Rankovic Sanela, Deshpande Akshay, Harel Shimon, Aiken Christopher, Rousso Itay
Ben-Gurion University of the Negev, Department of Physiology and Cell Biology, Beer Sheva, Israel.
Vanderbilt University Medical Center, Department of Pathology, Microbiology and Immunology, Nashville, TN.
J Virol. 2021 Apr 26;95(10). doi: 10.1128/JVI.00166-21. Epub 2021 Mar 10.
The HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. These reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.For successful infection, the HIV-1 genome, which is enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism of HIV-1 uncoating is a pivotal question of long standing. Using atomic force microscopy to analyze individual HIV-1 cores during reverse transcription, we observe a reproducible pattern of stiffness spikes. These spikes were shown to be associated with distinct stages of the reverse transcription reaction. Our findings suggest that these reverse-transcription-induced alterations gradually prepared the core for uncoating at the right time and location in target cells.
HIV核心由病毒基因组和相关蛋白质组成，被一个称为衣壳的锥形蛋白质外壳包裹。成功感染需要病毒基因组的逆转录以及衣壳在细胞内的拆解，这一过程称为脱壳。病毒衣壳的完整性对于逆转录至关重要，但在整合之前，必须打破病毒衣壳以释放新生的病毒DNA。我们采用原子力显微镜研究了在含有衣壳稳定宿主代谢物IP的体外逆转录反应过程中HIV-1核心的硬度变化。核心呈现出一系列硬度峰值，通常在逆转录开始后的10-30分钟、40-80分钟和120-160分钟之间出现多达三个峰值。添加逆转录酶（RT）抑制剂依非韦伦消除了这些峰值的出现以及随后衣壳的拆解，从而确定这两者均由逆转录导致。通过定时添加依非韦伦以及对RNAseH缺陷型RT突变体的分析，我们确定第一个硬度峰值需要负链强终止DNA合成，随后的峰值需要逆转录的后期阶段。额外的快速原子力显微镜成像实验揭示了核心中反复出现的形态变化，这些变化在时间上与观察到的硬度峰值相关。我们的研究揭示了病毒核心中离散的机械变化，这些变化可能与逆转录的特定阶段有关。这些由逆转录引起的衣壳变化逐渐重塑病毒核心，使其在靶细胞中为适时准确的脱壳做好准备。为了成功感染，包裹在衣壳内的HIV-1基因组必须逆转录成双链DNA并从衣壳中释放出来（这一过程称为脱壳）才能整合到靶细胞基因组中。HIV-1脱壳机制是一个长期存在的关键问题。利用原子力显微镜在逆转录过程中分析单个HIV-1核心，我们观察到一种可重复的硬度峰值模式。这些峰值与逆转录反应的不同阶段相关。我们的研究结果表明，这些由逆转录引起的改变逐渐使核心在靶细胞中的正确时间和位置做好脱壳准备。
