The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro . We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly. IMPORTANCE For successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.

The capsid-binding small molecule PF74 inhibits HIV-1 infection at early stages and perturbs uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. We recently introduced time-lapse atomic force microscopy to study the morphology and physical properties of HIV-1 cores during the course of reverse transcription. Here, we apply this AFM methodology to show that PF74 prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, but a cap-like structure dissociated from the narrow end of the core HIV-1. Our result provides direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice.

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP₆) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP₆and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.

The human immunodeficiency virus (HIV-1) capsid is a self-assembled protein shell that contains the viral genome. During the stages between viral entry into a host cell and nuclear import of the viral DNA, the capsid dissociates in a process known as uncoating, which leads to the release of the viral genetic material. Mutations that alter the stability of the capsid affect the uncoating rate and impair HIV-1 infectivity. To gain further insight into the role of capsid stability during uncoating, we used atomic force spectroscopy to quantify the stiffness of the capsid. Empty in vitro assemblies of wild type (WT) and mutant recombinant HIV-1 capsid protein (CA) as well as isolated WT and mutant HIV-1 cores (i.e., filled capsids) were analyzed. We find that hyperstable CA mutant assemblies (A204C, A14C/E45C, E45A and E45A/R132T) are significantly stiffer than WT assemblies. However, the hardening effect of disulfide crosslinking (A204C and A14C/E45C) is lower than that of hydrophobic interactions (E45A and E45A/R132T). Our results demonstrate that mutations that increase the intrinsic stability of the HIV-1 capsid have an increased stiffness of their lattice.

Advances in computational methods have led to considerable progress in the design of protein nanomaterials. However, nearly all nanoparticles designed so far exhibit strict point group symmetry, which limits structural diversity and precludes anisotropic functionalization. Here we describe a computational strategy for designing multicomponent bifaceted protein nanomaterials with two distinctly addressable sides. The method centres on docking pseudosymmetric hetero-oligomeric building blocks in architectures with dihedral symmetry and designing an asymmetric protein–protein interface between them. We obtain an initial 30-subunit assembly with pseudo-D ₅ symmetry and generate variants in which we alter the size and morphology of the bifaceted nanoparticles by designing extensions to one of the subunits. Functionalization of the two nanoparticle faces with protein minibinders enables the specific colocalization of two populations of polystyrene microparticles coated with the target protein receptors. The ability to accurately design anisotropic protein nanoparticles could be broadly useful in applications requiring the colocalization of distinct target moieties.

Computational design of self-assembling proteins has long relied on pre-existing structures and sequences, fundamentally limiting control over their structural and functional properties. Recent machine learning-based methods have transformed our ability to design functional small de novo proteins and oligomers, yet methods to design large de novo protein assemblies with structures tailored to specific applications are still underexplored. Here, we develop a generalizable method for designing de novo symmetric protein complexes that incorporate target functional motifs into their structures. We report 34 new protein nanoparticles that form on-target assemblies with cubic point group symmetries. The nanoparticles exhibit a wide variety of backbones that were designed with atom-level accuracy, as evidenced by several cryo-EM and crystal structures that reveal minimal deviations from the design models. We use the method to generate a de novo antigen-tailored nanoparticle vaccine that elicits robust immune responses in mice. These results establish a generalizable approach that can be used to design functional self-assembling protein complexes with structures tailored to specific applications.