Effects of Antiretroviral Therapy on Transcriptional Activity of HIV Proviruses

背景：我们和其他人最近发现，在抗逆转录病毒治疗（ART）期间，HIV感染可以通过启动ART之前可能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，特别是那些携带复制能力原病毒的克隆。这一发现可能表明，要治愈这种感染，必须根除一个重要的“活跃”HIV病毒库。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，至少一些克隆群体的成员在抗逆转录病毒治疗期间可以表达未剪接的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都经历了克隆扩增，并且由表达HIV RNA的成员组成。值得注意的是，HIV RNA的表达并不表明病毒正在进行复制，因为这些转录物中的大多数可能来自不能复制的原病毒。这一假设导致抗逆转录病毒治疗期间艾滋病毒持续存在的模型，由一小部分携带完整原病毒的扩增克隆细胞群组成，这些克隆群体中的一小部分成员表达艾滋病毒RNA，导致抗逆转录病毒治疗期间持续病毒血症中存在复制能力强的病毒颗粒的产生，并导致抗逆转录病毒治疗中断时迅速反弹。为了验证这一假设，我们已经开始在接受治疗和未接受治疗的个体中检测单个细胞中的HIV表达水平。目前尚不清楚在特定时间点有多少比例的受感染血细胞表达HIV RNA， HIV在单个细胞中的表达水平是多少，以及表达水平和谱是否随着抗逆转录病毒治疗而改变或保持不变。研究在抗逆转录病毒治疗之前和治疗期间表达HIV RNA的受感染细胞的比例及其在单细胞中的表达水平，将有助于更好地了解抗逆转录病毒治疗期间艾滋病毒的持续存在以及停止抗逆转录病毒治疗后艾滋病毒的反弹。为了解决这些重要问题，我们开发了单细胞HIV细胞相关RNA和DNA单基因组测序（CARD-SGS）方法（Wiegand等人，PNAS 114:E3659-E3668, 2017），该方法可用于确定(i)表达HIV RNA的HIV感染细胞总数的比例，（ii）单个细胞中HIV RNA的表达水平，（iii）扩增克隆中HIV RNA的表达水平，以及（iv）这些RNA与前病毒DNA的遗传关系。该试验采用改进的从总pbmc中分离细胞内RNA的方法，该方法由Hong等人（J. clinin）改进。hiv表达细胞的终点稀释。微生物学报。54:902-911,2016)。我们将使用CARD-SGS来研究抗逆转录病毒治疗过程中HIV表达谱的变化，以确定持续和反弹病毒血症的细胞来源，并研究构成HIV库重要组成部分的扩增前病毒的表达水平。____成就：我们之前报道了一种复制能力强的HIV-1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）。即使不是所有接受治疗的患者，也可能在某种程度上存在这种克隆。此外，我们还发现表达的群体包含相同序列的簇。相同细胞内RNA序列的群体表明，携带相同前病毒的多个细胞表达病毒RNA，或者单个细胞（或其后代）与其他感染细胞相比，正在经历更高水平的病毒表达。发现RNA变异都有缺陷，这意味着它们的表达要么来自增殖细胞群，要么来自单个细胞，而不是通过病毒复制的局部传播。我们与博士合作进行的研究结果。Jonathan Li（哈佛大学）和Brandon Keele （Leidos Biomedical Research, Inc.艾滋病和癌症病毒项目）认为，抗逆转录病毒治疗期间表达HIV RNA的扩增克隆可能是停止抗逆转录病毒治疗后反弹病毒血症的来源（Kearney et al.， J. Virol. 90:1369-1376, 2016）。我们获得了10名感染至少2年并接受分析性治疗中断（ATI）的ACTG研究参与者的单基因组序列。我们将所有10名参与者的反弹变异体与art前血浆中的变异体进行了比较，并将7/10名参与者的pbmc相关DNA和RNA与art前血浆中的变异体进行了比较。在这7名参与者中，我们在ART抑制期间检测到多个相同的DNA和HIV RNA序列与反弹的血浆HIV序列完全匹配，这表明克隆扩增的感染细胞可能是反弹病毒血症的来源，因此是HIV的储存库。此外，我们的研究表明，尽管一些表达细胞可能导致反弹病毒血症，但大多数细胞没有。这一发现以及对HIV RNA序列超突变的检测表明，缺陷HIV前病毒在抗逆转录病毒治疗期间得到表达，并可能解释之前关于HIV细胞RNA和血浆RNA水平之间弱关联的报道（Li等人，AIDS 30:343-353, 2016），因为大多数缺陷HIV前病毒不会产生病毒粒子。这项研究的结果有助于我们了解抗逆转录病毒治疗期间的HIV病毒库，并提出HIV病毒库包括增殖细胞群的假设，其中一些细胞在抗逆转录病毒治疗期间已经表达了HIV RNA。____我们目前的研究包括确定特定扩增克隆中表达HIV RNA的细胞比例，以及鉴定支持前病毒表达的细胞亚群（即中枢、过渡或效应记忆细胞）（Musick等人，Frontiers Microbiol）。，修改后接受)。为了解决这个问题，我们确定了在抗逆转录病毒治疗期间表达未剪接HIV-1细胞相关RNA的受感染细胞克隆中HIV-1前病毒的比例。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%的细胞含有未剪接的HIV-1 RNA。在效应记忆t细胞亚群中发现了最高水平的HIV-1 RNA。克隆中含有HIV-1 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。这项研究的结果表明，长期以来假设的细胞和前病毒激活水平之间的相关性可能并不准确，因此需要进一步的研究。

BACKGROUND: We and others recently discovered that HIV infection can be maintained during antiretroviral therapy (ART) by proliferation of cells that were likely infected prior to initiating ART. However, it is not known how commonly such clones express HIV RNA during ART, particularly those carrying replication-competent proviruses. Such a finding could reveal that there is an important "active" reservoir for HIV that must be eradicated to cure the infection. 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) indicate that at least some members of clonal populations can express unspliced RNA during ART and may, therefore, result in rebound viremia if ART is interrupted. We hypothesize 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. Of note, the expression of HIV RNA is not an indication of ongoing viral replication, as the majority of these transcripts likely result from proviruses that are replication incompetent. This hypothesis leads to a model of HIV persistence during ART as consisting of a small minority of expanded clonal populations of cells that carry intact proviruses, with a small fraction of the members of these clonal populations expressing HIV RNA, leading to the production of replication-competent virus particles present in persistent viremia during ART and leading to rapid rebound when ART is interrupted. To test this hypothesis, we have begun to examine HIV expression levels in single cells in both treated and untreated individuals. It is not known what fraction of infected blood cells express HIV RNA at a given point in time, to what levels HIV is expressed in single cells, and if expression levels and profiles change with ART or remain the same. Investigating the fraction of infected cells that express HIV RNA and their levels of expression in single cells both prior to and during ART will provide a better understanding of the HIV persistence during ART and its rebound after stopping ART. To address these important questions, we developed the single-cell HIV cell-associated RNA and DNA single-genome sequencing (CARD-SGS) method (Wiegand et al., PNAS 114:E3659-E3668, 2017), which can be used to determine (i) the fraction of total HIV-infected cells that express HIV RNA, (ii) the levels of HIV RNA expression in single cells, (iii) the levels of HIV RNA expression in expanded clones, and (iv) the genetic relationship of these RNAs to proviral DNA. This assay uses improved isolation of intracellular RNA from total PBMCs modified from Hong et al. (J. Clin. Microbiol. 54:902-911, 2016) and endpoint dilution of HIV-expressing cells. We will use CARD-SGS to investigate changes in the HIV expression profiles over the course of treatment with ART, to identify cellular sources of persistent and rebound viremia, and to investigate the expression levels in expanded proviruses that constitute an important part of the reservoir for HIV. ____ACCOMPLISHMENTS: We previously reported a replication-competent HIV-1 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). Such clones are likely to be present at some level in most if not all treated patients. In addition, we also found that the expressed populations contained clusters of identical sequences. Populations of identical intracellular RNA sequences suggest either that multiple cells carrying identical proviruses were expressing viral RNA or that single cells (or their descendants) were undergoing higher levels of viral expression in comparison to other infected cells. The finding that the RNA variants were all defective implies that their expression is either from a proliferating cell population or from a single cell, but not from local spread through viral replication. Results of our study conducted in collaboration with Drs. Jonathan Li (Harvard University) and Brandon Keele (AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc.) suggested that expanded clones expressing HIV RNA during ART may be a source for rebound viremia after stopping ART (Kearney et al., J. Virol. 90:1369-1376, 2016). We obtained single-genome sequences from 10 ACTG study participants infected for at least 2 years who underwent analytic treatment interruption (ATI). We compared the rebounding variants to those in pre-ART plasma in all 10 participants and with on-ART PBMC-associated DNA and RNA in 7/10 participants. In 3 of these 7participants, we detected multiple, identical DNA and HIV RNA sequences during suppression on ART that exactly matched rebounding plasma HIV sequences, suggesting that clonally expanded, infected cells may be a source of rebound viremia, and hence, a reservoir for HIV. Furthermore, our study showed that, although some expressing cells may have resulted in rebound viremia, most did not. This finding, along with the detection of hypermutated HIV RNA sequences, implies that defective HIV proviruses are expressed during ART and may explain previous reports on the weak association between levels of HIV cellular RNA and plasma RNA (Li et al., AIDS 30:343-353, 2016), as most defective HIV proviruses would not produce virions. The results of this study contribute to our understanding of the reservoir for HIV during ART and lead to the hypothesis that the HIV reservoir includes populations of proliferating cells, some of which are already expressing HIV RNA during ART. ____Our current studies include determining the fraction of cells within specific expanded clones that express HIV RNA and identifying the cell subsets that support proviral expression (i.e. central, transitional, or effector memory cells) (Musick et al., Frontiers Microbiol., accepted upon revision). To address this question, we determined the fraction of HIV-1 proviruses within infected cell clones that express unspliced HIV-1 cell-associated RNA during ART. In total, 34 different clones carrying either intact or defective proviruses were assessed. We found that about 3% of cells within clones contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory T-cell subset. The fraction of cells within clones that contained HIV-1 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. The results of this study suggest that the long-presumed correlation between the level of cellular and proviral activation may not be accurate and, therefore, requires further investigation.