Immature human immunodeficiency virus (HIV) virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. Using electron microscopy, we demonstrate that HIV virus-like particles (VLPs) assembled by the viral protein Gag and tagged at its C-terminus with the fluorescent protein Dendra2 have the same morphology and size as the VLPs assembled using only HIV Gag. We characterize the photophysical properties of Dendra2 and demonstrate that 60% of Dendra2 molecules can be photoswitched and reliably counted in our interferometric photoactivated localization microscopy (iPALM) setup. We further perform iPALM imaging on immobilized HIV Gag-Dendra2 VLPs and demonstrate that we can localize and count 900–1600 Dendra2 molecules within each immobilized VLP with a single-molecule localization precision better than (10 nm)3. Our molecular counts correspond to 1400–2400 Gag-Dendra2 proteins incorporated within each VLP. We further calculate temporal correlation functions of localization data, which we present as localization correlation analysis, and show dynamics within the lattice of immobilized VLPs in the timescale of 10–100 s. We further use our localization data to reconstruct time-lapse iPALM images of the Gag-Dendra2 lattice within the lumen of immobilized VLPs. The iPALM time-lapse images show significant lattice dynamics within the lumen of VLPs. Addition of disuccinimidyl suberate to the VLPs completely abrogated these dynamics as observed in both localization correlation analysis and time-lapse iPALM. In a complementary approach, we utilized HaXS8 cross-linking reactions between Halo and SNAP proteins and verified lattice dynamics in purified VLPs incorporating 10% Gag-SNAP, 10% Gag-Halo, and 80% Gag proteins. The HIV Gag lattice, along with the structural lattice of other enveloped viruses, has been mostly considered static. Our study provides an important tool to investigate the dynamics within these enveloped viruses.

中文翻译：

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定位相关分析和延时 iPALM 检测到的 HIV Gag 晶格动力学

未成熟的人类免疫缺陷病毒 (HIV) 病毒粒子具有锚定在其包膜腔内的 Gag 和 Gag-Pol 蛋白晶格。使用电子显微镜，我们证明由病毒蛋白 Gag 组装并在其 C 末端用荧光蛋白 Dendra2 标记的 HIV 病毒样颗粒 (VLP) 具有与仅使用 HIV Gag 组装的 VLP 相同的形态和大小。我们表征了 Dendra2 的光物理特性，并证明 60% 的 Dendra2 分子可以在我们的干涉光活化定位显微镜 (iPALM) 设置中进行光开关和可靠计数。我们进一步对固定化的 HIV Gag-Dendra2 VLP 进行 iPALM 成像，并证明我们可以在每个固定化 VLP 内定位和计数 900-1600 个 Dendra2 分子，单分子定位精度优于 (10 nm)3。我们的分子计数对应于每个 VLP 中包含的 1400–2400 个 Gag-Dendra2 蛋白。我们进一步计算定位数据的时间相关函数，我们将其呈现为定位相关分析，并在 10-100 秒的时间尺度内显示固定 VLP 晶格内的动态。我们进一步使用我们的定位数据来重建固定 VLP 管腔内 Gag-Dendra2 晶格的延时 iPALM 图像。iPALM 延时图像显示了 VLP 管腔内的显着晶格动力学。将辛二酸二琥珀酰亚胺基添加到 VLP 中完全消除了在定位相关性分析和延时 iPALM 中观察到的这些动态。在互补的方法中，我们在 Halo 和 SNAP 蛋白之间利用 HaXS8 交联反应，并验证了含有 10% Gag-SNAP、10% Gag-Halo 和 80% Gag 蛋白的纯化 VLP 中的晶格动力学。HIV Gag 晶格，连同其他包膜病毒的结构晶格，大多被认为是静态的。我们的研究提供了一个重要的工具来研究这些包膜病毒的动态。