Understanding Retroviral Reverse Transcription, Recombination, and Replication

在复制过程中，HIV-1将其包装的二聚体RNA基因组转化为DNA，并在感染事件中产生一个原病毒。在这一过程中，HIV-1需要保存其遗传信息，而宿主先天免疫反应试图消除能够产生感染性后代的原病毒的产生。HIV-1必须在病毒感染期间保护其基因组，同时在宿主细胞中导航。Vinay Pathak博士（HIV DRP）及其同事已经证明，保留94%衣壳（CA）蛋白的HIV-1核在整合前1.5小时进入细胞核并在整合位点附近分解（未被包裹）。然而，目前尚不清楚核衣壳是由于破裂而失去完整性，还是在衣壳解体前失去了少量CA。我们已经协助帕塔克医生的小组解决了这个问题。使用GFP含量标记，我们发现核衣壳直到整合前不久才保持其完整性，并且在衣壳相关宿主因子（mRuby-CPSF6）丢失之前平均1至3分钟丢失其GFP含量标记。这些观察结果表明完整的HIV-1衣壳是通过核孔输入的；逆转录发生在完整的衣壳中；整合前复合物与LEDGF/p75之间的相互作用，以及可能促进整合的其他宿主因子之间的相互作用，必须在衣壳完整性丧失和整合之间的短时间内发生。HIV-1将其基因组DNA整合到受感染细胞的染色体中，但它如何选择整合位点以及它们在三维核空间中的位置的影响尚不清楚。此外，我们还协助Pathak博士的部门研究转录活性HIV-1原病毒的核内位置。我们发现整合位点最初位于核膜附近，但经过几次细胞分裂后随机分布在整个细胞核中，这表明前病毒整合的染色体位点的位置是动态的。此外，我们观察到HIV-1核心在细胞核输入后不久定位于核斑，但转录活性原病毒位于核斑附近。总的来说，这些研究为HIV-1整合位点选择及其对转录活性的影响提供了见解。我们还协助Mary Kearney博士的小组（HIV DRP）建立了一个生物信息学管道，以确定在临床样本测序中鉴定的HIV-1前病毒基因组的完整性。

During replication, HIV-1 converts its packaged dimeric RNA genomes into DNA and generates one provirus in an infection event. In this process, HIV-1 needs to preserve its genetic information while the host innate immune response attempts to abolish the generation of proviruses capable of producing infectious progeny. HIV-1 must protect its genome during virus infection while navigating through the host cell. Dr. Vinay Pathak (HIV DRP) and colleague has shown that HIV-1 cores that retained 94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site 1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. We have assisted Dr. Pathak's section to address this question. Using a GFP content marker, we found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker on average of 1 to 3 min before loss of capsid-associated host factor (mRuby-CPSF6). These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration. HIV-1 integrates its genomic DNA into the chromosomes of the infected cell, but how it selects the site of integration and the impact of their location in the 3-dimensional nuclear space is not well understood. Additionally, we have assisted Dr. Pathak's section to investigate the intranuclear positions of transcriptionally active HIV-1 proviruses. We found that integration sites are first located near the nuclear envelope but become randomly distributed throughout the nucleus after a few cell divisions, indicating that the locations of the chromosomal sites of proviral integration are dynamic. Additionally, we observed that HIV-1 cores were localized to nuclear speckles shortly after nuclear import, but transcriptionally active proviruses were located adjacent to nuclear speckles. Overall, these studies provide insights into HIV-1 integration site selection and their effect on transcription activities. We have also assisted Dr. Mary Kearney's group (HIV DRP) to establish a bioinformatic pipeline to define the intactness of HIV-1 proviral genomes identified in sequencing of clinical samples.