Dynamics and Genetics of HIV Proviruses before and during Antiretroviral Therapy

背景：我们和其他人表明，尽管抗逆转录病毒治疗，艾滋病毒感染的细胞可以克隆性扩张和持续存在，前病毒整合部位可能会影响这种现象(Maldarelli等人，Science 345：179-183,2014；Wagner等人，Science 345：570-573,2014)。该项目的一个主要焦点是确定在ART期间持续存在的这种克隆性扩张的受感染细胞通常携带具有复制能力的前病毒，以及它们在哪些组织和细胞类型中持续存在。为了回答这个问题，我们开发了一种名为多置换扩增单基因组测序的新方法，它允许我们分析高度扩展克隆中的前病毒(Patro等人，PNAS116：25891-25899,2019年)。丙二醛-SGS检测将确定在抗逆转录病毒治疗期间持续存在的扩大细胞克隆中的艾滋病毒前病毒是否具有完整的序列或包含致命的突变或缺失。我们将进一步确定，当被激活时，这些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、完整的前病毒通常会进行克隆性扩张，这将意味着旨在治愈患者的策略将不仅要阻止病毒复制，还必须应对这些潜伏感染细胞的增殖。_成就：我们早在4年前就开始了对儿童艾滋病毒宿主的研究，最初的重点是在出生时感染艾滋病毒的儿童中是否存在扩大的、受感染的细胞克隆，以及这些细胞克隆是否在抗逆转录病毒治疗期间持续存在(Bale，Katusiime等人，mBio 12：e00568-21,2021)。我们发现，感染的T细胞克隆出现在儿童(不到2个月大)的早期，并且在治疗期间持续至少9年。这些发现表明，儿童中的艾滋病毒储存库是通过在开始治疗之前感染的细胞的克隆性扩增来维持的。6年多来，我们通过研究接受抗逆转录病毒治疗的儿童中最大的受感染T细胞克隆中的前病毒结构，对这些努力进行了跟踪，发现它们都是单独的ltrs(Botha等人，mBio，正在出版)。这些发现很重要，因为它们表明，整合后艾滋病毒LTRS的同源重组可能导致艾滋病毒基因的删除，只留下一段不活跃的残留物，可能是复制能力强的前病毒，对艾滋病毒储存库有贡献。这些发现表明，随着时间的推移，LtR重组可能显著地促进ART的储存层腐烂，这表明可能设计出一种新的治疗策略，利用或加速LtR重组。在一项相关的、正在进行的研究中，我们确定了从艾滋病毒携带者儿童身上收集的原始T细胞可以容纳整合的前病毒，有时是在克隆群体中，并且在一个例子中，隐藏的前病毒是序列完整的。这些发现改变了我们对T细胞生物学和HIV持久性的理解，因为受感染的幼稚细胞的增殖可能导致受感染的中央和效应记忆T细胞的自我更新池。描述这些发现的手稿正在准备中。另一项正在进行的、多方面的努力旨在确定前病毒种群的遗传学特征，该努力利用了来自捐赠者的样本，这些捐赠者在ART上有高水平的病毒血症，尽管病毒被完全抑制。在对这些样本进行的深入分析中产生的显著发现包括，观察到序列完整的前病毒不成比例地整合到编码锌指蛋白KRAb家族的基因的内含子区域，在这些样本中确定的10个最大的受感染T细胞克隆中，有6个是对从艾滋病毒儿童身上获得的样本进行的单独支持分析。为了促进这些努力，我们最近设计并发表了一种新的组合前病毒测序和整合位点检测方法，该方法允许更全面的前病毒特征，这是以前可能的(Joseph等人，J.Virol)。E0012222,2022)。

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 expanded cell 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: We began our investigations of the HIV reservoir in children more than 4 years ago, initially focusing on whether expanded, infected cell clones were present in children born with HIV infection and if these cell clones persisted during ART (Bale, Katusiime 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. We followed up on these efforts by investigating the proviral structures in the largest infected T cell clones in children on ART for more than 6 years, and found them all to be solo LTRs (Botha, et al., mBio, in press). 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 an inactive remnant of what might have been a replication competent provirus contributing to the HIV reservoir. These findings suggest that LTR recombination may significantly contribute to reservoir decay over time on ART, suggesting that a new curative strategy might be devised that exploits or accelerates LTR recombination. In a related, ongoing study, we have determined that naive T cells collected from children with HIV can harbor integrated proviruses, sometimes in clonal populations, and that in one instance, the harbored provirus was sequence-intact. These findings change our understanding of both T cell biology and HIV persistence, since the proliferation of infected naive cells may lead to a self-renewing pool of infected central and effector memory T cells. A manuscript describing these findings is in preparation. Another ongoing, multifaceted effort toward genetically characterizing provirus populations utilizes samples from donors who have high levels of viremia on ART despite full suppression of viral. Notable findings produced in this ongoing, in-depth analysis of these samples include the observation that sequence-intact proviruses are disproportionately found integrated into the intronic regions of genes encoding the KRAB family of zinc-finger proteins, and that of the 10 largest infected T cell clones identified in these samples, 6 are solo LTRs - supporting analysis of samples obtained from children with HIV. To facilitate these efforts, we have recently devised and published a new combination provirus sequencing and integration site detection assay that permits more comprehensive provirus characterization that was previously possible (Joseph et al., J. Virol. e0012222, 2022).