Inositol Hexakisphosphate (IP6) Accelerates Immature HIV-1 Gag Protein Assembly toward Kinetically Trapped Morphologies

Abstract

During the late stages of the HIV-1 lifecycle, immature virions are produced by the concerted activity of Gag polyproteins, primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains, which assemble into a spherical lattice, package viral genomic RNA, and deform the plasma membrane. Recently, inositol hexakisphosphate (IP6) has been identified as an essential assembly cofactor that efficiently produces both immature virions in vivo and immature virus-like particles in vitro. To date, however, several distinct mechanistic roles for IP6 have been proposed on the basis of independent functional, structural, and kinetic studies. In this work, we investigate the molecular influence of IP6 on the structural outcomes and dynamics of CA/SP1 assembly using coarse-grained (CG) molecular dynamics (MD) simulations and free energy calculations. Here, we derive a bottom-up, low-resolution, and implicit-solvent CG model of CA/SP1 and IP6, and simulate their assembly under conditions that emulate both in vitro and in vivo systems. Our analysis identifies IP6 as an assembly accelerant that promotes curvature generation and fissure-like defects throughout the lattice. Our findings suggest that IP6 induces kinetically trapped immature morphologies, which may be physiologically important for later stages of viral morphogenesis and potentially useful for virus-like particle technologies.

Temporal control by co-factors prevents kinetic trapping in retroviral Gag lattice assembly

“…Recent coarse-grained MD simulations support that IP6 can accelerate Gag-Gag interactions, capturing coordination of one IP6 to a hexamer (33). These simulations similarly found evidence of kinetic trapping with IP6(33), consistent with our finding above that fast activation of Gag by high concentrations of IP6 results in assembly that shows signs of kinetic trapping.…”

“…Recently, coarse-grained models of the immature HIV lattice have shown how IP6 stabilizes lattice assembly and speeds up binding with more molecular details, in ways that may promote defects in the structures (33). These coarse-grained models built on earlier work characterizing the specificity of Gag contacts needed for proper assembly (34), and the role of RNA and membrane binding in stabilizing Gag assembly (35).…”

Defects in the HIV immature lattice support essential lattice remodeling within budded virions

“…Our models here contain pairwise interactions, and cooperativity enters only in that the formation of a completed cycle (whether a hexamer or a higher-order cycle of multiple hexamers) is significantly more stable, because it requires two bond breaking events. However, coordination of the hexamer by IP6 can produce conformational changes(48) or kinetic effects(30) that could change the stability of hexamer contacts between say, a dimer vs a 5-mer. We did not include this additional cooperativity to keep the model as simple as possible; we expect that added cooperativity in hexamer formation would change the prefactors in the quantitative relationship we predict between the hexamer free energies and the first-passage times, as intermediates would be biased away from smaller fragments.…”

“…More generally, modeling stages of viral assembly has been critical for establishing the regimes of energetic and kinetic parameters that distinguish successful assembly from malformed or kinetically trapped intermediates, such as in viral capsid assembly (57)(58)(59)(60). Computational models of self-assembly can be used to assess how additional complexity encountered in vivo, such as macromolecular co-factors (30,31,61), crowding (59,62), and changes to membrane-to-surface geometry (24), could help to promote or suppress assembly relative to in vitro conditions. Our reactiondiffusion model developed here provides an open-source and extensible resource (34) to study preceding and following steps in the Gag assembly pathway with the addition of co-factors.…”

“…Molecular dynamics simulations of incomplete hexamers along the immature lattice gap-edge demonstrated conformational changes in Gag monomers that indicate lower stability and likely targets for protease cleavage (10). Coarse-grained simulations of lattice assembly in solution (30) and on membranes (31) have identified the importance of co-factors, including the membrane, RNA, and IP6 in stabilizing hexamer formation and growth. With our reaction-diffusion approach, we have access to longer timescales despite the large system size (~2500 monomers), and precise control over the association kinetics.…”