Newton, L. S. | bioRxiv Science

bioRxiv2026

Allosteric capsid inhibitors and their escape mutants drive HIV-1 sensing

Small-molecule capsid inhibitors suppress HIV-1 infectivity by binding to capsid at the same site as FG motif-bearing host cofactors Sec24C, NUP153, CPSF6 and disordered nucleoporins residing in the nuclear pore complex central channel. We have used rational design to develop inhibitors called "allosteres" that target this pocket and inhibit HIV-1 infectivity. X-ray crystal structures of capsid/inhibitor complexes, reveal allosteric shifts upon inhibitor binding in the capsid C-terminal domain which impact the capsid lattice three-fold symmetry axis. Consistent with an uncoating mechanism, we find that allosteres cause HIV-1 to trigger innate immune response dependent on viral DNA and DNA sensor cGAS. Allosteres exhibit a similar loss of potency against clinically induced Lenacapavir resistance mutants but, strikingly, we find that HIV-1 bearing key resistance mutations induces innate immune activation in the absence of inhibitor. We hypothesise that resistant mutant sensitivity to cGAS contributes to reduction of HIV-1 transmission during Lenacapavir use in prophylaxis. Our work expands the physicochemical space and scaffold range for HIV capsid targeting inhibitors, provides mechanistic details of inhibition and facilitates improved inhibitor design.

microbiology↗

bioRxiv2025

A modified cyclosporine enhances lentivector transduction ex vivo and in vivo by degrading IFITM3

Intrinsic innate immune barriers have evolved to suppress viral infection and can reduce effective gene delivery in gene therapy. We have developed BG147, a novel cyclosporine A analogue, optimised via structure-guided design to prevent inhibition of HIV cofactor Cyclophilin A and to specifically inhibit interferon-induced transmembrane proteins (IFITM1-3). BG147 enhances VSV-G pseudotyped lentiviral vector transduction ex vivo in hematopoietic stem and progenitor cells (HSPCs) and in in vivo ocular gene therapy of photoreceptor cells in mice. Upon BG147 treatment, IFITM proteins are mislocalised and degraded through lysosomal acidification-dependent pathways. IFITM3 levels functionally return in cells 96 h after BG147 washout. BG147 promises to transform ex vivo and in vivo eye gene therapies by transiently inhibiting intrinsic immune barriers mediated by IFITM proteins to enhance a wide range of protocols. One Sentence SummaryModified cyclosporine, BG147, enhances lentivector gene therapy transduction, ex vivo in HSPC and in vivo in mouse photoreceptors, by degrading IFITM3.

cell biology↗

bioRxiv2024

An integrative characterisation of proline cis and trans conformers in a disordered peptide

Intrinsically disordered proteins (IDPs) often contain proline residues, which undergo cis/trans isomerisation. While molecular dynamics (MD) simulations have the potential to fully characterise the proline cis and trans sub-ensembles, they are limited by the slow timescales of isomerisation and force field inaccuracies. Nuclear magnetic resonance (NMR) spectroscopy can report on ensemble-averaged observables for both the cis and trans proline states, but a full atomistic characterisation of these sub-ensembles is challenging. Given the importance of proline cis/trans isomerisation for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle X-ray scattering (SAXS) to characterise the two sub-ensembles of the ORF6 C-terminal region (ORF6CTR) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimised for disordered proteins. Each simulation was run for an accumulated time of 180-220 {micro}s until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamics simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamics simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6CTR are extremely dynamic and that interdisciplinary approaches combining both multi-scale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterised by either approach in isolation. SIGNIFICANCEThis study employs MD simulations (with metadynamics), NMR spectroscopy, and SAXS to elucidate the individual cis and trans proline conformations of ORF6CTR from SARS-CoV-2. The good agreement on proline cis/trans populations observed in experiments (NMR) and those calculated from simulations in the AMBER03ws force field (with SAXS reweighting) showcases the efficiency of this interdisciplinary approach, which can be used to characterise highly dynamic disordered protein states, even for very slow processes. Furthermore, our study emphasises the importance of considering both computational and experimental methodologies to gain a more holistic understanding of highly dynamic proteins. The presented integrative approach sets a precedent for future studies aiming to explore complex and dynamic biological systems with slow transitions such as proline isomerisations.

biophysics↗