Structural Convergence between Cryo-EM and NMR Reveals Intersubunit Interactions Critical for HIV-1 Capsid Function

Abstract

Summary Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two and three-dimensional arrays provided a molecular model of the capsid protein (CA) hexamer and revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits extremely well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a novel CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

“…Third, our cryo-EM and cosedimentation experiments reveal that the PRY/SPRY rh domain retains the ability to bind HIV-1 CA helical tubes without breaking the assembly. Importantly, our crystal structure of PRY/SPRY rh , the crystal structure of the HIV-1 CA hexamer lattice (36,51), and the cryo-EM maps of HIV-1 capsid and helical tubes (18,38) together establish a structural framework for further detailed investigation of this lattice-specific interaction.…”

“…In addition, these CA tubes are well separated (Fig. 3D), which differs from the bundled appearance of the CA tubes without TRIM5α (38). In contrast, using MBP-PRY/SPRY hu in the same assay, the CA tubes remain bundled (Fig.…”

Dynamic allostery governs cyclophilin A–HIV capsid interplay

“…In particular, we compared wild-type and A92E and G94D escape mutants of the NL4-3 strain as well as wildtype HXB2 CA alone and in complex with CypA. As discussed previously (14,15), tubular assemblies recapitulate the hexameric lattice, the predominant symmetry arrangement of the conical HIV-1 capsid core, illustrated in Fig. 1A.…”

“…We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone 1 H- 15 N and 1 H- 13 C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories.…”

Monte Carlo Simulations of HIV Capsid Protein Homodimer

“…The self-assembly of the CA proteins has been extensively studied using X-ray, NMR, and cryo-electron microscopy (Cryo-EM). [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] Remarkably, a complete atomic model of the capsid was recently obtained by Cryo-EM combined with all-atom simulations. 17 Other theoretical and computational studies [27][28][29][30] also provided important insight into the structure and the assembling process for the HIV capsid.…”