High-resolution structures of HIV-1 Gag cleavage mutants determine structural switch for virus maturation

Abstract

HIV-1 maturation occurs via multiple proteolytic cleavages of the Gag polyprotein, causing rearrangement of the virus particle required for infectivity. Cleavage results in beta-hairpin formation at the N terminus of the CA (capsid) protein and loss of a six-helix bundle formed by the C terminus of CA and the neighboring SP1 peptide. How individual cleavages contribute to changes in protein structure and interactions, and how the mature, conical capsid forms, are poorly understood. Here, we employed cryoelectron tomography to determine morphology and high-resolution CA lattice structures for HIV-1 derivatives in which Gag cleavage sites are mutated. These analyses prompt us to revise current models for the crucial maturation switch. Unlike previously proposed, cleavage on either terminus of CA was sufficient, in principle, for lattice maturation, while complete processing was needed for conical capsid formation. We conclude that destabilization of the six-helix bundle, rather than beta-hairpin formation, represents the main determinant of structural maturation.

Keywords: capsid; cryoelectron tomography; maturation; retrovirus; subtomogram averaging.

Fig. 1.

Biochemical characterization of mutant virus-like particles used in this study. (A) Schematic illustration of the proteolytic cleavages involved in HIV-1 Gag maturation, ordered by relative rate as determined using purified PR in solution (23). Schematic representations of typical immature and mature viral morphologies are displayed to the left of the corresponding stages in the proteolytic cleavage cascade. (B) Schematic representation of Gag cleavage patterns in each of the cleavage mutants used. Mutated sites that cannot be cleaved are denoted with crossed out, dashed lines. Sites where cleavage occurs as in wild-type Gag are denoted by solid lines. Brackets under the schematics indicate the CA-containing products that remain unprocessed and their corresponding molecular mass. (C) SDS/PAGE analysis of virus preparations used for the structural analyses. Particles were purified from the supernatant of transfected HEK293T cells by ultracentrifugation through an iodixanol gradient. Samples were separated by SDS/PAGE (12.5% acrylamide, 30:1 acrylamide/bisacrylamide), and proteins were visualized by silver staining. Numbers to the left indicate the position of molecular mass standards (in kilodaltons). Purified recombinant Gag protein was used as a standard to estimate particle concentration. (D) Quantitative immunoblot analysis of MA-CA and MA-SP1 band intensities. Samples were separated by SDS/PAGE (12% acrylamide, 200:1 acrylamide/bisacrylamide), and proteins were detected by quantitative immunoblot (LI-COR Biotechnology) using a polyclonal antiserum raised against recombinant CA.

Fig. 2.

(A–D, i–xx) Representative viral morphologies for each of the cleavage mutants. Orthoslices through tomograms show representative examples of the viral morphologies observed in the different datasets. The frequency of each phenotype in the corresponding dataset is shown above each of the panels, as well as the percentage of the respective dataset that each absolute frequency represents. Color bars underneath each set of orthoslices represent the percentage of viral particles displaying immature (green), partially mature (yellow), mature (red), or undefined (black) morphology. Scale bars, 50 nm.

Fig. 3.

Structure of the immature Gag CA layer. Isosurface representations of the structure of the immature Gag CA layer in each cleavage mutant, compared with the previously published wild-type (WT) immature structure (EMD-4017) (10). (A) View perpendicular to the lattice. One immature CA domain is highlighted in color for emphasis (cyan, CA-NTD; orange, CA-CTD). (B) Comparison of the density N-terminal to helix 1 in the CA-NTD density in each of the maps. In each case, PDB coordinates of helix 1 and its upstream linker (red) were rigid-body-docked into the density map. For WT Gag, MA-SP1, and MA-CA, model 4 from an NMR solution structure of MA-CA (PDB ID code 16LN) was used. For CA-NC, helix 1 and the upstream beta-hairpin from a mature CA crystal structure (PDB ID code 5HGK) were used. (C) As in B, but with density maps low-pass-filtered to 6 Å to more readily show the larger density upstream of helix 1 in CA-NC, consistent with the presence of a beta-hairpin. (D) Comparison of the CA-SP1 six-helix bundle density in each immature structure. The helical bundle in all structures appears to have the same length, ending at residue T371. Additional disordered density is visible downstream of the bundle in WT Gag and CA-NC, corresponding to the C-terminal part of SP1 and downstream residues.

Fig. 4.

Structure of the mature CA hexamer. Isosurface representations of the structure of the mature CA hexamer in each cleavage mutant, compared with the previously published wild-type (WT) CA hexamer structure (EMD-3465) (11). A structural model of mature CA with the beta-hairpin in the closed conformation (PDB ID code 5MCX) was rigid-body-docked into the electron densities for WT CA, CA-NC, and CA-SP1, with the CA-NTD shown in cyan, the CA-CTD shown in orange, and the beta-hairpin shown in red. For MA-CA, the CA-NTD is a separately docked NMR solution structure (PDB ID code 1L6N) that contains a linear MA-CA linker (red) instead of a beta-hairpin motif. The resolutions of the maps differ: WT CA, 6.8 Å; MA-CA, 8.3 Å; CA-NC, 9.7 Å; CA-SP1, 7.9 Å. (A) View of the lattice down its sixfold symmetry axis from the CA-NTD end. A density corresponding to the beta-hairpin is seen in WT CA, CA-NC, and CA-SP1, but not in MA-CA. (B) Rotation of 90° to view the hexamer viewed perpendicular to the CA lattice and cut open to better visualize the beta-hairpin and helix 1. The CA-SP1 six-helix bundle seen in the immature lattice is absent in the mature lattice, and no density was seen for residues 354–378.

Fig. 5.

Structure of the mature CA pentamer. Comparison between previously obtained wild-type (WT) CA pentamer structure (EMD-3466) and the CA-NC and CA-SP1 pentamer structures obtained here. (A) Isosurface representations of the mature pentamer maps in the WT virus and in the CA-NC and CA-SP1 cleavage mutants, viewed from outside the core. (B) Orthoslice of 5.4-Å thickness through each of the maps, viewed parallel to the plane of the pentamer. Although the resolution of the CA-NC and CA-SP1 pentamers is limited due to the very small number of subvolumes averaged, the overall pentamer structure in both of these mutants is similar to the WT CA pentamer, indicating that a CA pentamer is present. (C) Perpendicular orthoslice to the view in B, through the fivefold symmetry axis, showing the curvature of each pentamer. Scale bars, 10 nm.

Fig. 6.

(A–C, i–xxvii) Lattice map representations of aligned subtomograms used to generate the structures, illustrating the different viral morphologies observed in the samples. Scale bars, 50 nm. (A) Computational slices of 5.4-Å thickness through the tomogram to illustrate representative viruses for each morphology. (B) Lattice maps overlaid on the corresponding tomographic slice. Each hexagon or pentagon corresponds to a single subtomogram, centered on a CA domain hexamer or pentamer, respectively. These geometric objects are displayed at the coordinates and orientations of the center of each subvolume determined during alignment. They provide a representation of the arrangement of hexameric subunits in the immature lattice and of the hexameric and pentameric subunits in mature CA lattices. Immature hexamers are displayed on a color scale of red to green, from low to high CCC. A corresponding magenta-to-cyan color scale is used for mature hexamers. Mature pentamers are shown in blue. Only one pentamer is clearly visible in the illustrated lattice maps (arrow in xxiv). (C) Lattice maps displayed without the corresponding tomographic slice.