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. 2009 Feb 5;457(7230):694-8.
doi: 10.1038/nature07724.

Visualization of a missing link in retrovirus capsid assembly

Giovanni Cardone  1 , John G Purdy, Naiqian Cheng, Rebecca C Craven, Alasdair C Steven
Affiliations

Visualization of a missing link in retrovirus capsid assembly

Giovanni Cardone et al. Nature. .

Abstract

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.

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Figures

Figure 1
Figure 1
RSV CA protein assembles in vitro in 0.5M phosphate buffer into small isometric particles. a, Cryo-electron micrograph of capsids; most are ~ 17 nm in diameter, while a few are ~ 30 nm - white circles. Bar, 50 nm. b – d, Averaged images. b, 17-nm capsids, unclassified. c, 17-nm capsids, projecting the 3-fold view. d, 30-nm capsids, unclassified, showing two concentric shells. Bar, 10 nm.
Figure 2
Figure 2
Three-dimensional reconstruction of the RSV CA T=1 capsid. a, Surface rendering colored radially, red to blue, viewed along a 2-fold axis of symmetry. Bar, 5 nm. b, Segmented view of a pentamer and surrounds, colored by subunit. An additional subunit from an adjacent pentamer is purple. The center-to-center spacing between pentamers is 8.7 nm at mid-floor. c, Two views of a single subunit (left, right), with fitted pseudo-atomic model. The density is contoured at 3σ. Fitted were an NTD (orange, residues 1 to 147) and a CTD (blue, residues 152 to 230).
Figure 3
Figure 3
The 30-nm RSV-CA double-layer capsid. a, Surface rendering of cryo-EM reconstruction showing the outer T=3 capsid (yellow) and the inner T=1 capsid (green), exposed in b by removal of the front half of the outer layer. Bar, 10 nm. Neighboring hexamers are 10.2 nm apart, center-to-center, at mid-floor, while the spacing between hexamers and pentamers is 9.5 nm. c, Pseudo-atomic model of the T=3 capsid. An asymmetric unit contains three CA subunits: P1 in pentamers (red); and H1 (blue) and H2 (green) in hexamers. In the T=3 shell, pentamers and hexamers interact via the CTD-CTD interface between P1 and H1, while adjacent hexamers interact via H2. d, Interactions between the two layers. NTDs of the T=1 capsid (orange) contact CTDs of H1 subunits in the T=3 capsid (blue). Highlighted in yellow is the loop between helices 4 and 5 in the NTD, and in cyan, the major homology region in the CTD.
Figure 4
Figure 4
Pseudo-atomic models of the RSV-CA subunit, pentamer, and hexamer. a shows the relative positions and orientations of the NTD (orange) and CTD (blue), as disposed in the T=1 capsid. The dashed line connects the C-terminus of the NTD to the N-terminus of the CTD. b and c show axial views of the pentamer and hexamer, respectively.
Figure 5
Figure 5
Inter-subunit interactions in the T=1 capsid, represented in stereo views. Each subunit interacts with three adjacent subunits via three interfaces. a, NTD-NTD interface. In each pentamer is a ring of five NTDs. The pentagon marks the 5-fold axis. Helices 1 and 2 of one subunit are close to helices 1 and 3 of the adjacent subunit. b, Neighboring CA pentamers interact via a CTD-CTD dimer interface, mediated by association of helices 9, but presumably also involving the 310 helix. The helices 9 cross at ~45°, their closest point being at the N-termini of these helices (residue A184). c, NTD-CTD interface. The CTD of one subunit lies under the NTD of the next subunit in the same pentamer. The CTD residues closest to this interface are in the middle of helix 8, around R170, and at the start of helix 11, around E217. The NTD residues involved are at the start of helix 4.
Figure 6
Figure 6
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