Mechanisms of HIV-1 Assembly

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Abstract

Despite intense study for decades, major questions regarding the assembly pathway of HIV-1 are still unresolved. In vitro, the protein component of the viral capsid, Gag, is in a monomer-dimer equilibrium in solution, but addition of virtually any nucleic acid leads to highly efficient assembly of virus-like particles (VLPs). While cryo-EM studies of VLPs have elucidated the structure of the capsid domain and flanking regions of Gag, the structures of the remaining more flexible Gag domains have remained less clear. Previously, in collaboration with Dr. Alan Rein (NCI), we discovered a heretofore unknown strong dimerization mechanism of Gag apparently mediated by a nucleic acid-induced conformational change in Gag. This allosterically induced Gag-Gag interaction is stronger than interactions at any other known Gag-Gag interface, and therefore is likely to constitute the initial oligomerization step leading to assembly of the virion. In order to elucidate the structure of this initial complex in more detail, we have initiated a collaboration with Dr Lewis Kay, University of Toronto, jointly with the Rein laboratory, to determine NMR structures of the Gag nucleocapsid domain in complex with hexanucleotides under conditions that promote Gag nucleocapsid dimerization. In addition, we intend to follow the formation of larger oligomers and assembly intermediates taking advantage of the size resolution of sedimentation velocity analytical ultracentrifugation. Pilot experiments were carried out previously to explore two different strategies. The first is aimed at the binding of Gag to short oligonucleotides that permit scaffolding a distinct small number of Gag molecules. This should reveal the interplay between Gag scaffolding and its conformational changes and dimerization. The second strategy targets the entire size-distribution of oligomers leading up to VLPs through layering Gag in the centrifugal field in situ onto buffer containing inositol hexakisphosphate, which promotes spontaneous VLP assembly. In the report period we have revisited and refined the experimental designs to be used for pursuing these approaches. From experiments with SARS-CoV-2 nucleocaspid binding to oligonucleotides of different lengths we have gained expertise in the behavior of these short nucleic acids in analytical ultracentrifugation and thermal unfolding experiments, which will expedite the planned experiments of Gag interactions. Besides the supramolecular assembly pathway, another open question of Gag/nucleic acid interactions relates to the origin of the specificity of Gag for viral RNA over host nucleic acid. To this end, we have designed hydrodynamic experiments to probe the lifetime of Gag/RNA complexes as well as the presence of conformational changes in the complex. This will be facilitated by new sample holders with longer pathlength, for which we have assembled prototypes that are in the process of validation.