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. 1998 Jun 23;95(13):7299-304.
doi: 10.1073/pnas.95.13.7299.

Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms

M Yeager  1 , E M Wilson-Kubalek, S G Weiner, P O Brown, A Rein
Affiliations

Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms

M Yeager et al. Proc Natl Acad Sci U S A. .

Abstract

We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR-) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR- MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR- nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR- MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of approximately 45 A and a length of approximately 200 A. Because of the pleomorphic shape and paracrystalline packing of the Gag-RNA complexes, we raise the possibility that assembly of MuLV is driven by protein-RNA, as well as protein-protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.

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Figures

Figure 1
Figure 1
Frozen-hydrated images of immature, protease-deficient (PR−) (a, b) and mature, wild-type (WT) (c) MuLV particles. Both particle types are bounded by a lipid bilayer envelope (E) and display a variation in diameter. The inner membrane leaflet was thicker than the outer leaflet, which we attribute at least in part to the matrix protein (M). Both particles displayed a core region (C), which was rounded in the PR− MuLV and polygonal in the WT MuLV. The surface of the core in the PR− MuLV displayed a characteristic track-like structure (T). Occasionally, a second inner track (IT) could be identified that was concentric or continuous with the outer fringe. Rotavirus particles (labeled RV) (≈1,000 Å diameter) retained their constant diameter and spherical shape during ultracentrifugation with the PR− MuLV (d) and WT MuLV (e).
Figure 2
Figure 2
Size distribution for 67 PR− MuLV and 32 WT-MuLV particles. PR− MuLV and WT MuLV have comparable diameters (Do) (PR− MuLV 1,260 ± 114 Å, WT MuLV 1,239 ± 139 Å). The irregular polygonal shape of the core in the WT MuLV precluded measurement of the diameter. The more rounded core in the PR− MuLV was readily measured and yielded a core diameter (Di) of 958 ± 102 Å.
Figure 3
Figure 3
Montage of frozen-hydrated images of immature PR− MuLV (a) and mature WT MuLV (b) that have been masked from the original negative, centered, and background subtracted. In rare instances, PR− MuLV particles were identified that displayed a core layer manifesting discrete diffraction spots (Inset in a).
Figure 4
Figure 4
Circularly averaged radial density profiles for the most rounded PR− MuLV (a) and WT MuLV (b) displayed in Fig. 3. The locations of the membrane envelope (E), the track pattern (T), and the inner track pattern (IT) are indicated. The lipid bilayer envelope (E) has a constant distance between the phosphate head groups (peak-to-peak distance 37.5 ± 3.3 Å; n = 8) for both particles, but the inner leaflet seems to have a lower density in the PR− MuLV compared with the WT MuLV. We attribute the shoulder of density on the inner leaflet of the bilayer to the region occupied by the matrix protein (M), which has a thickness of 40 ± 9 Å. The track region (T) corresponds to the outer surface of the core and is not well defined in the WT MuLV particles. For the PR− MuLV, there was considerable variation in the lucent region between the track and matrix regions (43 ± 16 Å, n = 4). Consequently, the radial location of the track pattern varies from particle to particle.
Figure 5
Figure 5
Rotational correlation analysis of PR− MuLV images. Sectors were extracted from PR− MuLV that displayed a well defined track pattern. Image analysis revealed 56- to 64-fold rotational symmetry, corresponding to the repeat pattern for the striations in the track pattern (Right). This rotational symmetry gives a spacing between the ties on the tracks of 44 ± 4 Å, and the peak-to-peak separation of the tracks was also ≈44 Å. Note the variation in the radial thickness of the region between the tracks and the lipid envelope.
Figure 6
Figure 6
Circularly averaged Fourier transforms (FFTs) for the core regions of PR− MuLV (solid line) and WT MuLV (dotted line). The FFT for the PR− MuLV particles displays well defined peaks centered at 0.022 and 0.032 Å−1, corresponding to spacings of 45.4, and 31.8 Å. In contrast, the FFT for the WT MuLV displays two broad peaks centered at 0.015 Å−1 and 0.025 Å, corresponding to spacings of 67 and 40 Å, respectively, in real space.
Figure 7
Figure 7
Schematic model for the packing of the Gag polyprotein in immature murine leukemia virus. The rotationally averaged image of a single particle is shown to the right. The thicker inner leaflet of the lipid bilayer envelope is attributed to the matrix protein (MA), which is known to be anchored to the membrane via a myristoyl group. The low density zone between the MA region and the tracks is assigned to the location of the pp12 protein, which is known to be rich in proline and is likely to be disordered. The ordered track-like structure is assigned to the location of the capsid protein but could also include contributions from the nucleocapsid protein together with bound RNA.
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