Atomic-level modelling of the HIV capsid

Abstract

The mature capsids of human immunodeficiency virus type 1 (HIV-1) and other retroviruses are fullerene shells, composed of the viral CA protein, that enclose the viral genome and facilitate its delivery into new host cells. Retroviral CA proteins contain independently folded amino (N)- and carboxy (C)-terminal domains (NTD and CTD) that are connected by a flexible linker. The NTD forms either hexameric or pentameric rings, whereas the CTD forms symmetric homodimers that connect the rings into a hexagonal lattice. We previously used a disulphide crosslinking strategy to enable isolation and crystallization of soluble HIV-1 CA hexamers. Here we use the same approach to solve the X-ray structure of the HIV-1 CA pentamer at 2.5 Å resolution. Two mutant CA proteins with engineered disulphides at different positions (P17C/T19C and N21C/A22C) converged onto the same quaternary structure, indicating that the disulphide-crosslinked proteins recapitulate the structure of the native pentamer. Assembly of the quasi-equivalent hexamers and pentamers requires remarkably subtle rearrangements in subunit interactions, and appears to be controlled by an electrostatic switch that favours hexamers over pentamers. This study completes the gallery of substructures describing the components of the HIV-1 capsid and enables atomic-level modelling of the complete capsid. Rigid-body rotations around two assembly interfaces appear sufficient to generate the full range of continuously varying lattice curvature in the fullerene cone.

Figure 1. Structure of the disulfide-stabilized HIV-1 CA pentamer and comparison with the hexamer

a, Top view of the pentamer, with the NTD colored in orange and the CTD in blue. Helices are represented as cylinders. b, c, Top view (b) and side view (c) of the pentamer, with the helices as ribbons. Each subunit is in a different color. d, e, f, Equivalent views of the hexamer (PDB code: 3H4E). The yellow spheres in (a) and (d) indicate the positions of the pentamer-stabilizing (N21C/A22C) and hexamer-stabilizing (A14C/E45C) disulfide bonds, respectively.

Figure 2. Comparison of the pentamer and hexamer interactions

Both oligomers are created by quasi-equivalent packing of each of the two domains of one CA subunit (colored in green) with the NTD of a second subunit (blue). a, Stereoview superposition of the pentamer interface (dark colors) and the hexamer interface (light colors). The structures are superimposed on the blue NTD. b, Close-up view of representative NTD-CTD contact regions in the pentamer and hexamer. Key residues are shown explicitly and labeled, with hydrogen bonds colored in yellow. c, Comparison of crystallographically independent NTD-CTD interfaces in the pentamer, superimposed on the NTD. Flexion of the two domains is indicated by the black double-headed arrow, and occurs about molecular pivots composed of intermolecular helix-capping hydrogen bonds. The relevant side chains are shown explicitly and labeled, and hydrogen bonds are indicated by yellow lines.

Figure 3. Quasi-equivalence in the pentameric and hexameric NTD rings

a, b, Top views of the pentameric (a) and hexameric (b) NTD rings, with each subunit in a different color. Subunits in the pentamer and hexamer are shown in darker and lighter shades, respectively. The angles subtended by adjacent domains are shown explicitly for the blue and orange subunits. One of the repeating 3-helix units is outlined in black. c, Close-up view of the pentameric and hexameric repeat units, superimposed on helices 1 and 3 of the blue subunit. The aliphatic residues that form a small hydrophobic core are shown explicitly and labeled. d, Illustration of the “rotation” between adjacent subunits, in going from the hexamer to the pentamer. The approximate position of the rotation axis is indicated by the red dot. Note that this axis is parallel to neither the pentameric nor hexameric symmetry axes.

Figure 4. Model of the HIV-1 capsid

a, Stereoview of a backbone-only fullerene cone model composed of 1,056 CA subunits. The hexamers, pentamers, and dimers are colored in orange, yellow, and blue, respectively. Note that the capsid displays a variably curved surface. In the body of the cone, curvature changes continually, and this was modeled by means of subunit flexion at the NTD-CTD interface. Pentamers alter the trajectory of the hexagonal lattice and create regions of sharp curvature (i.e., declinations). Exactly 12 declinations are required to close a hexagonal lattice. Our modeling suggests that formation of the declinations entails a flexible CTD dimer. Note also that the CTD subunits surrounding the local 3-fold axes are in close proximity, consistent with the finding that this site constitutes a fourth set of capsid-stabilizing interactions. b, Graph showing the extent by which the intersubunit distances across the modeled NTD-CTD interfaces deviate from the expected value, as a function of cone length (deviation = distance_modeled − distance_expected, where distance_expected = 9.0 Å, and refers to the average separation of hydrogen-bonded pairs in the X-ray structures of the hexameric and pentameric NTD-CTD interfaces) (see Methods for details). Note that 99% of the NTD-CTD distances in the model are within 1 A of the expected value. This suggests that the model is of good quality, in light of the sizeable number of constraints imposed on the subunit interactions.