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.
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机译：未成熟的人免疫缺陷病毒（HIV）病毒粒子具有锚定的GAG和GAG-POL蛋白，锚定在其封套的内腔上。使用电子显微镜，我们证明了由病毒蛋白堵塞组装的HIV病毒样颗粒（VLP）并用荧光蛋白DENDRA2标记在其C末端具有相同的形态和尺寸，因为仅使用HIV GAG组装的VLP组装。我们表征了Dendra2的光物理性质，并证明60％的Dendra2分子可以是光纤和可靠地计数在我们的干涉式光活化的定位显微镜（IPALM）设置中。我们进一步在固定的HIV GAG-DENDRE2 VLP上进行IPALM成像，并证明我们可以在每个固定的VLP内定位和计数900-1600dendra2分子，单分子定位精度优于（10nm）3。我们的分子计数对应于1400-2400 GAG-DENDRA2蛋白，其在每个VLP中掺入。我们进一步计算定位数据的时间相关函数，我们作为定位相关性分析，并在10-100秒的时间尺寸下显示固定的VLP的晶格内的动态。我们进一步使用本地化数据来重建在固定VLP的内腔内GAG-DENDRA2格子的时间流逝IPALM图像。 IPALM时间流逝图像显示VLP的内腔内的显着晶格动力学。在本地化相关性分析和延时IPALM中观察到的这些动态，加入vlps的四琥珀酰亚胺酰亚胺酰亚胺酰亚胺。以互补方法，我们在卤素和捕获蛋白之间使用HaxS8交联反应，并在纯化的VLP中验证了含有10％呕吐，10％GAG-卤素和80％GAG蛋白的晶格动力学。 HIV GAG格子以及其他封闭病毒的结构晶格以及主要被认为是静态的。我们的研究提供了调查这些包络病毒内的动态的重要工具。
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