Dynamics and Genetics of HIV Proviruses before and during Antiretroviral Therapy

背景：我们和其他人发现，尽管抗逆转录病毒治疗，hiv感染的细胞仍然可以克隆扩增和持续存在，并且前病毒整合位点可能影响这一现象（Maldarelli等人，Science 345:179-183, 2014； Wagner等人，Science 345:570-573, 2014）。该项目的一个主要重点是确定在抗逆转录病毒治疗期间持续存在的这种克隆扩增的受感染细胞携带复制能力原病毒的普遍程度，以及它们持续存在于哪些组织和细胞类型中。为了回答这个问题，我们开发了一种称为多位移扩增单基因组测序（MDA-SGS）测定的新方法，该方法使我们能够分析高度扩增克隆中的原病毒（Patro等人，PNAS 116:25891-25899, 2019）。MDA-SGS检测将确定在抗逆转录病毒治疗期间持续存在的扩增克隆中的HIV前病毒是否具有完整的序列，还是包含致命的突变或缺失。我们将进一步确定，当被激活时，这种克隆是否能够产生传染性病毒粒子。如果我们证明感染细胞中潜伏的、完整的前病毒通常经历克隆扩增，这将意味着旨在治愈患者的策略不仅要阻止病毒复制，还要应对这些潜伏感染细胞的增殖。____成就：去年，为了调查儿童中的艾滋病毒库，我们进行了研究，以确定出生时感染艾滋病毒的儿童中是否存在扩大的受感染细胞克隆，以及这些细胞克隆在抗逆转录病毒治疗期间是否持续存在（Bale， Katsuimme等人，mBio 12:e00568- 21,2021）。我们发现感染的T细胞克隆在儿童（小于2个月大）早期出现，并且在治疗期间持续至少9年。这些发现表明，儿童体内的HIV库是通过治疗前感染细胞的克隆扩增来维持的。今年，我们扩大了这些研究，以解决与出生时携带艾滋病毒的儿童有关的另外两个问题。首先，我们研究了接受抗逆转录病毒治疗超过6年的儿童中最大的感染T细胞克隆的原病毒结构，发现它们都只包含单独的ltr。这些发现很重要，因为它们表明整合后HIV LTR的同源重组可能导致HIV基因的缺失，只留下一个LTR。如果正确的话，那么有理由假设一些完整的HIV基因组将通过这种机制随着时间的推移而被删除，并且这种机制可能能够用于治疗HIV。这项研究的结果目前正在整理成一份手稿。我们在这一领域的第二个成就是一项发现，即可以在几乎所有出生时就携带艾滋病毒的儿童身上发现受感染的初始细胞，而且至少在一些儿童身上发现了受感染的初始细胞的克隆。这些发现改变了我们对T细胞生物学和HIV持久性的理解，因为受感染的初始细胞的增殖可以导致受感染的中枢和效应记忆T细胞的自我更新池。我们目前正在确定儿童受感染的幼稚细胞克隆中的原病毒结构。____我们之前建立了两个新的公共数据库，用于存储和注释HIV前病毒序列及其在宿主基因组中的整合位点，特别是那些在抗逆转录病毒治疗供体中持续存在的序列。这些数据库（PSD; https://psd.cancer.gov和RID； https://rid.ncifcrf.gov/index.php）可用于对抗逆转录病毒治疗中持续存在的原病毒进行荟萃分析，以更好地了解艾滋病毒库并为治疗策略提供信息。今年，我们从接受抗逆转录病毒治疗的捐赠者那里收集了4870个全长HIV病毒。世界各地的调查人员每天访问PSD约25次。我们将RID增加到6,300,189个艾滋病毒整合点。全世界的调查人员每天访问RID大约45次。____我们开发了两个新的生物信息学管道来注释和检测抗逆转录病毒治疗期间持续存在的完整HIV原病毒（Wright等人，Retrovirology 18:16, 2021）。细胞10,2021)。我们的管道是独一无二的，因为它们可以整合到现有的管道中，从下一代测序实验（Illumina和Oxford Nanopore）中提取和比对HIV序列。今年，我们还改进了检测在治疗期间持续存在的一些HIV原病毒序列倒置的管道。____与匹兹堡大学的John Mellors合作，我们使用一种新的整合位点测定方法研究了持续存在于ART上的原病毒的遗传结构，该方法可以捕获HIV LTR、gag、env和nef序列。这种检测方法是对以前的HIV整合位点检测方法的改进，而以前的HIV整合位点检测方法不能捕获HIV遗传信息。我们发现，在持续治疗的HIV前病毒中，有很大一部分具有不对称的ltr，这可能阻止了ltr的重组，并允许这些前病毒在治疗中持续数年(Joseph等人，J. Virol。e0012222, 2022)。____在我们与John Mellors合作的基础上，我们开发了一种基于qpcr的检测方法，可测量接受抗逆转录病毒治疗的供者体内HIV感染细胞克隆的大小，并可用于研究这些细胞克隆在治疗过程中的动态（Brandt等人）。病毒。13,2021)。我们发现，大多数受感染的细胞克隆在治疗过程中高度稳定，包括那些携带完整HIV原病毒的细胞。然而，一些带有完整HIV原病毒的感染T细胞克隆随着时间的推移而变大，而一些在纵向样本中变小。____我们与塔夫茨大学的John Coffin和NCI的HIV动力学和复制项目的Steve Hughes合作，对长期抗逆转录病毒治疗的个体中持续存在的HIV前病毒的选择压力进行了迄今为止最彻底的调查（Coffin等）。中华医学杂志。17:e1009141,2021)。我们发现6个不同基因的整合事件可以导致HIV前病毒的持久性选择。这些基因是原癌基因，如BACH2和STAT5B，整合事件增强T细胞的增殖或促进其存活。这一发现表明，在治疗过程中存在一个自我更新的HIV感染细胞库，并表明仅停止病毒复制不足以治愈HIV感染。

BACKGROUND: We and others showed that HIV-infected cells can clonally expand and persist despite ART, and that the proviral integration site may influence this phenomenon (Maldarelli et al., Science 345:179-183, 2014; Wagner et al., Science 345:570-573, 2014). A major focus of this project is to determine how commonly such clonally expanded, infected cells that persist during ART carry replication-competent proviruses and in which tissues and cell types they persist. To answer this question, we developed a novel method called the multiple-displacement amplification single-genome sequencing (MDA-SGS) assay, which allows us to analyze the proviruses in highly expanded clones (Patro et al., PNAS 116:25891-25899, 2019). The MDA-SGS assay will determine if HIV proviruses in expanding clones that persist during ART have intact sequences or contain lethal mutations or deletions. We will further determine if, when activated, such clones are capable of producing infectious virions. If we show that latent, intact proviruses in infected cells commonly undergo clonal expansion, it will mean that strategies intended to cure patients will have to not only block viral replication, but also cope with the proliferation of these latently infected cells. ____ACCOMPLISHMENTS: Last year, to investigate the HIV reservoir in children, we conducted studies to determine if expanded, infected cell clones were present in children born with HIV infection and if these cell clones persisted during ART (Bale, Katsuimme et al., mBio 12:e00568-21, 2021). We found that infected T cell clones arose early in children (less than 2 months old) and that they persisted for at least 9 years during treatment. These findings demonstrated that the HIV reservoir in children is maintained by clonal expansion of cells that were infected prior to initiating treatment. This year, we expanded these studies to address two additional questions pertaining to children born with HIV. First, we investigated the proviral structures in the largest infected T cell clones in children on ART for more than 6 years and found all of them to contain only solo LTRs. These findings are important because they suggest that homologous recombination of the HIV LTRs after integration may lead to the deletion of HIV genes, leaving only a single LTR. If correct, then it is reasonable to assume that some intact HIV genomes will be deleted over time through this mechanism and that this mechanism may be able to be exploited towards a cure for HIV. The results of this study are currently being assembled into a manuscript. Our second accomplishment in this area is a finding that infected naive cells can be identified in virtually all children born with HIV and that, at least in some, clones of infected naive cells are found. These findings change our understanding of T cell biology and of HIV persistence, since, the proliferation of infected naive cells can lead to a self-renewing pool of infected central and effector memory T cells. We are currently in the process of defining the proviral structures in the clones of infected naive cells in children. ____We previously established two new public database to store and annotate HIV proviral sequences and their integration sites into the host genome, especially those that persist in donors on ART. These databases (PSD; https://psd.cancer.gov and RID; https://rid.ncifcrf.gov/index.php ) are available for meta-analyses of proviruses that persist on ART to better understand the HIV reservoir and to inform strategies towards a cure. This year, we grew the PSD to 4,870 full-length HIV proviruses collected from donors on ART. The PSD is accessed about 25 times daily by investigators worldwide. We grew the RID to 6,300,189 HIV integration sites. The RID is accessed about 45 times daily from investigators worldwide. ____We developed two new bioinformatic pipelines to annotate and detect intact HIV proviruses that persist during ART (Wright et al., Retrovirology 18:16, 2021) (Wright, et al. Cells 10, 2021). Our pipelines are unique because they can be incorporated into existing pipelines to extract and align HIV sequence reads from next-generation sequencing experiments (Illumina and Oxford Nanopore). This year, we also improved the pipelines to detect sequence inversions in some HIV proviruses that persist during treatment. ____In collaboration with John Mellors (University of Pittsburgh), we investigated the genetic structure of proviruses that persist on ART using a new integration sites assay that captures HIV LTR, gag, env, and nef sequences. This assay is an improvement on the previous HIV integration sites assay which is not capable of capturing HIV genetic information. We found that a high fraction of HIV proviruses that persist on treatment have asymmetric LTRs, possibly preventing recombination of the LTRs and allowing these proviruses to persist for years on treatment (Joseph et al., J. Virol. e0012222, 2022). ____ To build on our collaboration with John Mellors, we developed a qPCR-based assay that measures the size of HIV infected cell clones in donors on ART and can be used to investigate the dynamics of these cell clones over the course of treatment (Brandt, et al. Viruses. 13, 2021). We found that most infected cell clones are highly stable over the course of treatment, including those with intact HIV proviruses. However, a few infected T cell clones with intact HIV proviruses grew larger over time and a few diminished in size over longitudinal samples. ____ In collaboration with John Coffin (Tufts University) and Steve Hughes (HIV Dynamics and Replication Program, NCI), we conducted the most thorough investigation to date of selection pressures for and against HIV proviruses that persist in individuals on long-term ART (Coffin, et al. PLoS Pathog. 17: e1009141, 2021). We found that integration events into 6 different genes can results in selection for persistence of HIV proviruses. Integration events into these genes, which are proto-oncogenes such as BACH2 and STAT5B, enhance the proliferation of the T cells or promote their survival. This finding demonstrates that there is a self-renewing pool of HIV infected cells during treatment and show that halting viral replication alone will not be sufficient to cure HIV infection.