Magic Angle Spinning NMR Reveals Sequence-Dependent Structural Plasticity, Dynamics, and the Spacer Peptide 1 Conformation in HIV-1 Capsid Protein Assemblies

Abstract

A key stage in HIV-1 maturation towards an infectious virion requires sequential proteolytic cleavage of the Gag polyprotein leading to the formation of a conical capsid core that encloses the viral RNA genome and a small complement of proteins. The final step of this process involves severing the SP1 peptide from the CA-SP1 maturation intermediate, which triggers the condensation of the CA protein into the capsid shell. The details of the overall mechanism, including the conformation of the SP1 peptide in CA-SP1, are still under intense debate. In this report, we examine tubular assemblies of CA and the CA-SP1 maturation intermediates using Magic Angle Spinning NMR spectroscopy. At magnetic fields of 19.9 T and above, outstanding-quality 2D and 3D MAS NMR spectra were obtained for tubular CA and CA-SP1 assemblies yield, permitting resonance assignments for subsequent detailed structural characterization. Dipolar- and scalar-based correlation experiments unequivocally indicate that SP1 peptide is in a random coil conformation and mobile in the assembled CA-SP1. Analysis of two CA protein sequence variants reveals that, unexpectedly, the conformations of the SP1 tail, the functionally important CypA loop, and the loop preceding helix 8 are modulated by residue variations at distal sites. These findings provide support for the role of SP1 as a trigger of the disassembly of the immature CA capsid for its subsequent de novo reassembly into mature cores, and establish the importance of sequence-dependent conformational plasticity in CA assembly.

Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy

“…It has been proposed that SP1 has helical propensity (9), and low-resolution cryo-electron microscopy has suggested that SP1 forms an intermolecular six-helix bundle in immature HIV-1 capsid (29,35), although the resolution is insufficient to permit any definitive conclusions in this regard. An unstructured SP1, on the other hand, is supported by solution NMR studies on ΔGag (this work) and the smaller CA-SP1-NC (16) and CA CTD -SP1-NC (36) fragments, and by solid-state NMR studies on tubular assemblies formed by the shorter CA-SP1 construct (37). Understanding the conformational transition of SP1 from an unstructured motif in monomeric/dimeric ΔGag to a helical conformation in an assembled Gag lattice, if this does indeed occur, will require atomic resolution structures of immature Gag assemblies.…”

Dynamic allostery governs cyclophilin A–HIV capsid interplay

“…In the presence of high concentration of NaCl (0.5-2.4 M), the CA protein assembles into tubes (14). As shown in Fig.…”

“…CA (HXB2, NL4-3, NL4-3/A92E, and NL4-3/G94D variants) and CypA were expressed and purified by using the same protocol as reported (14,41). Tubular assemblies of CA protein were prepared and their morphology characterized as described (14) and detailed in SI Appendix, SI Text.…”

“…1). We also uncovered that, in the HIV-1 CA-SP1 maturation intermediate, dynamic disorder in the SP1 peptide plays an important role in the final step of virus maturation, permitting condensation of CA into the cores of infectious virions (14).…”

“…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.…”