Effect of Lenacapavir and PF74 on HIV-1 capsid morphology: EM images

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 c..., Negative stain electron microscopy. 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 capsid..., , # 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/) ,来那卡帕韦（Lenacapavir，GS-6207；简称LEN）是一种强效HIV-1衣壳抑制剂，已获批用于治疗多重耐药感染。LEN可结合至六聚体中相邻衣壳（capsid, CA）蛋白之间的疏水口袋，稳定衣壳晶格，但目前对其调控HIV-1衣壳的完整机制尚未完全阐明。本研究采用融合了绿色荧光蛋白（GFP）的CA蛋白（GFP-CA）或液相GFP内容物标记物（cmGFP）对HIV-1衣壳进行标记，以评估LEN对HIV-1衣壳的影响。经GFP-CA标记而非cmGFP标记的HIV-1核心可通过抗GFP抗体进行免疫染色，且对衣壳结合型宿主限制因子MX2的敏感性更低，这表明GFP-CA可整合至衣壳晶格中，可作为衣壳晶格稳定性的标记物，而cmGFP则可作为核心完整性的指示指标。LEN处理分离的HIV-1核心后，可引发剂量依赖性的cmGFP信号丢失，同时保留GFP-CA信号，结果表明LEN可破坏核心完整性，但稳定衣壳晶格。本研究随后开展了负染电子显微镜实验：将成熟核心（5 μL，组分19）与5 μL含LEN（终浓度分别为1、10或100 nM）或PF74（终浓度10 μM）的HS+IP6缓冲液在0.2 mL聚合酶链式反应（PCR）管中于37℃孵育20 min。将5 μL HIV核心样品滴涂至经碳膜包被的200目或400目铜网（Electron Microscopy Sciences）。使用前通过辉光放电使铜网表面亲水。孵育1 min后，用Whatman滤纸吸去未结合的样品，并用2滴2%醋酸铀酰染色1 min。吸去多余染液后，将铜网自然晾干，使用日立7800透射电子显微镜在80 kV电压下观察，并采用5056×2960像素的电荷耦合器件（CCD）相机（NanoSprint 15B，Advanced Microscopy Techniques）进行成像。观察到的衣壳形态可分为四类：完整无缺、窄端断裂、其他类型断裂以及异常形态。实验采用盲法分析，统计衣壳的数量及占比…… # 来那卡帕韦与PF74对HIV-1衣壳形态的影响：电镜图像 [https://doi.org/10.5061/dryad.70rxwdc7z](https://doi.org/10.5061/dryad.70rxwdc7z) ## 数据与文件结构说明 本数据集包含通过电子显微镜采集的HIV-1衣壳图像。已移除识别信息的原始图像被整理为TIFF图像栈。文件"EM_Raw_Images.xlsx"包含每个检测到的衣壳的相关详情，包括样品名称、图像栈名称、图像编号以及衣壳形态。本研究共开展两次实验，总计分析了1819个衣壳。 ### 文件与变量 #### 文件：EM_Raw_Images.xlsx **描述**：本文件包含每个已分析HIV-1衣壳对应的TIFF图像栈名称与图像编号。 #### 文件：Exp#_Stack#.tif **描述**：原始图像栈。元数据详见上述Excel文件。 ## 代码与软件 可使用ImageJ查看图像。ImageJ的下载地址为：[https://imagej.net/ij/](https://imagej.net/ij/)