Dynamic regulation of HIV-1 capsid interaction with the restriction factor TRIM5α identified by magic-angle spinning NMR and molecular dynamics simulations

Abstract

The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid's hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid's morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.

Keywords: CA protein assemblies; HIV-1 capsid; HIV-AIDS; TRIM5α; magic-angle spinning NMR.

Fig. 1.

(A) Structure of a HIV-1 conical capsid and its building blocks. (B) Domain structure of TRIM5α. (C) TEM images of tubular assemblies of cross-linked hexamer CA (A14C/E45C/W184A/M185A) assemblies (Left), cone-like CA A204C assemblies (Middle), and cone-like coassemblies containing natural abundance CA A204C and 10% cross-linked U-¹³C,¹⁵N-pentamer CA (N21C/A22C/W184A/M185A) (Right). (D) Selected regions of ¹³C-¹³C correlation spectra of tubular hexamer CA assemblies (black), CA A204C assemblies (magenta), and coassemblies of A204C/cross-linked pentamer CA (light green). The first contour is set at 3.5 times the noise RMSD. (E) Pelleting assay of cross-linked hexamer CA/TRIM5α CC-SPRY tubular coassemblies and TEM of cross-linked hexamer CA tubular assemblies in the presence of TRIM5α CC-SPRY. P, pellet; S, supernatant. (Scale bars: 100 nm.)

Fig. 2.

(A) ¹⁵N^H-¹³C^α REDOR dephasing curves (Left) and ¹H-¹³C^α DIPSHIFT line shapes (Right) for tubular assemblies of U-¹³C,¹⁵N cross-linked hexamers (black), cone-like assemblies of CA A204C (magenta), and cone-like coassemblies of 10:1 natural abundance A204C/U-¹³C,¹⁵N cross-linked pentamers (light green). The best-fitted ¹H-¹³C^α dipolar line shapes are shown in gray, and the dipolar coupling constants are indicated. (B) RMSFs of CA oligomers [Protein Data Bank (PDB) ID code 3J3Q] over the 1.2-μs MD simulations vs. residue number. Hexamer and pentamer atoms are shown in black and light green, respectively. The RMSFs were calculated after minimizing the translational and rotational movements of the CA oligomers. The error bars represent RMSF variations over the entire ensemble of all hexamers and pentamers. (C) RMSF of CA hexamers (Top) and pentamers (Bottom) mapped onto the all-atom CA model and presented in histograms. The translational and rotational degrees of freedom of the whole capsid, rather than individual CA oligomers, were removed before RMSF calculations. (D) Summary of chemical shift and peak intensity differences between the tubular CA assemblies of cross-linked hexamers in the absence and presence of TRIM5α CC-SPRY, mapped onto an isolated CA monomer (PDB ID code 3J34). (E) Superpositions of select regions of the 2D ¹³C-¹³C correlation spectra of free (gray) and TRIM5α CC-SPRY–bound (cyan) tubular assembly of cross-linked CA hexamers. The first contour is set at three times the noise level. The 1D traces illustrate peak width and/or intensity changes. (F) Normalized peak intensity ratios extracted from the ¹³C-¹³C correlation spectra of TRIM5α CC-SPRY–bound CA tubular assemblies vs. TRIM5α CC-SPRY–free assemblies. Ratios >1 SD above or below the average (dashed line) are considered significant (outside the gray box). (G) ¹³C chemical shift changes of CA on binding of TRIM5α CC-SPRY, extracted from ¹³C-¹³C correlation spectra. The dashed line at 0.2 ppm marks the boundary between ¹³C chemical shift differences >1 SD above the average.

Fig. 3.

Structural changes in the CA NTD on TRIM5α CC-SPRY binding. (A) Superposition of select regions of the ¹³C-¹³C combined $\text{R}{2}_{\nu }^{\text{n}}$-driven (CORD) correlation spectra of free (gray) and TRIM5α CC-SPRY–bound (cyan) tubular assembly of cross-linked CA hexamers. Affected resonances are labeled by residue name and number. (B) Structural mapping of residues that experience chemical shift changes (cyan) onto the CA structure (PDB ID code 3J34).

Fig. 4.

Chemical shift perturbations on TRIM5α CC-SPRY binding and structural mapping onto the CA hexameric building block at the NTD-NTD interface. (A) Superposition of select regions in the ¹³C-¹³C CORD correlation spectra of free (gray) and TRIM5α CC-SPRY–bound (cyan) tubular assemblies of cross-linked CA hexamers. Affected resonances are labeled with residue name and number. (B and C) Top (B) and side (C) views of the CA hexamer (PDB ID code 3J34) in ribbon representation. Residues whose C^α or C^β resonances are affected are colored cyan in one monomer and in magenta in a neighboring monomer. (D) Expansion of the NTD-NTD interface, highlighting those residues that experience chemical shift changes. Affected residues are shown in stick representation and are labeled with residue name and number.

Fig. 5.

Chemical shift changes observed for resonances associated with the major homology region. (A) Superposition of select regions of the ¹³C-¹³C CORD correlation spectra of free (gray) and TRIM5α CC-SPRY–bound (cyan) tubular assemblies of cross-linked CA hexamers. Affected resonances are labeled with residue name and number. (B) Space-filling representation of a trimer of hexamers in tubular CA assemblies (PDB ID code 3J34) with two CA monomer chains at the interhexameric interface in ribbon representation. Residues near the interface whose resonances are affected by TRIM5α CC-SPRY binding are shown in magenta in one monomer and in cyan in the other monomer. (C) CTD-CTD interface in the tubular CA assembly (PDB ID code 3J34). (D) Side view of the hexameric subunit in the tubular CA assembly (PDB ID code 3J34), highlighting affected residues in the major homology region (MHR) and neighboring residues. (E) Expansion of MHR with affected residues shown in cyan stick representation.