Helical Conformation in the CA-SP1 Junction of the Immature HIV-1 Lattice Determined from Solid-State NMR of Virus-like Particles

Abstract

Maturation of HIV-1 requires disassembly of the Gag polyprotein lattice, which lines the viral membrane in the immature state, and subsequent assembly of the mature capsid protein lattice, which encloses viral RNA in the mature state. Metastability of the immature lattice has been proposed to depend on the existence of a structurally ordered, α-helical segment spanning the junction between capsid (CA) and spacer peptide 1 (SP1) subunits of Gag, a segment that is dynamically disordered in the mature capsid lattice. We report solid state nuclear magnetic resonance (ssNMR) measurements on the immature lattice in noncrystalline, spherical virus-like particles (VLPs) derived from Gag. The ssNMR data provide definitive evidence for this critical α-helical segment in the VLPs. Differences in ssNMR chemical shifts and signal intensities between immature and mature lattice assemblies also support a major rearrangement of intermolecular interactions in the maturation process, consistent with recent models from electron cryomicroscopy and X-ray crystallography.

In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

“…A potent maturation inhibitor, Bevirimat (BVM), blocks CA-SP1 cleavage and prevents formation of the mature conical capsid [19][20][21][22] . Recent structural and mutational studies have indicated that the junction between CA and SP1 could act as a molecular switch to regulate immature Gag assmebly and protease cleavage [23][24][25][26] . Structural analyses of the Gag lattice in mutant viruses that have impaired cleavage of Gag at specific sites suggest that processing is ordered and that the RNA/protein complex (RNP) may maintain a link with the remaining Gag lattice after cleavage 27 .…”

Strategies for identifying dynamic regions in protein complexes: flexibility changes accompany methylation in chemotaxis receptor signaling states

“…Solid-state NMR is ideal for studies of large protein complexes and membrane proteins because it is not reliant on long range order, solubility, or rapid tumbling [5]. In the past decade, solid-state NMR has been used to study a variety of biological systems such as HIV-1 capsid protein assemblies [6][7][8][9][10][11], 300 kDa GB1-antibody complexes [12,13], and membrane-bound influenza M2 proton channels [14][15][16]. Such NMR studies have also provided measurements of protein dynamics [17][18][19] and identification of disordered regions [20][21][22][23][24].Research in recent years on intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) has highlighted the importance of disordered regions in the function of many proteins.…”

MicroED Structures of HIV-1 Gag CTD-SP1 Reveal Binding Interactions with the Maturation Inhibitor Bevirimat

“…In 2016, compelling evidence for the predicted 6-helix bundle structure of the junction helices was provided by electron cryotomography and subtomogram averaging of ΔMA-Gag, non-enveloped virus-like particles (VLPs) 16 , X-ray crystallography of a Gag construct comprised of the C-terminal domain (CTD) of CA and SP1 17 , and solid-state NMR spectroscopy of ΔMA-Gag VLPs 18 . The cryoEM and X-ray structures revealed that the protease cleavage sites are sequestered on the interior of the 6-helix bundle.…”