HIV-1 Capsid Protein Research Articles

Kinetic Implications of IP6 Anion Binding on the Molecular Switch of the HIV-1 Capsid Assembly.

HIV-1 capsid proteins (CA) self-assemble into a fullerene-shaped capsid, enabling cellular transport and nuclear entry of the viral genome. A structural switch comprising the Thr-Val-Gly-Gly (TVGG) motif either assumes a disordered coil or a 310 helix conformation to regulate hexamer or pentamer assembly, respectively. The cellular polyanion inositol hexakisphosphate (IP6) binds to a positively charged pore of CA capsomers rich in arginine and lysine residues mediated by electrostatic interactions. Both IP6 binding and TVGG coil-to-helix transition are essential for pentamer formation. However, the connection between IP6 binding and TVGG conformational switch remains unclear. Using extensive atomistic simulations, we show that IP6 imparts structural order at the central ring, which results in multiple kinetically controlled events leading to the coil- to-helix conformational change of the TVGG motif. IP6 facilitates the helix-to-coil transition by allowing the formation of intermediate conformations. Our results identify the key kinetic role of IP6 in HIV-1 pentamer formation.

Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM

The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface. Using this approach, we successfully enriched the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) on cryoEM grids through biotinylation and resolved its structure. We further utilized this affinity method to tether the biotin-tagged dsDNA to selectively enrich a stable MCM3-ATP-dsDNA complex for cryoEM structure determination. Intriguingly, both MCM3 apo and dsDNA bound structures exhibit left-handed open spiral conformations, distinct from other reported MCM structures. The large open gate is sufficient to accommodate a dsDNA which could potentially be melted. The value of mspSA affinity method was further demonstrated by mitigating the issue of preferential angular distribution of HIV-1 capsid protein hexamer and RNA polymerase II elongation complex from Saccharomyces cerevisiae.

High-Efficiency Trifluoromethyl-Methionine Incorporation into Cyclophilin A by Cell-Free Synthesis for 19F NMR Studies.

Fluorine-19 NMR spectroscopy has emerged as a powerful tool for studying protein structure, dynamics, and interactions. Of particular interest is the exploitation of trifluoromethyl (tfm) groups, given their high sensitivity and superior transverse relaxation properties, compared to single fluorine atoms. However, biosynthetic incorporation of tfm-bearing amino acids remains challenging due to cytotoxicity and incompatibility with natural tRNA synthetases. Here, we report on overcoming this challenge using cell-free synthesis, incorporating trifluoromethyl-methionine (tfmM) into the protein Cyclophilin A (CypA) with remarkably high efficiency, impossible via biosynthetic means. Importantly, we demonstrate that tfmM CypA binds a native substrate, the N-terminal domain of HIV-1 capsid protein (HIV-1 CA-NTD), and retains peptidyl prolyl cis/trans isomerase activity. It also binds the peptide inhibitor Cyclosporine A (CsA) with the same affinity as non-labeled, wild-type CypA. Furthermore, we show that 19F isotope shifts and 19F solvent paramagnetic relaxation enhancements (PREs) provide valuable structural information on surface exposure. Taken together, our study illustrates that tfmM can readily be incorporated into proteins at very high levels by cell-free synthesis without disturbing protein structure and function, significantly expanding the scope of 19F NMR spectroscopy for studying protein structure and dynamics.

"Pseudosubstrate Envelope"/Free Energy Perturbation-Guided Design and Mechanistic Investigations of Benzothiazole HIV Capsid Modulators with High Ligand Efficiency.

Based on our proposed "pseudosubstrate envelope" concept, 25 benzothiazole-bearing HIV capsid protein (CA) modulators were designed and synthesized under the guidance of free energy perturbation technology. The most potent compound, IC-1k, exhibited an EC50 of 2.69 nM against HIV-1, being 393 times more potent than the positive control PF74. Notably, IC-1k emerged as the highest ligand efficiency (LE = 0.32) HIV CA modulator, surpassing that of the approved drug lenacapavir (LE = 0.21). Surface plasmon resonance assay and crystallographic analysis confirmed that IC-1k targeted HIV-1 CA within the chemical space of the "pseudosubstrate envelope". Further mechanistic studies revealed a dual-stage inhibition profile: IC-1k disrupted early-stage capsid-host-factor interactions and promoted late-stage capsid misassembly. Preliminary pharmacokinetic evaluations demonstrated significantly improved metabolic stability in human liver microsomes for IC-1k (T1/2 = 91.3 min) compared to PF74 (T1/2 = 0.7 min), alongside a favorable safety profile. Overall, IC-1k presents a promising lead compound for further optimization.

G-quadruplex formation in RNA aptamers selected for binding to HIV-1 capsid.

HIV-1 capsid protein (CA) is essential for viral replication and interacts with numerous host factors to facilitate successful infection. Thus, CA is an integral target for the study of virus-host dynamics and therapeutic development. The multifaceted functions of CA stem from the ability of CA to assemble into distinct structural components that come together to form the mature capsid core. Each structural component, including monomers, pentamers, and hexamers, presents a variety of solvent-accessible surfaces. However, the structure-function relationships of these components that facilitate replication and virus-host interactions have yet to be fully elucidated. A major challenge is the genetic fragility of CA, which precludes the use of many common methods. To overcome these constraints, we identified CA-targeting aptamers with binding specificity for either the mature CA hexamer lattice alone or both the CA hexamer lattice and soluble CA hexamer. To enable utilization of these aptamers as molecular tools for the study of CA structure-function relationships in cells, understanding the higher-order structures of these aptamers is required. While our initial work on a subset of aptamers included predictive and qualitative biochemical characterizations that provided insight into aptamer secondary structures, these approaches were insufficient for determining more complex non-canonical architectures. Here, we further clarify aptamer structural motifs using focused, quantitative biophysical approaches, primarily through the use of multi-effective spectroscopic methods and thermodynamic analyses. Aptamer L15.20.1 displayed particularly strong, unambiguous indications of stable RNA G-quadruplex (rG4) formation under physiological conditions in a region of the aptamer also previously shown to be necessary for CA-aptamer interactions. Non-canonical structures, such as the rG4, have distinct chemical signatures and interfaces that may support downstream applications without the need for complex modifications or labels that may negatively affect aptamer folding. Thus, aptamer representative L15.20.1, containing a putative rG4 in a region likely required for aptamer binding to CA with probable function under cellular conditions, may be a particularly useful tool for the study of HIV-1 CA.

Rare-Event Sampling using a Reinforcement Learning-Based Weighted Ensemble Method.

Despite the power of path sampling strategies in enabling simulations of rare events, such strategies have not reached their full potential. A common challenge that remains is the identification of a progress coordinate that captures the slow relevant motions of a rare event. Here we have developed a weighted ensemble (WE) path sampling strategy that exploits reinforcement learning to automatically identify an effective progress coordinate among a set of potential coordinates during a simulation. We apply our WE strategy with reinforcement learning to three benchmark systems: (i) an egg carton-shaped toy potential, (ii) an S-shaped toy potential, and (iii) a dimer of the HIV-1 capsid protein (C-terminal domain). To enable rapid testing of the latter system at the atomic level, we employed discrete-state synthetic molecular dynamics trajectories using a generative, fine-grained Markov state model that was based on extensive conventional simulations. Our results demonstrate that using concepts from reinforcement learning with a weighted ensemble of trajectories automatically identifies relevant progress co-ordinates among multiple candidates at a given time during a simulation. Due to the rigorous weighting of trajectories, the simulations maintain rigorous kinetics.

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.

Quinazolinone-based subchemotypes for targeting HIV-1 capsid protein: design and synthesis

Quinazolinone-based subchemotypes for targeting HIV-1 capsid protein: design and synthesis

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.

WEDAP: A Python Package for Streamlined Plotting of Molecular Simulation Data.

Given the growing interest in path sampling methods for extending the time scales of molecular dynamics (MD) simulations, there has been great interest in software tools that streamline the generation of plots for monitoring the progress of large-scale simulations. Here, we present the WEDAP Python package for simplifying the analysis of data generated from either conventional MD simulations or the weighted ensemble (WE) path sampling method, as implemented in the widely used WESTPA software package. WEDAP facilitates (i) the parsing of WE simulation data stored in highly compressed, hierarchical HDF5 files and (ii) incorporates trajectory weights from WE simulations into all generated plots. Our Python package consists of multiple user-friendly interfaces: a command-line interface, a graphical user interface, and a Python application programming interface. We demonstrate the plotting features of WEDAP through a series of examples using data from WE and conventional MD simulations that focus on the HIV-1 capsid protein's C-terminal domain dimer as a showcase system. The source code for WEDAP is freely available on GitHub at https://github.com/chonglab-pitt/wedap.

Arg18 Substitutions Reveal the Capacity of the HIV-1 Capsid Protein for Non-Fullerene Assembly.

In the fullerene cone HIV-1 capsid, the central channels of the hexameric and pentameric capsomers each contain a ring of arginine (Arg18) residues that perform essential roles in capsid assembly and function. In both the hexamer and pentamer, the Arg18 rings coordinate inositol hexakisphosphate, an assembly and stability factor for the capsid. Previously, it was shown that amino-acid substitutions of Arg18 can promote pentamer incorporation into capsid-like particles (CLPs) that spontaneously assemble in vitro under high-salt conditions. Here, we show that these Arg18 mutant CLPs contain a non-canonical pentamer conformation and distinct lattice characteristics that do not follow the fullerene geometry of retroviral capsids. The Arg18 mutant pentamers resemble the hexamer in intra-oligomeric contacts and form a unique tetramer-of-pentamers that allows for incorporation of an octahedral vertex with a cross-shaped opening in the hexagonal capsid lattice. Our findings highlight an unexpected degree of structural plasticity in HIV-1 capsid assembly.

HIV-1 Capsid Rapidly Induces Long-Lived CPSF6 Puncta in Non-Dividing Cells, but Similar Puncta Already Exist in Uninfected T-Cells.

The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

AbstractIn the realm of high‐throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post‐screening. Our innovation introduces a tag‐free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity‐enhancing derivatization method, utilized in tandem with nano LC‐MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

A Tag-Free Platform for Synthesis and Screening of Cyclic Peptide Libraries.

In the realm of high-throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post-screening. Our innovation introduces a tag-free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity-enhancing derivatization method, utilized in tandem with nano LC-MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

Discovery of low-molecular-weight phenylalanine derivatives as novel HIV capsid modulators with improved antiretroviral activity and metabolic stability.

The HIV capsid (CA) protein is a promising target for anti-AIDS treatment due to its critical involvement in viral replication. Herein, we utilized the well-documented CA inhibitor PF74 as our lead compound and designed a series of low-molecular-weight phenylalanine derivatives. Among them, compound 7t exhibited remarkable antiviral activity with a high selection index (EC50 = 0.040 µM, SI = 2815), surpassing that of PF74 (EC50 = 0.50 µM, SI = 258). Furthermore, when evaluated against the HIV-2 strain, 7t (EC50 = 0.13 µM) demonstrated approximately 14-fold higher potency than that of PF74 (EC50 = 1.76 µM). Insights obtained from surface plasmon resonance (SPR) revealed that 7t exhibited stronger target affinity to the CA hexamer and monomer in comparison to PF74. The potential interactions between 7t and the HIV-1 CA were further elucidated using molecular docking and molecular dynamics simulations, providing a plausible explanation for the enhanced target affinity with 7t over PF74. Moreover, the metabolic stability assay demonstrated that 7t (T1/2 = 77.0 min) significantly outperforms PF74 (T1/2 = 0.7 min) in human liver microsome, exhibiting an improvement factor of 110-fold. In conclusion, 7t emerges as a promising drug candidate warranting further investigation.

Design and Synthesis of New GS-6207 Subtypes for Targeting HIV-1 Capsid Protein.

HIV-1 capsid protein (CA) is the molecular target of the recently FDA-approved long acting injectable (LAI) drug lenacapavir (GS-6207). The quick emergence of CA mutations resistant to GS-6207 necessitates the design and synthesis of novel sub-chemotypes. We have conducted the structure-based design of two new sub-chemotypes combining the scaffold of GS-6207 and the N-terminal cap of PF74 analogs, the other important CA-targeting chemotype. The design was validated via induced-fit molecular docking. More importantly, we have worked out a general synthetic route to allow the modular synthesis of novel GS-6207 subtypes. Significantly, the desired stereochemistry of the skeleton C2 was confirmed via an X-ray crystal structure of the key synthetic intermediate 22a. Although the newly synthesized analogs did not show significant potency, our efforts herein will facilitate the future design and synthesis of novel subtypes with improved potency.

Identification of clickable HIV-1 capsid-targeting probes for viral replication inhibition

Of the targets for HIV-1 therapeutics, the capsid core is a relatively unexploited but alluring drug target due to its indispensable roles throughout virus replication. Because of this, we aimed to identify "clickable" covalent modifiers of the HIV-1 capsid protein (CA) for future functionalization. We screened a library of fluorosulfate compounds that can undergo sulfur(VI) fluoride exchange (SuFEx) reactions, and five compounds were identified as hits. These molecules were further characterized for antiviral effects. Several compounds impacted invitro capsid assembly. One compound, BBS-103, covalently bound CA via a SuFEx reaction to Tyr145 and had antiviral activity in cell-based assays by perturbing virus production, but not uncoating. The covalent binding of compounds that target the HIV-1 capsid could aid in the future design of antiretroviral drugs or chemical probes that will help study aspects of HIV-1 replication.

HIV-1 capsid and viral DNA integration.

HIV-1 capsid protein (CA)-independently or by recruiting host factors-mediates several key steps of virus replication in the cytoplasm and nucleus of the target cell. Research in the recent years have established that CA is multifunctional and genetically fragile of all the HIV-1 proteins. Accordingly, CA has emerged as a validated and high priority therapeutic target, and the first CA-targeting antiviral drug was recently approved for treating multi-drug resistant HIV-1 infection. However, development of next generation CA inhibitors depends on a better understanding of CA's known roles, as well as probing of CA's novel roles, in HIV-1 replication. In this timely review, we present an updated overview of the current state of our understanding of CA's multifunctional role in HIV-1 replication-with a special emphasis on CA's newfound post-nuclear roles, highlight the pressing knowledge gaps, and discuss directions for future research.

Biochemical Detection of Capsid in the Nucleus During HIV-1 Infection.

Recent evidence has shown that uncoating and reverse transcription precede nuclear import. These recent breakthroughs have been made possible through the development of innovative biochemical and imaging techniques. This method outlines the biochemical assay used for detecting the presence of the HIV-1 core in the nuclear compartment. In this procedure, human cells are infected with HIV-1NL4-3, with or without the inclusion of PF74, a small molecule that inhibits core entry into the nuclear compartment. Subsequently, cells are separated into cytosolic and nuclear fractions. To assess whether the capsid protein has reached the nuclear compartment, cytosolic and nuclear fractions are subjected to Western blot analysis, utilizing antibodies specific to the HIV-1 capsid protein p24. To validate the true origin of these fractions, Western blot analysis employing antibodies against cytosolic and nuclear markers arealso performed. In summary, this assay provides a reliable and efficient means to detect the presence of the HIV-1 capsid protein in the nucleus during infection under various conditions.

Multidisciplinary studies with mutated HIV-1 capsid proteins reveal structural mechanisms of lattice stabilization

HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CANTD-CANTD inter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal β-hairpin. (3) Altered conformations of E45Aa α9-helices compared to WT, E45A/R132T, WTPF74, WTNup153, and WTCPSF6 decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.