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， 在感染事件中产生一个前病毒。在这个过程中，HIV-1需要保持其 遗传信息，而宿主先天免疫反应试图消除这一代 能够产生传染性后代的前病毒。HIV-1必须保护其基因组， 病毒感染，同时通过宿主细胞导航。Vinay Pathak博士（艾滋病毒DRP）和 我的同事发现，HIV-1核心保留了94%的衣壳（CA）蛋白 进入细胞核并在整合位点附近解体（未涂覆）， 一体化然而，无论是核衣壳由于破裂或小的破裂而失去了完整性， 衣壳解体前CA的损失尚不清楚。我们协助帕萨克医生的部门 解决这个问题。使用GFP含量标记，我们发现核衣壳保留了 它们的完整性直到整合前不久，并且平均失去了它们的GFP含量标记 1至3分钟后，Captain相关宿主因子（mRuby-CPSF 6）消失。这些观察结果 这意味着完整HIV-1衣壳通过核孔输入;逆转录 发生在完整的衣壳中;并且整合前复合物和 LEDGF/p75和可能的其他促进整合的宿主因子，必须发生在 衣壳完整性丧失和整合之间的时间短。HIV-1整合了其 基因组DNA进入受感染细胞的染色体，但它如何选择的网站， 集成和影响他们的位置在三维核空间是不好的 明白此外，我们还协助Pathak博士的部门调查了 转录活性HIV-1前病毒的核内位置。我们发现， 位点首先位于核膜附近，然后随机分布在整个 细胞分裂后的细胞核，表明染色体位点的位置， 是动态的。此外，我们观察到HIV-1核心定位于 核导入后不久，核斑点，但转录活性前病毒， 位于核斑点附近。总的来说，这些研究提供了对HIV-1的深入了解。 整合位点选择及其对转录活性影响。我们还协助 玛丽科尔尼博士的小组（艾滋病毒DRP）建立一个生物信息学管道，以定义 在临床样品测序中鉴定的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.