Temporal control by cofactors prevents kinetic trapping in retroviral Gag lattice assembly.

For retroviruses like HIV to proliferate, they must form virions shaped by the self-assembly of Gag polyproteins into a rigid lattice. This immature Gag lattice has been structurally characterized and reconstituted in vitro, revealing the sensitivity of lattice assembly to multiple cofactors. Due to this sensitivity, the energetic criterion for forming stable lattices is unknown, as are their corresponding rates. Here, we use a reaction-diffusion model designed from the cryo-ET structure of the immature Gag lattice to map a phase diagram of assembly outcomes controlled by experimentally constrained rates and free energies, over experimentally relevant timescales. We find that productive assembly of complete lattices in bulk solution is extraordinarily difficult due to the large size of this ∼3700 monomer complex. Multiple Gag lattices nucleate before growth can complete, resulting in loss of free monomers and frequent kinetic trapping. We therefore derive a time-dependent protocol to titrate or "activate" the Gag monomers slowly within the solution volume, mimicking the biological roles of cofactors. This general strategy works remarkably well, yielding productive growth of self-assembled lattices for multiple interaction strengths and binding rates. By comparing to the in vitro assembly kinetics, we can estimate bounds on rates of Gag binding to Gag and the cellular cofactor IP6. Our results show that Gag binding to IP6 can provide the additional time delay necessary to support smooth growth of the immature lattice with relatively fast assembly kinetics, mostly avoiding kinetic traps. Our work provides a foundation for predicting and disrupting formation of the immature Gag lattice via targeting specific protein-protein binding interactions.

Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation.

Human immunodeficiency virus type 1 (HIV-1) assembly occurs on the inner leaflet of the host cell plasma membrane, incorporating the essential viral envelope glycoprotein (Env) within a budding lattice of HIV-1 Gag structural proteins. The mechanism by which Env incorporates into viral particles remains poorly understood. To determine the mechanism of recruitment of Env to assembly sites, we interrogate the subviral angular distribution of Env on cell-associated virus using multicolor, three-dimensional (3D) superresolution microscopy. We demonstrate that, in a manner dependent on cell type and on the long cytoplasmic tail of Env, the distribution of Env is biased toward the necks of cell-associated particles. We postulate that this neck-biased distribution is regulated by vesicular retention and steric complementarity of Env during independent Gag lattice formation.

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

Assembly and maturation of the human immunodeficiency virus type 1 (HIV-1) are governed by the Gag polyprotein. Here we study the conformation and dynamics of a large HIV-1 Gag fragment comprising the matrix, capsid, spacer peptide 1 and nucleocapsid domains (referred to as ΔGag) by heteronuclear multidimensional NMR spectroscopy. In solution, ΔGag exists in a dynamic equilibrium between monomeric and dimeric states. In the presence of nucleic acids and at low ionic strength ΔGag assembles into immature virus-like particles. The structured domains of ΔGag (matrix, the N- and C-terminal domains of capsid, and the N- and C-terminal zinc knuckles of nucleocapsid) retain their fold and reorient semi-independently of one another; the linkers connecting the structural domains, including spacer peptide 1 that connects capsid to nucleocapsid, are intrinsically disordered. Structural changes in ΔGag upon proteolytic processing by HIV-1 protease, monitored by NMR in real-time, demonstrate that the conformational transition of the N-terminal 13 residues of capsid from an intrinsically disordered coil to a ß-hairpin upon cleavage at the matrix|capsid junction occurs five times faster than cleavage at the capsid|spacer peptide 1 junction. Finally, nucleic acids interact with both nucleocapsid and matrix domains, and proteolytic processing at the spacer peptide 1|nucleocapsid junction by HIV-1 protease is accelerated in the presence of single-stranded DNA.

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis.

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release--akin to its role in vesicle formation--and is not restricted to severing the thin membrane tether.