Three-dimensional structure of HIV-1 virus-like particles by electron cryotomography

Abstract

While the structures of nearly every HIV-1 protein are known in atomic detail from X-ray crystallography and NMR spectroscopy, many questions remain about how the individual proteins are arranged in the mature infectious viral particle. Here, we report the three-dimensional structures of individual HIV-1 virus-like particles (VLPs) as obtained by electron cryotomography. These reconstructions revealed that while the structures and positions of the conical cores within each VLP were unique, they exhibited several surprisingly consistent features, including similarities in the size and shape of the wide end of the capsid (the "base"), uniform positioning of the base and other regions of the capsid 11nm away from the envelope/MA layer, a cone angle that typically varied from 24 degrees to 18 degrees around the long axis of the cone, and an internal density (presumably part of the NC/RNA complex) cupped within the base. Multiple and nested capsids were observed. These results support the fullerene cone model for the viral capsid, indicate that viral maturation involves a free re-organization of the capsid shell rather than a continuous condensation, imply that capsid assembly is both concentration-driven and template-driven, suggest that specific interactions exist between the capsid and the adjacent envelope/MA and NC/RNA layers, and show that a particular capsid shape is favored strongly in-vivo.

Figure 1.

Primary data and processing steps. Top: purified HIV-1 virus-like particles were frozen in thin films across EM grids. Series of images were recorded of many such fields of frozen particles as the sample was tilted incrementally about one axis and then a second, orthogonal axis. One experimental image from one tilt series is shown. The roughly spherical objects are mostly HIV-1 VLPs (including those labeled 1B, 1C, 1E, 1G, 1K, and 1M), but some apparently empty liposomes are present in the sample (Empty) as well as other irregular forms (Irregular). The small black dots are 10 nm gold fiducials used to align the tilt series for reconstruction, and the large arc appearing across the upper right corner is the edge of a circular hole in a supporting carbon film. Middle: Three-dimensional reconstructions of the fields of VLPs were calculated from the various series of tilted images. A single section 1.34 nm thick through the middle of the reconstruction corresponding to the image above is shown. Because this is a single section, some objects that are seen in the projection image above (for instance many of the gold beads) do not appear. Bottom: Individual VLPs were selected from the reconstruction and denoised, and then the boundaries of the lipid bilayer and capsid were semi-automatically delineated (segmented). Three examples (1E, 1C, and 1K) are shown. In each pair, the left image shows a single section through the middle of an extracted, denoised VLP. The right image shows three-dimensional renditions of our interpretation of the capsids in red, shown in place within the denoised section, with the area enclosed by the lipid bilayer in blue.

Figure 2.

Montage of all segmented HIV-1 VLPs. A total of 26 VLPs were segmented and aligned along their principal axes. Three views are shown for each VLP, presenting from left to right axial, sagittal, and coronal perspectives, respectively. Three-dimensional renditions of the capsid surfaces are shown in red for cones, orange for rods, and yellow for all others, placed within a denoised two-dimensional section through the middle of the VLP, with the area enclosed by the lipid bilayer in blue. Because each VLP was extracted as a cube from a larger three-dimensional reconstruction and then aligned to the others, the boundaries of the images appear as randomly oriented, clipped squares. Each capsid is identified by a number and a letter, where the number signifies which field of particles it came from. Capsids 3gR and 3gC are part of the same, double-capsid VLP. The edges of the boxes containing each VLP image here and in all other Figures represent 160 nm.

Figure 3.

Nested capsids. Two VLPs, (a)–(c) 10a and (d)–(f) 1g, presented nested capsids. (a) and (d) show sagittal sections, (b) and (e) show coronal sections, and (c) and (f) show three-dimensional renditions as in Figure 2, except that the outer capsid surface has been made transparent to reveal the inner capsid, and in the case of VLP 1g ((f)), the surfaces of all five capsids present are rendered.

Figure 4.

Cone angle variation in 3D. The cone angle (in degrees) for the 16 conical capsids is plotted as a function of rotation around the cone axis from 0–180°. The methodology is depicted in the inset. For each measurement, straight lines (blue in inset) were fit to the edges of the cone between the base and the tip. These lines defined the cone angle as the cone was rotated around its long axis. The resulting values were plotted by assigning the rotation position corresponding to the maximum cone angle as 0°. The unique VLP identifiers are shown in the legend. The three black curves are the corresponding cone angles for three different theoretical fullerene cones modeled to mimic HIV-1 capsids, as described.^,

Figure 5.

Conserved patterns of density in VLPs. The 16 VLPs with conical capsids were aligned using (a), (b), (e) and (f) the bases or (c), (d), (g) and (h) the tips. The top panels show (a) and (c) sagittal and (b) and (d) coronal sections through the capsids and lipid bilayer surfaces, with the same color code for each VLP as in Figure 4. The bottom panels show again (e) and (g) sagittal and (f) and (h) coronal sections through the corresponding three-dimensional average VLP. In (e), the magenta arrow points towards the edge of the average bilayer/matrix layer, the green arrow points towards the edge of the average capsid, and the yellow arrow points towards the presumed average internal RNP density. In (g) and (h), the yellow arrows point to the missing average cap density. Notable features include the surprisingly uniform, asymmetric capsid base ((b)); the uniform cone angles (particularly (b) and (d)); the uniform distance between the capsid base and the bilayer/matrix layer ((a) and (b)); the preponderance of internal density within the base as opposed to the tip of the average capsids ((e)–(h)); the conserved internal density lining the inside of the average capsid base ((e) in particular); and the lack of clear density in the cap of the average capsid tip ((g) and (h)). The very dark density in the upper right corner of (f) outside the VLP is an artefact due to gold fiducials in one of the reconstructions.