Motions on the Millisecond Time Scale and Multiple Conformations of HIV-1 Capsid Protein: Implications for Structural Polymorphism of CA Assemblies

Abstract

The capsid protein (CA) of human immunodeficiency virus 1 (HIV-1) assembles into a cone-like structure that encloses the viral RNA genome. Interestingly, significant heterogeneity in shape and organization of capsids can be observed in mature HIV-1 virions. In vitro, CA also exhibits structural polymorphism and can assemble into various morphologies, such as cones, tubes, and spheres. Many intermolecular contacts that are critical for CA assembly are formed by its C-terminal domain (CTD), a dimerization domain, which was found to adopt different orientations in several X-ray and NMR structures of the CTD dimer and full-length CA proteins. Tyr145 (Y145), residue two in our CTD construct used for NMR structure determination, but not present in the crystallographic constructs, was found to be crucial for infectivity and engaged in numerous interactions at the CTD dimer interface. Here we investigate the origin of CA structural plasticity using solid-state NMR and solution NMR spectroscopy. In the solid state, the hinge region connecting the NTD and CTD is flexible on the millisecond time scale, as evidenced by the backbone motions of Y145 in CA conical assemblies and in two CTD constructs (137−231 and 142−231), allowing the protein to access multiple conformations essential for pleimorphic capsid assemblies. In solution, the CTD dimer exists as two major conformers, whose relative populations differ for the different CTD constructs. In the longer CTD (144−231) construct that contains the hinge region between the NTD and CTD, the populations of the two conformers are likely determined by the protonation state of the E175 side chain that is located at the dimer interface and within hydrogen-bonding distance of the W184 side chain on the other monomer. At pH 6.5, the major conformer exhibits the same dimer interface as full-length CA. In the short CTD (150−231) construct, no pH-dependent conformational shift is observed. These findings suggest that the presence of structural plasticity at the CTD dimer interface permits pleiotropic HIV-1 capsid assembly, resulting in varied capsid morphologies.

Dynamic allostery governs cyclophilin A–HIV capsid interplay

“…As reported previously by us, such motions are seen in the hinge region of CA (residues 144-148) and are directly connected to the ability of the CA protein to assemble into pleiomorphic conical capsids (12). Dynamics in this time regime can be detected qualitatively from the peak intensities in the heteronuclear correlation spectra because such motions would interfere with CP, decoupling, or MAS, causing reductions in peak intensity or complete peak disappearance.…”

“…This dynamic regulation certainly also applies to HIV-1 because numerous dynamic processes are associated with HIV-1 assembly, disassembly, release, and maturation (10,11). For example, we previously demonstrated that internal conformational dynamics of the CA protein and its structural plasticity determine its ability to assemble into pleiomorphic conical capsids (12,13) (Fig. 1).…”

Recoupling of chemical shift anisotropy by R-symmetry sequences in magic angle spinning NMR spectroscopy

“…The hinge region is conformationally flexible on millisecond timescales, 39,[48][49][50][51] and as we have recently demonstrated, these millisecond motions are related to conformational plasticity of CA, permitting its assembly into different morphologies (i.e., conical, tubular, and spherical). 51 We have recorded the 15 N σ 1 -CSA line shapes for all Tyr residues in U- 13 shapes are shown in Figure 7 and they are in excellent agreement with each other. The 15 N reduced anisotropies, δ σ , are similar for all tyrosine residues, and close to the rigid limit value (δ σ = 110 ppm 52 ).…”

Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

“…Although a previous nuclear magnetic resonance (NMR) analysis of the RSV CA, CA-S, and CA-SP proteins (53) did not detect any obvious effect of SP on the structure of the alpha-helical bundle, a detailed analysis of protein dynamics will be needed for the detection of any such structural effect of SP. Retroviral capsid proteins are characterized by an unusual degree of internal dynamics in regions that are important to their assembly/disassembly capabilities, e.g., the CTD and the flexible loop region on the outside surface of the NTD (62)(63)(64). In particular, a recent solid-state NMR comparison of assembled HIV-1 CA and CA-SP1 proteins provided detailed evidence that the presence of SP1 causes long-distance structural effects within these regions of the CTD and the NTD and provided strong support for this interpretation of the RSV CA-SP results presented here (65).…”