DOI: 10.5061/dryad.18931zd49
title: Data from: Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure

description: The HIV-1 capsid has emerged as a tractable target for antiretroviral
 therapy. Lenacapavir, developed by Gilead Sciences, is the first
 capsid-targeting drug approved for medical use. Here we investigate the
 effect of Lenacapavir on HIV capsid stability and uncoating. We employ a
 single-particle approach that simultaneously measures capsid content
 release and lattice persistence. We demonstrate that Lenacapavir’s potent
 antiviral activity is predominantly due to lethal hyperstabilisation of
 the capsid lattice and resultant loss of compartmentalisation. This study
 highlights that disrupting capsid metastability is a powerful strategy for
 the development of novel antivirals.
description: The raw TIRF microscopy image stacks were collected using
 Micromanager (https://micro-manager.org/) and saved in TIFF format
 conforming to the OME standard
 (https://www.openmicroscopy.org/).
description: # Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces
 capsid failure
 [https://doi.org/10.5061/dryad.18931zd49](https://doi.org/10.5061/dryad.18931zd49) **Primary Depositor:** James Walsh **Institution:** University of New South Wales **Email:** [james.walsh@phys.unsw.edu.au](mailto:james.walsh@phys.unsw.edu.au) **ORCID:** 0000-0003-0447-2323 **Date:** The date of microscopy data acquisition is specified in the file name of each TIFF image stack, typically in the format YYYYMMDD. **Location:** All data was collected at UNSW Sydney. **Secondary Contact:** Till Boecking **Email:** [till.boecking@unsw.edu.au](mailto:till.boecking@unsw.edu.au) **ORCID:** 0000-0003-1165-3122 **Funding:** National Health and Medical Research Council (APP1182212), National Health and Medical Research Council (APP1194263), Australian Research Council (DP160101874) and Australian Research Council (DP200102871), Wellcome Trust (214344/Z/18/Z). This repository contains data collected for the paper "Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure" by K. M. Rifat Faysal et al. (eLife 2024), including fluorescence microscopy image stacks (Figures 2, 3, 4, 7, 8), drug dose-response curves of virus infection and cDNA synthesis (Figures 5, 7) and absorbance measurements of CA assembly (Figure 6). The data is organised to correspond to the figures in the paper. All methods are included in the methods section of the paper. The raw TIRF microscopy image stacks were collected using Micromanager ([https://micro-manager.org/](https://micro-manager.org/)) and saved in TIFF format conforming to the OME standard ([https://www.openmicroscopy.org/](https://www.openmicroscopy.org/)). ## Description of the data and file structure **Figures 2&3:** Survival analysis of capsid opening and CypA paint analysis of CA lattice stability in the absence and presence of LEN and IP6. Single-particle uncoating image stacks (dual-colour and single-colour experiments) were collected and analysed by K.M. Rifat Faysal. The image stacks were acquired with a rate of 1 frame/6 s. Each folder corresponds to a specific experiment acquired with conditions as described in the list below and contains a TIFF file (OME standard) with dual-channel (iGFP + CypA paint) or single-channel (iGFP only) data as indicated in the list below. The following experiments are shown in the survival curves in Fig 2D: 0 nM LEN, ID #31; 500 nM LEN, ID #6. The survival curves shown in Fig 3A and B are calculated from pooled data of all experiments for a given condition as listed below. **Figure 4:** Measuring the kinetics of Lenacapavir binding using competition with fluorescently labelled CPSF6 peptide. This figure combines two experiments collected using two different labels for the CPSF6 peptide: Atto643 and AlexaFluor568. The data for each label is zipped separately to enable it to be uploaded. The data for AlexaFluor568 is split into three zip files to enable them to be uploaded. These three files should all be unzipped into the same folder. For each file name, the number after the P indicates the PDMS chip it was imaged on, C is the channel within that chip and F is the field of view. The concentration of Lenacapavir used is denoted by the number before the nm or pm, in nanomolar or picomolar concentrations respectively. Concentrations that are not integer concentrations use a “p” in place of a decimal place, for example, 1.5 nM is labelled \_1p5_nm. The frame rate is given in seconds per frame by the value before SPF. **Figure 5&7:** Tab-delimited text files of dose response-curves plotting (i) relative infectivity of a GFP-reporter virus in Jurkat cells as a function of drug concentration and (ii) viral cDNA synthesis in Jurkat cells as a function of drug concentration. The file “Infectivity (LEN with and without preincubation).txt” contains the LEN dose-infectivity data for Figure 5 (with and without preincubation of the reporter virus with LEN). Empty cells correspond to conditions not tested in the experiment. Column 1: Concentration of lenacapavir in nM Columns 2-7: Relative infectivity comparing virions exposed to lenacapavir from the beginning of the infection experiment (“0 h preincubation”, columns 2, 4, 6) and virions also preincubated with lenacapavir for 48 h before the infection experiment (“48 h preincubation”, columns 3, 5, 7). Data from three independent experiments: Exp A (columns 2, 3), Exp B (columns 4, 5) and Exp C (columns 6, 7).  Column 8: Relative infectivity of virions exposed to lenacapavir from the beginning of the infection experiment (“0 h preincubation”). This data was collected in an experiment comparing lenacapvir and PF74. It is the same as the data in column 2 of the file “Infectivity (LEN vs PF74).txt”.   The file “Infectivity (LEN vs PF74).txt” contains the PF74 dose-infectivity data for Figure 7. Empty cells correspond to conditions not tested in the experiment. Column 1: Concentration of drug in nM Column 2: Relative infectivity of virions in the presence of lenacapavir during the infection experiment (GS-6207). Column 3: Relative infectivity of virions in the presence of PF74 during the infection experiment (GS-6207). The file “vDNA synthesis (LEN).txt” contains the LEN dose-vDNA synthesis data for Figure 5 (with and without preincubation of the reporter virus with LEN). Column 1: Concentration of lenacapavir in nM Column 2-3: Mean (column 2) and standard deviation (column 3) of the level of total viral DNA (determined by qPCR) from cells infected with virions that were pretreated with lenacapavir for 48 h before the infection experiment (“48 h preincubation”). Column 4-5: Mean (column 4) and standard deviation (column 5) of the level of total viral DNA (determined by qPCR) from cells infected with virions that were exposed to lenacapavir from the beginning of the infection experiment (“0 h preincubation”). **Figure 6:** Tab-delimited text files of absorbance measurements as a function of time for CA assembly reactions conducted in the presence and absence of LEN and IP6. The conditions for each assembly reaction are provided in the header of the corresponding data column. **Figures 7&8:** CypA paint analysis of CA lattice stability in the presence of PF74, BI-2 and CPSF6 peptide. Dual-colour (iGFP + CypA paint) single-particle uncoating image stacks were collected and analysed by C. L. Márquez. Each folder corresponds to a specific experiment with conditions as described in the list below and contains two TIFF files corresponding to the iGFP channel (file name appended by “_2”) and the CypA paint channel (file name appended by “_1” or “_3”). ### Specific Conditions **Figures 2&3:** Condition: 0 nM LEN. Foldername: oct-27-2020-exp1-igfp-pspax2-fresh-dly-cypa-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 4. Condition: 0 nM LEN. Foldername: oct-27-2020-exp6-igfp-pspax2-fresh-dly-cypa-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 9. Condition: 0 nM LEN. Foldername: oct-27-2020-exp7-igfp-pspax2-fresh-dly-cypa-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 10. Condition: 0 nM LEN. Foldername: april-28-21-exp2-iGFP-psPAX2-cypa647-dly [Channels: iGFP + CypA paint]. Experiment ID: 19. Condition: 0 nM LEN. Foldername: may23-21-exp1-igfp-pspax2-slo-cypa-paint [Channels: iGFP + CypA paint]. Experiment ID: 31. Condition: 0 nM LEN. Foldername: jun10-2021-exp1-igfp-pspax2-gs6207-CypA-paint [Channels: iGFP + CypA paint]. Experiment ID: 44. Condition: 0 nM LEN. Foldername: jun10-2021-exp2-igfp-pspax2-gs6207-CypA-paint [Channels: iGFP + CypA paint]. Experiment ID: 45. Condition: 0 nM LEN. Foldername: jun30-21-exp1-igfp-pspax2-cypa-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 67. Condition: 0 nM LEN. Foldername: jun30-21-exp2-igfp-pspax2-cypa-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 68. Condition: 0.5 nM LEN. Foldername: oct29-2020-exp2-igfp-pspax2-fresh-dly-cypa-paint-gs-6207-500pM_1 [Channels: iGFP + CypA paint]. Experiment ID: 12. Condition: 0.5 nM LEN. Foldername: jun1-2021-exp1-igfp-pspax2-gs6207-500pM_1 [Channels: iGFP]. Experiment ID: 32. Condition: 0.5 nM LEN. Foldername: jun1-2021-exp2-igfp-pspax2-gs6207-500pM_1 [Channels: iGFP]. Experiment ID: 33. Condition: 0.5 nM LEN. Foldername: jun1-2021-exp5-igfp-pspax2-gs6207-500pM_1 [Channels: iGFP]. Experiment ID: 37. Condition: 5 nM LEN. Foldername: oct29-2020-exp1-igfp-pspax2-fresh-dly-cypa-paint-gs-6207-5nM_2 [Channels: iGFP + CypA paint]. Experiment ID: 11. Condition: 5 nM LEN. Foldername: jun10-2021-exp3-igfp-pspax2-gs6207-5nM-CypA-paint [Channels: iGFP + CypA paint]. Experiment ID: 46. Condition: 5 nM LEN. Foldername: jun10-2021-exp4-igfp-pspax2-gs6207-5nM-CypA-paint [Channels: iGFP + CypA paint]. Experiment ID: 47. Condition: 5 nM LEN. Foldername: jun10-2021-exp5-igfp-pspax2-gs6207-5nM-CypA-paint [Channels: iGFP + CypA paint]. Experiment ID: 48. Condition: 50 nM LEN. Foldername: oct-27-2020-exp4-igfp-pspax2-fresh-dly-cypa-paint-gs6207-50nM_1 [Channels: iGFP + CypA paint]. Experiment ID: 7. Condition: 50 nM LEN. Foldername: jun1-2021-exp3-igfp-pspax2-gs6207-50nM_1 [Channels: iGFP]. Experiment ID: 34. Condition: 50 nM LEN. Foldername: jun1-2021-exp4-igfp-pspax2-gs6207-50nM_1 [Channels: iGFP]. Experiment ID: 35. Condition: 50 nM LEN. Foldername: jun1-2021-exp4-igfp-pspax2-gs6207-50nM_2 [Channels: iGFP]. Experiment ID: 36. Condition: 50 nM LEN. Foldername: jun1-2021-exp6-igfp-pspax2-gs6207-50nM_1 [Channels: iGFP]. Experiment ID: 38. Condition: 500 nM LEN. Foldername: oct-27-2020-exp3-igfp-pspax2-fresh-dly-cypa-paint-gs6207-500nM_1 [Channels: iGFP + CypA paint]. Experiment ID: 6. Condition: 500 nM LEN. Foldername: jun1-2021-exp7-igfp-pspax2-gs6207-500nM_1 [Channels: iGFP]. Experiment ID: 39. Condition: 500 nM LEN. Foldername: jun1-2021-exp8-igfp-pspax2-gs6207-500nM_1 [Channels: iGFP]. Experiment ID: 40. Condition: 500 nM LEN. Foldername: jun16-2021-exp1-igfp-pspax2-gs6207-500nM-CypA-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 55. Condition: 0 nM LEN + 100 µM IP6. Foldername: april-28-21-exp3-iGFP-psPAX2-cypa647-ip6-100uM-dly [Channels: iGFP + CypA paint]. Experiment ID: 20. Condition: 0 nM LEN + 100 µM IP6. Foldername: april-28-21-exp4-iGFP-psPAX2-cypa647-ip6-100uM-dly [Channels: iGFP + CypA paint]. Experiment ID: 21. Condition: 0 nM LEN + 100 µM IP6. Foldername: april-28-21-exp7-iGFP-psPAX2-cypa647-ip6-100uM-dly [Channels: iGFP + CypA paint]. Experiment ID: 24. Condition: 5 nM LEN + 100 µM IP6. Foldername: jun10-2021-exp8-igfp-pspax2-gs6207-5nM-IP6-100uM-CypA-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 51. Condition: 5 nM LEN + 100 µM IP6. Foldername: jun10-2021-exp9-igfp-pspax2-gs6207-1nM-IP6-100uM-CypA-paint_2 [Channels: iGFP + CypA paint]. Experiment ID: 53. Condition: 50 nM LEN + 100 µM IP6. Foldername: jun10-2021-exp11-igfp-pspax2-gs6207-50nM-IP6-100uM-CypA-paint_1 [Channels: iGFP + CypA paint]. Experiment ID: 54. Condition: 500 nM LEN + 100 µM IP6. Foldername: april-28-21-exp5-iGFP-psPAX2-cypa647-ip6-100uM-gs6207-500nM-dly [Channels: iGFP + CypA paint]. Experiment ID: 22. Condition: 5 nM LEN, 4h preincubation. Foldername: jun7-2021-exp1-igfp-pspax2-gs6207-5nM-preincubation_1 [Channels: iGFP]. Experiment ID: 41. Condition: 5 nM LEN, 4h preincubation. Foldername: jun7-2021-exp2-igfp-pspax2-gs6207-5nM-preincubation_1 [Channels: iGFP]. Experiment ID: 42. Condition: 5 nM LEN, 4h preincubation. Foldername: jun7-2021-exp3-igfp-pspax2-gs6207-5nM-preincubation_1 [Channels: iGFP]. Experiment ID: 43. **Figures 7&8:** Figure 7A-Graph 1. Condition: control. Foldername: 20170910_P1C2a -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 39. Figure 7A-Graph 2. Condition: 0.1 µM PF74. Foldername: 20180123_P1C3a -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 16.9. Figure 7A-Graph 3. Condition: 0.5 µM PF74. Foldername: 20180123_P1C4a -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 16.9. Figure 7A-Graph 4. Condition: 1 µM PF74. Foldername: 20180123_P1C2a -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 16.9. Figure 7A-Graph 5. Condition: 5 µM PF74. Foldername: 20180123_P1C5a -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 16.9. Figure 7A-Graph 6. Condition: 10 µM PF74. Foldername: 20180123_P2C1a -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 16.9. Figure 7B. Condition: 10 µM PF74 . Foldername: 20180925_P1C2a -- Number of frames (frame rate): 800 (1 frame/6 s); single-molecule intensity calibration value: 15.36. Figure 7C. Condition: 50 µM BI2. Foldername: 20170910_P3C2aa -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 39. Figure 8A-Graph 1. Condition: 1 µM unlabeled CPSF6 peptide. Foldername: 20171207_P1C5-posBa -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 19.96. Figure 8A-Graph 2. Condition: 5 µM unlabeled CPSF6 peptide. Foldername: 20171207_P1C4a -- Number of frames (frame rate): 800 (1 frame/s); single-molecule intensity calibration value: 19.96. Figure 8A-Graph 3. Condition: 10 µM unlabeled CPSF6 peptide. Foldername: 20171207_P1C3a -- Number of frames (frame rate): 800 (1 frame/s)+100 (1 frame/10s); single-molecule intensity calibration value: 19.96. Figure 8A-Graph 4. Condition: 100 µM unlabeled CPSF6 peptide. Foldername: 20171207_P1C2a -- Number of frames (frame rate): 800 (1 frame/s)+100 (1 frame/10s); single-molecule intensity calibration value: 19.96. Figure 8C. Condition: 100 µM CPSF6 peptide. Foldername: 20180705_P2C4a -- Number of frames (frame rate): 800 (1 frame/6 s); single-molecule intensity calibration value: 13.63. ## Code/Software This data was used to generate single-particle localizations intensity traces using JIM ([https://github.com/lilbutsa/JIM-Immobilized-Microscopy-Suite](https://github.com/lilbutsa/JIM-Immobilized-Microscopy-Suite)). Analysis of traces was then performed using Matlab ([https://au.mathworks.com/products/matlab.html](https://au.mathworks.com/products/matlab.html)).