DOI: 10.5061/dryad.70rxwdc7z
title: Effect of Lenacapavir and PF74 on HIV-1 capsid morphology: EM images

description: Lenacapavir (GS-6207; LEN) is a potent HIV-1 capsid inhibitor approved for
 treating multidrug-resistant infection. LEN binds to a hydrophobic pocket
 between neighboring capsid (CA) proteins in hexamers and stabilizes the
 capsid lattice, but its effect on HIV-1 capsids is not fully understood.
 Here, we labeled HIV-1 capsids with green fluorescent protein fused to CA
 (GFP-CA) or a fluid-phase GFP content marker (cmGFP) to assess LEN's
 impact on HIV-1 capsids. HIV-1 cores labeled with GFP-CA, but not cmGFP,
 could be immunostained with an anti-GFP antibody and were less sensitive
 to the capsid-binding host restriction factor MX2, demonstrating that
 GFP-CA is incorporated into the capsid lattice and is a marker for capsid
 lattice stability whereas cmGFP is an indicator of core integrity. LEN
 treatment of isolated HIV-1 cores resulted in a dose-dependent loss of
 cmGFP signal while preserving the GFP-CA signal, indicating that LEN
 disrupts core integrity but stabilizes the capsid lattice. In contrast,
 capsid inhibitor PF-3450074 (PF74) induced loss of core integrity and the
 capsid lattice. Electron microscopy of LEN- or PF74-treated viral cores
 revealed frequent breakage at the narrow end of the capsid and other
 morphological changes. Our results suggest that LEN treatment does not
 prevent nuclear envelope docking but inhibits nuclear import of cores with
 or without loss of core integrity. In contrast, PF74 treatment blocks
 nuclear import by inhibiting the nuclear envelope docking of viral cores,
 highlighting their different mechanisms of nuclear import inhibition.
description: <strong>Negative stain electron microscopy.
 </strong>Mature cores (5 µL of fraction 19) were incubated with 5 µL
 HS+IP6 buffer containing LEN (1, 10, or 100 nM final concentration) or
 PF74 (10 µM final concentration) in a 0.2 mL PCR tube for 20 min at 37ºC.
 HIV core specimen (5 µL) was applied to carbon film coated 200 or 400 mesh
 copper grids (Electron Microscopy Sciences). Grids were rendered
 hydrophilic by glow discharge prior to usage. After a 1 min incubation,
 the unbound specimen was wicked off with Whatman filter paper and stained
 with 2 droplets of 2% uranyl acetate for 1 min. After wicking off excess
 stain, grids were air dried and examined using a Hitachi 7800 transmission
 electron microscope at 80kV and imaged using a 5056X2960 pixel CCD camera
 (NanoSprint 15B, Advanced Microscopy Techniques). The observed capsid
 morphologies were categorized into four types: apparently intact, broken
 at the narrow end, broken (other), and aberrant. Samples were blinded and
 the number and percentage of capsids in each category was determined for
 each sample condition.
description: # Effect of Lenacapavir and PF74 on HIV-1 capsid morphology: EM images
 [https://doi.org/10.5061/dryad.70rxwdc7z](https://doi.org/10.5061/dryad.70rxwdc7z) ## Description of the data and file structure Images of HIV-1 capsids were captured using electron microscopy. The raw images, with identifying information removed, were organized into TIFF stacks. The file "EM_Raw_Images.xlsx" includes details such as the sample name, image stack name, image number, and capsid morphology for each detected capsid. In total, 1,819 capsids were analyzed from two experiments. ### Files and variables #### File: EM\_Raw\_Images.xlsx **Description:** The TIFF stack name and image number for each analyzed HIV-1 capsid are provided here. #### File: Exp#\_Stack#.tif **Description:** raw image stacks. See Excel file for metadata ## Code/software ImageJ can be used to view the images. ImageJ can be downloaded at: [https://imagej.net/ij/](https://imagej.net/ij/)