Effects of Antiretroviral Therapy on Transcriptional Activity of HIV Proviruses

背景：我们和其他人发现，在抗逆转录病毒治疗（ART）期间，HIV感染可以通过启动ART之前被感染的细胞增殖来维持。然而，尚不清楚这些克隆在抗逆转录病毒治疗期间表达HIV RNA的普遍程度。扩展克隆可能是持续性病毒血症的来源（Maldarelli et al., Science 345:179-183, 2014; Simonetti et al., PNAS 113:1883-1888, 2016）的研究结果表明，至少一些克隆群体成员在抗逆转录病毒治疗期间可以表达HIV未剪接RNA，因此，如果抗逆转录病毒治疗中断，可能会导致反弹病毒血症。我们假设，在接受抗逆转录病毒治疗的个体中持续存在的大多数感染细胞经历了克隆扩增，并且由表达HIV RNA的成员组成。为了验证这一假设，我们使用我们的单细胞HIV细胞相关RNA和DNA单基因组测序（CARD-SGS）方法检测了治疗和未治疗个体中单细胞中的HIV表达水平（Wiegand等人，PNAS 114:E3659-E3668, 2017）。在抗逆转录病毒治疗期间，我们确定了受感染细胞克隆中表达HIV未剪接RNA的HIV前病毒的比例(Musick等人，Front。微生物学报，2019,10:2204)。总共评估了34个携带完整或缺陷原病毒的不同克隆。我们发现克隆中约3%的细胞含有HIV未剪接RNA。在效应记忆t细胞亚群中发现了最高水平的HIV RNA。克隆中含有HIV RNA的细胞比例在完整原病毒（中位数2.3%）和缺陷原病毒（中位数3.5%）的克隆中没有差异（p=0.2）。然而，在含有多种耐药突变的前病毒细胞中发现了更高的RNA含量和水平，包括那些导致反弹病毒血症的前病毒。这些发现表明，扩增T细胞克隆中的绝大多数HIV-1前病毒，包括完整的前病毒，可能在任何给定时间都是转录沉默的，这意味着感染的T细胞可能能够被激活增殖而不诱导整合前病毒的表达，或者，可能能够在没有细胞激活的情况下增殖。____成就：我们之前报道了一种复制能力强的HIV变体，它是由高度扩增的细胞克隆产生的，并持续存在于接受ART治疗的HIV感染供体的血浆中（Simonetti等人，PNAS 113:1883-1888, 2016）。在2020年，我们将这一案例研究扩展到另外5名通过抗逆转录病毒疗法抑制病毒血症的供者，并证明了携带和表达具有复制能力的HIV的克隆在供者中很常见（Halvas等人，J. Clin）。中国农业科学，2013,(3):557 - 557。为了验证细胞可以在不诱导整合前病毒表达的情况下分化和增殖的假设，我们将收集自ART完全抑制病毒血症患者的pbmc分为静息（DR-）和激活（DR+）亚群。我们从亚群中分离出单个受感染的细胞，并测量含有HIV RNA的部分和每个单细胞中HIV RNA的水平。我们的数据显示，静息细胞和活化细胞中HIV RNA的比例和水平没有差异，这表明尽管增殖和活化，持续接受抗逆转录病毒治疗的感染细胞能够维持前病毒潜伏期（Groebner等人，Conference on Retroviruses and Opportunistic Infections, 2021）。这项研究的结果目前正在整理成一份手稿。_____因为我们之前的研究表明，只有一小部分持续抗逆转录病毒治疗的感染细胞具有转录活性的原病毒，我们假设5'非翻译区的HIV启动子可能含有DNA甲基化的CpG岛。这种甲基化可能通过抑制HIV转录因子的结合来阻止HIV病毒的表达。为了验证这一假设，我们分离了先前在体内被证明是转录沉默的单个HIV前病毒，用硫酸氢盐处理它们以脱胺化未甲基化的CpG岛，并量化了保留其活性的鸟嘌呤的数量（表明它们被甲基化）。虽然我们在HIV编码区内发现了甲基化的CpG岛，但我们很少在转录沉默的原病毒的HIV启动子中发现甲基化的鸟嘌呤。我们的研究结果表明，HIV启动子的甲基化不是HIV持续存在的机制。我们于2021年在《病毒》杂志上发表了这些发现（Boltz et al., Viruses 13:799）。2021)。由于没有发现HIV启动子的甲基化是抗逆转录病毒治疗供者HIV潜伏期的机制，我们研究了HIV环境基因中编码的反义基因（Ast）的表达（Sklutuis等）。逆转录病毒和机会性感染会议，2022年)。体外研究表明，HIV编码一个反义基因，该基因招募并保留了EZH2， EZH2是HIV- 15 ' ltr中多色素抑制复合物-2 （PRC2）的一个组成部分。EZH2催化组蛋白H3上赖氨酸27的三甲基化，组蛋白H3是一种抑制性表观遗传标记，可促进核小体组装并抑制HIV-1转录。这一机制表明Ast作为lncRNA促进HIV- 15 ' ltr的表观遗传沉默，以诱导和维持HIV感染细胞中的病毒潜伏期。为了解决这些发现，我们的实验室使用数字反转录PCR技术探索了从ART抑制病毒血症的供体分离的单个感染细胞中Ast的表达水平。我们发现在给定时间点，每100个pbmc中有26个HIV Ast拷贝。这一发现证实了在抗逆转录病毒疗法抑制病毒血症的供体中存在可检测到的Ast水平，并提示其作为一种调节RNA的潜在作用。我们的Ast研究结果正在准备发表。____在没有抗逆转录病毒治疗的情况下，艾滋病毒感染者的血浆病毒血症水平各不相同。决定个体血浆病毒血症水平的因素尚不完全清楚，但可能与原病毒表达、免疫控制和病毒适应性水平有关。超过90%的艾滋病患者在没有抗逆转录病毒治疗的情况下对HIV-1复制的自然控制有限（非控制者）；然而，一小部分PLWH（5%）自然控制HIV-1复制水平，这反映在较低水平的血浆病毒血症（控制者）上。然而，使用CARD-SGS分析（如上所述），我们的数据显示，与病毒血症控制者（通过测量单个感染细胞中未剪接的HIV-1 RNA水平来确定）相比，非控制者中具有转录活性前病毒的感染细胞比例并不高。相反，在病毒血症控制者中，受感染的细胞总数较少。这些发现表明，HIV复制的自然控制不是由于这些个体中潜伏感染细胞的比例较高，而是由于更有效的免疫反应（即NK细胞和/或CTL反应），它们有效地识别和杀死表达HIV RNA的感染细胞。这项工作在逆转录病毒和机会性感染会议（CROI）上进行了介绍，并提交了一份手稿，目前正在接受同行评审。我们未来的研究将描述HIV控制者和非控制者的免疫反应。

BACKGROUND: We and others discovered that HIV infection can be maintained during antiretroviral therapy (ART) by the proliferation of cells that were infected prior to initiating ART. However, it was not known how commonly such clones express HIV RNA during ART. The findings that expanded clones can be the source of persistent viremia (Maldarelli et al., Science 345:179-183, 2014; Simonetti et al., PNAS 113:1883-1888, 2016) indicated that at least some members of clonal populations can express HIV unspliced RNA during ART and may, therefore, result in rebound viremia if ART is interrupted. We hypothesized that the majority of infected cells that persist in individuals on ART have undergone clonal expansion and are composed of members that express HIV RNA. To test this hypothesis, we examined HIV expression levels in single cells in both treated and untreated individuals using our single-cell HIV cell-associated RNA and DNA single-genome sequencing (CARD-SGS) method (Wiegand et al., PNAS 114:E3659-E3668, 2017). We determined the fraction of HIV proviruses within infected cell clones that express HIV unspliced RNA during ART (Musick et al., Front. Microbiol. 10:2204, 2019). In total, 34 different clones carrying either intact or defective proviruses were assessed. We found that about 3% of cells within clones contained HIV unspliced RNA. Highest levels of HIV RNA were found in the effector memory T-cell subset. The fraction of cells within clones that contained HIV RNA was not different in clones with intact (median 2.3%) vs. defective (median 3.5%) proviruses (p=0.2). However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia. These findings show that the vast majority of HIV-1 proviruses within expanded T-cell clones, including intact proviruses, may be transcriptionally silent at any given time, implying that infected T cells may be able to be activated to proliferate without inducing the expression of the integrated provirus or, alternatively, may be able to proliferate without cellular activation. ____ACCOMPLISHMENTS: We previously reported a replication-competent HIV variant that is produced by a highly expanded cell clone and persists in the plasma of an HIV-infected donor treated with ART (Simonetti et al., PNAS 113:1883-1888, 2016). In 2020, we expanded this case study to 5 additional donors with their viremia suppressed on ART and demonstrated that clones carrying and expressing replication-competent HIV are common among donors (Halvas et al., J. Clin. Invest. 130:5847-5857, 2020). To follow up on the hypothesis that cells may differentiate and proliferate without inducing the expression of the integrated provirus, we sorted PBMCs collected from patients with viremia fully suppressed on ART into resting (DR-) and activated (DR+) subsets. We isolated single infected cells from the subsets and measured the fraction that have HIV RNA and the levels of HIV RNA in each of the single cells. Our data show that the fraction and levels of HIV RNA are not different in the resting and activated cells, indicating that infected cells that persist on ART are able to maintain proviral latency despite proliferation and activation (Groebner et al., Conference on Retroviruses and Opportunistic Infections, 2021). The results of this study are currently being assembled into a manuscript. _____Because our previous studies showed that only a small fraction of infected cells that persist on ART have proviruses that are transcriptionally active, we hypothesized that the HIV promotor in the 5' untranslated region may contain CpG islands that are DNA methylated. Such methylation may prevent the expression of the HIV provirus by inhibiting the binding of the HIV transcription factors. To test this hypothesis, we isolated single HIV proviruses that were previously shown to be transcriptionally silent in vivo, treated them with bisulfate to deaminate unmethylated CpG islands, and quantified the number of guanines that retained their animation (indicating that they were methylated). Although we found CpG islands within HIV coding regions that were methylated, we rarely identified methylated guanines within the HIV promotor of transcriptionally silent proviruses. Our findings show that methylation of the HIV promotor is not a mechanism for HIV persistence. We published these findings in Viruses in 2021 (Boltz et al., Viruses 13:799. 2021). ___Since methylation of the HIV promoter was not found to be a mechanism for HIV latency in donors on ART, we investigated the expression of an antisense gene (Ast) encoded within the HIV env gene (Sklutuis, et al. Conference on Retroviruses and Opportunistic Infections, 2022). In vitro studies showed that HIV encodes an antisense gene that recruits and retains EZH2, a component of the polychrome repressor complex-2 (PRC2) to the HIV-1 5'LTR. EZH2 catalyzes the trimethylation of lysine 27 on histone H3, a suppressive epigenetic mark that promotes nucleosome assembly and suppresses of HIV-1 transcription. This mechanism suggests that Ast acts as a lncRNA in promoting epigenetic silencing of the HIV-1 5'LTR to induce and maintain viral latency in HIV infected cells. To address these findings, our laboratory explored the expression levels of Ast in single infected cells isolated from donors with viremia suppressed on ART using digital reverse transcription PCR. We found a median of 26 copies of HIV Ast per 100 PBMCs at a given point in time. This finding confirmed the presence of detectable Ast levels in donors with their viremia suppressed on ART and suggests its potential role as a regulatory RNA. The results of our Ast study are being prepared for publication. ____ In the absence of ART, levels of plasma viremia vary in people living with HIV (PLWH). What determines levels of plasma viremia within individuals is not fully understood but is likely associated with levels of proviral expression, immune-control, and viral fitness. More than 90% of PLWH have limited natural control of HIV-1 replication without ART (non-controllers); however, a small fraction of PLWH (5%) naturally control levels of HIV-1 replication, which is reflected by lower levels of plasma viremia (controllers). However, using the CARD-SGS assay (described above), our data reveal that there is not a higher fraction of infected cells with transcriptionally active proviruses in non-controllers compared to viremic controllers (determined by measuring levels of unspliced HIV-1 RNA in single infected cells). Rather, there are fewer total infected cells in viremic controllers. These findings suggest that natural control of HIV replication is not due to a higher fraction of latently infected cells in these individuals but is due to more potent immune responses (i.e. NK cell and/or CTL responses) that efficiently recognize and kill infected cells that express HIV RNA. This work was presented at the Conference on Retroviruses and Opportunistic Infections (CROI) and a manuscript was submitted and is currently under peer review. Our future studies will characterize the immune responses in HIV controllers and non-controllers.