Structural Basis for Alternative Self-Assembly Pathways Leading to Different Human Immunodeficiency Virus Capsid-Like Nanoparticles.

The mechanisms that underlie the spontaneous and faithful assembly of virus particles are guiding the design of self-assembling protein-based nanostructures for biomedical or nanotechnological uses. In this study, the human immunodeficiency virus (HIV-1) capsid was used as a model to investigate what molecular feature(s) may determine whether a protein nanoparticle with the intended architecture, instead of an aberrant particle, will be self-assembled in vitro. Attempts of using the HIV-1 capsid protein CA for achieving in vitro the self-assembly of cone-shaped nanoparticles that contain CA hexamers and pentamers, similar to authentic viral capsids, had typically yielded hexamer-only tubular particles. We hypothesized that a reduction in the stability of a transient major assembly intermediate, a trimer of CA dimers (ToD), will increase the propensity of CA to assemble in vitro into cone-shaped particles instead of tubes. Certain amino acid substitutions at CA-CA interfaces strongly favored in vitro the assembly of cone-shaped nanoparticles that resembled authentic HIV-1 capsids. All-atom MD simulations indicated that ToDs formed by CA mutants with increased propensity for assembly into cone-shaped particles are destabilized relative to ToDs formed by wt CA or by another mutant that assembles into tubes. The results also indicated that ToD destabilization is mediated by conformational distortion of different CA-CA interfaces, which removes some interprotein interactions within the ToD. A model is proposed to rationalize the linkage between reduced ToD stability and increased propensity for the formation of CA pentamers during particle growth in vitro, favoring the assembly of cone-shaped HIV-1 capsid-like nanoparticles.

HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

A Branched SELEX Approach Identifies RNA Aptamers That Bind Distinct HIV-1 Capsid Structural Components.

The HIV-1 capsid protein (CA) assumes distinct structural forms during replication, each presenting unique, solvent-accessible surfaces that facilitate multifaceted functions and host factor interactions. However, functional contributions of individual CA structures remain unclear, as evaluation of CA presents several technical challenges. To address this knowledge gap, we identified CA-targeting aptamers with different structural specificities, which emerged through a branched SELEX approach using an aptamer library previously selected to bind the CA hexamer lattice. Subsets were either highly specific for the CA lattice or bound both the CA lattice and CA hexamer. We then evaluated four representatives to reveal aptamer regions required for binding, highlighting interesting structural features and challenges in aptamer structure determination. Further, we demonstrate binding to biologically relevant CA structural forms and aptamer-mediated affinity purification of CA from cell lysates without virus or host modification, supporting the development of structural form-specific aptamers as exciting new tools for the study of CA.

Human Immunodeficiency Virus Type 1 Gag Polyprotein Modulates Membrane Physical Properties like a Surfactant: Potential Implications for Virus Assembly.

Human immunodeficiency virus (HIV) assembly at an infected cell's plasma membrane requires membrane deformation to organize the near-spherical shape of an immature virus. While the cellular expression of HIV Gag is sufficient to initiate budding of virus-like particles, how Gag generates membrane curvature is not fully understood. Using highly curved lipid nanotubes, we have investigated the physicochemical basis of the membrane activity of recombinant nonmyristoylated Gag-Δp6. Gag protein, upon adsorption onto the membrane, resulted in the shape changes of both charged and uncharged nanotubes. This shape change was more pronounced in the presence of charged lipids, especially phosphatidylinositol bisphosphate (PI(4,5)P2). We found that Gag modified the interfacial tension of phospholipid bilayer membranes, as judged by comparison with the effects of amphipathic peptides and nonionic detergent. Bioinformatic analysis demonstrated that a region of the capsid and SP1 domains junction of Gag is structurally similar to the amphipathic peptide magainin-1. This region accounts for integral changes in the physical properties of the membrane upon Gag adsorption, as we showed with the synthetic CA-SP1 junction peptide. Phenomenologically, membrane-adsorbed Gag could diminish the energetic cost of increasing the membrane area in a way similar to foam formation. We propose that Gag acts as a surface-active substance at the HIV budding site that softens the membrane at the place of Gag adsorption, lowering the energy for membrane bending. Finally, our experimental data and theoretical considerations give a lipid-centric view and common mechanism by which proteins could bend membranes, despite not having intrinsic curvature in their molecular surfaces or assemblies.

Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

Cationic Residues of the HIV-1 Nucleocapsid Protein Enable DNA Condensation to Maintain Viral Core Particle Stability during Reverse Transcription.

The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC's basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC's high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC's basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects.

Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site.

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.

Investigating the Intramolecular Competition of Different RNA Binding Motifs for Neomycin B by Native Top-Down Mass Spectrometry.

The ongoing search for small molecule drugs that target ribonucleic acids (RNA) is complicated by a limited understanding of the principles that govern RNA-small molecule interactions. Here we have used stoichiometry-resolved native top-down mass spectrometry (MS) to study the binding of neomycin B to small model hairpin RNAs, an unstructured RNA, and a viral RNA construct. For 15-22 nt model RNAs with hairpin structure, we found that neomycin B binding to hairpin loops relies on interactions with both the nucleobases and the 2'-OH groups, and that a simple 5' or 3' overhang can introduce an additional binding motif. For a 47 nt RNA construct derived from stem IA of the human immunodeficiency virus 1 (HIV-1) rev response element (RRE) RNA, native top-down MS identified four different binding motifs, of which the purine-rich internal loop showed the highest affinity for neomycin B. Stoichiometry-resolved binding site mapping by native top-down MS allows for a new perspective on binding specificity, and has the potential to reveal unexpected principles of small molecule binding to RNA.

Computational Insights into the Conformational Dynamics of HIV-1 Vpr in a Lipid Bilayer for Ion Channel Modeling.

HIV-1 Vpr is a multifunctional accessory protein consisting of 96 amino acids that play a critical role in viral pathogenesis. Among its diverse range of activities, Vpr can create a cation-selective ion channel within the plasma membrane. However, the oligomeric state of this channel has not yet been elucidated. In this study, we investigated the conformational dynamics of Vpr helices to model the ion channel topology. First, we employed a series of multiscale simulations to investigate the specific structure of monomeric Vpr in a membrane model. During the lipid bilayer self-assembly coarse grain simulation, the C-terminal helix (residues 56-77) effectively formed the transmembrane region, while the N-terminal helix exhibited an amphipathic nature by associating horizontally with a single leaflet. All-atom molecular dynamics (MD) simulations of full-length Vpr inside a phospholipid bilayer show that the C-terminal helix remains very stable inside the bilayer core in a vertical orientation. Subsequently, using the predicted C-terminal helix orientation and conformation, various oligomeric states (ranging from tetramer to heptamer) possibly forming the Vpr ion channel were built and further evaluated. Among these models, the pentameric form exhibited consistent stability in MD simulations and displayed a compatible conformation for a water-assisted ion transport mechanism. This study provides structural insights into the ion channel activity of the Vpr protein and the foundation for developing therapeutics against HIV-1 Vpr-related conditions.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

A two-step mechanism for the binding of the HIV-1 MPER epitope by the 10E8 antibody onto biosensor-supported lipid bilayers.

HIV-1 antibodies targeting the carboxy-terminal area of the membrane-proximal external region (ctMPER) are close to exerting viral pan-neutralization. Here, we reconstituted the ctMPER epitope as the N-terminal extremity of the Env glycoprotein transmembrane domain helix and immobilized it onto biosensor-supported lipid bilayers. We assessed the binding mechanism of anti-MPER antibody 10E8 through Surface Plasmon Resonance, and found, through equilibrium and kinetic binding analyses as a function of bilayer thickness, peptide length, and paratope mutations, that 10E8 engages first with the epitope peptide (encounter), limited by ctMPER helix accessibility at the membrane surface, and then inserts into the lipid bilayer assisted by favorable Fab-membrane interactions (docking). This mechanistic information may help in devising new strategies to develop more efficient MPER-targeting vaccines.

Hexagonal Lattices of HIV Capsid Proteins Explored by Simulations Based on a Thermodynamically Consistent Model.

HIV capsid proteins (CAs) may self-assemble into a variety of shapes under in vivo and in vitro conditions. Here, we employed simulations based on a residue-level coarse-grained (CG) model with full conformational flexibility to investigate hexagonal lattices, which are the underlying structural pattern for CA aggregations. Facilitated by enhanced sampling simulations to rigorously calculate CA dimerization and polymerization affinities, we calibrated our model to reproduce the experimentally measured affinities. Using the calibrated model, we performed unbiased simulations on several large systems consisting of 1512 CA subunits, allowing reversible binding and unbinding of the CAs in a thermodynamically consistent manner. In one simulation, a preassembled hexagonal CA sheet developed spontaneous curvatures reminiscent of those observed in experiments, and the edges of the sheet exhibited local curvatures larger than those of the interior. In other simulations starting with randomly distributed CAs at different concentrations, existing CA assemblies grew by binding free capsomeres to the edges and by merging with other assemblies. At high CA concentrations, rapid establishment of predominant aggregates was followed by much slower adjustments toward more regular hexagonal lattices, with increasing numbers of intact CA hexamers and pentamers being formed. Our approach of adapting a general CG model to specific systems by using experimental binding data represents a practical and effective strategy for simulating and elucidating intricate protein aggregations.

HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.

Molecular Determinants of PQBP1 Binding to the HIV-1 Capsid Lattice.

Human immunodeficiency virus type 1 (HIV-1) stimulates innate immune responses upon infection, including cyclic GMP-AMP synthase (cGAS) signaling that results in type I interferon production. HIV-1-induced activation of cGAS requires the host cell factor polyglutamine binding protein 1 (PQBP1), an intrinsically disordered protein that bridges capsid recognition and cGAS recruitment. However, the molecular details of PQBP1 interactions with the HIV-1 capsid and their functional implications remain poorly understood. Here, we show that PQBP1 binds to HIV-1 capsids through charge complementing contacts between acidic residues in the N-terminal region of PQBP1 and an arginine ring in the central channel of the HIV-1 CA hexamer that makes up the viral capsid. These studies reveal the molecular details of PQBP1's primary interaction with the HIV-1 capsid and suggest that additional elements are likely to contribute to stable capsid binding.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

Intermediate conformations of CD4-bound HIV-1 Env heterotrimers.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

IMPORTANCE: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells.

The envelope (Env) glycoproteins on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAbs) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes, including V2i, the gp120-g41 interface, and gp41-MPER, are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by the pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where the virus-mAb mix was pre-incubated/not pre-incubated for 1 hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein mediates viral entry and is the sole target of neutralizing antibodies. Our data suggest that antibody epitopes including V2q (e.g., PG9, PGT145), CD4bs (e.g., VRC01, 3BNC117), and V3 (2219, 2557) are masked on HIV-1 particles. The PG9 and 2219 epitopes became accessible for binding after conformational unmasking was induced by the pre-binding of select mAbs. Attempts to understand the masking mechanism led to the revelation that interaction between virus and host cells is needed to sensitize the virions for neutralization by broadly neutralizing antibodies (bNAbs). These data provide insight on how bNAbs may gain access to these occluded epitopes to exert their neutralization effects and block HIV-1 infection. These findings have important implications for the way we evaluate the neutralizing efficacy of antibodies and can potentially guide vaccine design.

Asymmetric conformations of cleaved HIV-1 envelope glycoprotein trimers in styrene-maleic acid lipid nanoparticles.

During virus entry, the pretriggered human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer initially transits into a default intermediate state (DIS) that remains structurally uncharacterized. Here, we present cryo-EM structures at near-atomic resolution of two cleaved full-length HIV-1 Env trimers purified from cell membranes in styrene-maleic acid lipid nanoparticles without antibodies or receptors. The cleaved Env trimers exhibited tighter subunit packing than uncleaved trimers. Cleaved and uncleaved Env trimers assumed remarkably consistent yet distinct asymmetric conformations, with one smaller and two larger opening angles. Breaking conformational symmetry is allosterically coupled with dynamic helical transformations of the gp41 N-terminal heptad repeat (HR1N) regions in two protomers and with trimer tilting in the membrane. The broken symmetry of the DIS potentially assists Env binding to two CD4 receptors-while resisting antibody binding-and promotes extension of the gp41 HR1 helical coiled-coil, which relocates the fusion peptide closer to the target cell membrane.

HIV-1 Group M Capsid Amino Acid Variability: Implications for Sequence Quality Control of Genotypic Resistance Testing.

BACKGROUND: With the approval of the HIV-1 capsid inhibitor, lenacapavir, capsid sequencing will be required for managing lenacapavir-experienced individuals with detectable viremia. Successful sequence interpretation will require examining new capsid sequences in the context of previously published sequence data. METHODS: We analyzed published HIV-1 group M capsid sequences from 21,012 capsid-inhibitor naïve individuals to characterize amino acid variability at each position and influence of subtype and cytotoxic T lymphocyte (CTL) selection pressure. We determined the distributions of usual mutations, defined as amino acid differences from the group M consensus, with a prevalence ≥ 0.1%. Co-evolving mutations were identified using a phylogenetically-informed Bayesian graphical model method. RESULTS: 162 (70.1%) positions had no usual mutations (45.9%) or only conservative usual mutations with a positive BLOSUM62 score (24.2%). Variability correlated independently with subtype-specific amino acid occurrence (Spearman rho = 0.83; p < 1 × 10-9) and the number of times positions were reported to contain an HLA-associated polymorphism, an indicator of CTL pressure (rho = 0.43; p = 0.0002). CONCLUSIONS: Knowing the distribution of usual capsid mutations is essential for sequence quality control. Comparing capsid sequences from lenacapavir-treated and lenacapavir-naïve individuals will enable the identification of additional mutations potentially associated with lenacapavir therapy.