithovirus and Mimivirus particles in (C). Black open boxes on the particles show the area used to statistically calculate the nucleoid density in (D). (C) Relative density line profiles (λ⁻¹, see Methods) of Pithovirus and Mimivirus particles calculated from measurements in the red and blue bands in (B). The profile shows that the values of the central areas (a–b and c–d) on each viral particle reach a plateau, and the calculated density of Pithovirus was relatively lower than that of Mimivirus. (D) Statistics of the estimated relative nucleoid density of Pithovirus and Mimivirus particles. The relative nucleoid density (λ⁻¹, see Methods) was calculated with each particle size (the particle width in Pithovirus; the capsid diameter in Mimivirus) in a central area of 150 nm × 150 nm and averaged in 9 Pithovirus and 36 Mimivirus particles. The nucleoid density of Pithovirus particle was estimated to be ~0.77 times the density of Mimivirus particles.