Dynamics and Genetics of HIV Proviruses before and during Antiretroviral Therapy

背景：尽管抗逆转录病毒治疗（ART）能够抑制感染患者的HIV复制，防止疾病进展，但如果ART中断，病毒会持续存在于一个稳定的库中，并反弹到治疗前的水平。终身治疗是昂贵的，并且有出现耐药性和毒副作用的风险。因此，确定消除储存库的策略是艾滋病毒研究的主要优先事项。最近，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）。在一个病例中，经扩增的原病毒与抗逆转录病毒治疗期间持续性病毒血症中可检测到的单一病毒变体相匹配；此外，我们发现克隆扩增细胞产生的病毒颗粒具有复制能力（Simonetti et al., PNAS 113:1883-1888, 2016）。这是一个例子，血液中传染性病毒的来源已被追踪到携带潜伏性原病毒的受感染细胞的克隆。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。____虽然患者之间存在相当大的差异，但通过PCR检测到的携带HIV前病毒的静息CD4+ T细胞的频率已经非常粗略地估计为平均约1000个细胞中有1个细胞；然而，据报道，携带复制能力原病毒的潜伏感染细胞数量要低得多（Ho et al., Cell 155:540-551, 2013）。这种差异是由于存在大量有缺陷的原病毒。Ya-Chi Ho及其同事描述了静息CD4+ T细胞中的前病毒，这些前病毒在单轮最大T细胞激活后不会被诱导产生复制能力强的病毒（Ho et al., Cell 155:540-551, 2013）。这些原病毒中几乎有一半具有阻止复制的大的内部缺失，而另外三分之一被宿主限制因子APOBEC3G致死性超突变。其他缺陷和进一步的分析使缺陷原病毒的比例高达98% （Bruner等人，在2016年逆转录病毒和机会性感染会议上的报告）。此外，Ho等人发现，一些完整的原病毒在第二轮激活后能够产生感染性病毒粒子（Cell 155:540-551, 2013），即使它们没有被先前的激活诱导。这一结果引发了Maldarelli等人和Wagner等人所描述的扩展克隆携带完整且具有复制能力的原病毒的普遍程度的问题。为了回答这个问题，我们开发了一种称为全长整合原病毒单基因组测序（FLIP-SGS）的新方法，该方法使我们能够分析高度扩展的克隆中的原病毒，如dr。Frank Maldarelli和Stephen Hughes （HIV动力学和复制项目）使用整合位点测定（ISA）。FLIP-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原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：我们发现（Kearney et al., PLoS Pathog. 10:e1004010, 2014），抗逆转录病毒治疗前最初多样化的血浆病毒种群随着时间的推移变得更加均匀，在许多患者中出现相同序列的突变，正如其他人最初报道的那样（Bailey et al.， J. Virol. 80:6441-6457, 2006）。这一观察结果表明，在抗逆转录病毒治疗期间，受感染细胞的克隆扩增和死亡之间存在动态平衡。目前尚不清楚扩增的克隆如何普遍携带具有复制能力的前病毒，或者它们是否携带具有CTL逃逸突变的HIV前病毒，这可能使它们具有生存优势。然而，到目前为止，我们已经发现了几个这样的病例（Simonetti et al., PNAS 113:1883-1888, 2016; Bui et al., PLoS Pathog. 13:e1006230, 2016; Patro et al.， PNAS，修订后接受）。我们目前的研究旨在确定扩增克隆是否在所有感染个体中维持HIV-1储库。____为了确定持续性病毒血症的可能来源并研究抗逆转录病毒治疗对病毒复制的影响，我们分析了10名HIV-1感染儿童的纵向样本中的HIV群体，这些儿童在病毒多样性较低时开始抗逆转录病毒治疗（van Zyl等，J. Clin）。投资管理。127:3827-3834,2017)。8名儿童在10个月大时开始抗逆转录病毒治疗，血浆病毒血症被抑制了7-9年。在抑制病毒血症前，两名儿童的病毒血症控制分别为15个月和30个月。后两名儿童显示出病毒进化的明显证据，而通过多种分析方法，抗逆转录病毒药物抑制的8名儿童中没有一名显示出病毒进化。这表明，在抗逆转录病毒治疗中，血液中持续的HIV复制并不常见，因此，它不是治愈HIV-1感染的重大障碍。____为了研究抗逆转录病毒治疗期间淋巴结中HIV-1复制维持HIV-1储存库的可能性，我们在5个供体中寻找经过长达13年病毒抑制后病毒复制的证据（McManus et al.， J. clinin）。投资。打印前的Epub格式)。我们分析了抗逆转录病毒治疗前和治疗期间淋巴结和外周血中的前病毒群体，评估了单个淋巴结和血细胞中的病毒RNA表达水平，并分析了配对淋巴结和血液样本中的前病毒整合位点。在抗逆转录病毒治疗期间收集的淋巴结和血液样本中发现了具有相同序列、相同整合位点和相似RNA表达水平的原病毒，并且在血液或淋巴结中没有检测到与治疗前人群有显著差异的单一序列。这些发现表明，所有可检测到的持续性HIV-1感染都与抗逆转录病毒治疗前感染的细胞克隆增殖在淋巴结中的维持一致，而不是在抗逆转录病毒治疗期间持续的病毒复制。____我们将FLIP-SGS方法应用于五个接受art治疗的供者的淋巴结和PBMCs，以确定两个区室中相同的亚基因组序列组是受感染细胞克隆扩增的结果还是病毒的遗传瓶颈（Patro等人，PNAS，经修订后接受）。我们发现，在抗逆转录病毒治疗开始前和治疗失败后，细胞扩增和病毒遗传瓶颈都可能导致相同的原病毒序列。我们鉴定了一个携带完整前病毒的扩增t细胞克隆，该病毒与先前通过病毒生长试验检测到的变异相匹配，并鉴定了具有野生型和耐药缺陷前病毒的扩增克隆。我们还发现来自同一供体的两个克隆携带相同的原病毒，除了非重叠缺失，我们可以从中推断完整的亲本原病毒的序列。因此，FLIP-SGS可用于“病毒古生物学”，以更好地了解患者体内HIV-1的进化，并确定扩增克隆（包括具有耐药突变的克隆）内原病毒的克隆性和结构。重要的是，我们证明了标准SGS观察到的相同序列并不总是足以建立原克隆。

BACKGROUND: Despite the ability of antiretroviral therapy (ART) to inhibit HIV replication in infected patients, preventing disease progression, the virus persists in a stable reservoir and rebounds to pretherapy levels if ART is interrupted. Lifelong therapy is expensive and risks the emergence of drug resistance and toxic side effects. Consequently, identification of strategies to eliminate the reservoir is a major priority of HIV research. Recently, 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). In one case, an expanded provirus was shown to match the single viral variant present at detectable levels in the persistent viremia during ART; furthermore, we showed that the virus particles produced by the clonally expanded cells were replication competent (Simonetti et al., PNAS 113:1883-1888, 2016). This is one example where the source of infectious virus in blood has been traced to a clone of infected cells carrying a mostly latent provirus. 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. ____Although there is considerable patient-to-patient variation, the frequency of resting CD4+ T cells that harbor HIV proviruses detectable by PCR has been very roughly estimated to average about 1 cell in 1000; however, the number of latently infected cells carrying replication-competent proviruses has been reported to be much lower (Ho et al., Cell 155:540-551, 2013). The difference is due to the presence of a large number of defective proviruses. Ya-Chi Ho and colleagues described the proviruses in resting CD4+ T cells that were not induced to produce replication-competent virus after a single round of maximal T-cell activation (Ho et al., Cell 155:540-551, 2013). Almost half of these proviruses had large internal deletions that preclude replication, while another third were lethally hypermutated by the host restriction factor APOBEC3G. Other defects and further analyses brought the fraction of defective proviruses up to 98% (Bruner et al., presentation at 2016 Conference on Retroviruses and Opportunistic Infections). Additionally, Ho et al. found that some of the intact proviruses were capable of producing infectious virions following a second round of activation (Cell 155:540-551, 2013), even though they had not been induced by the prior activation. This result leads to the question of how commonly the expanding clones described by Maldarelli et al. and Wagner et al. carry intact and replication-competent proviruses. To answer this question, we developed a novel method called the full-length integrated proviral single-genome sequencing (FLIP-SGS) assay, which allows us to analyze the proviruses in highly expanded clones like those identified by Drs. Frank Maldarelli and Stephen Hughes (HIV Dynamics and Replication Program) using the integration sites assay (ISA). The FLIP-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: We found (Kearney et al., PLoS Pathog. 10:e1004010, 2014) that the initially diverse pre-ART plasma virus population becomes more homogeneous with time, with the appearance, in many patients, of rakes of identical sequence, as originally reported by others (Bailey et al., J. Virol. 80:6441-6457, 2006). This observation suggests that there is a dynamic balance between the clonal expansion and death of infected cells during ART. It is not yet known how commonly expanded clones carry replication-competent proviruses or if they carry HIV proviruses with CTL escape mutations that may give them a survival advantage. However, to date we have identified several such cases (Simonetti et al., PNAS 113:1883-1888, 2016; Bui et al., PLoS Pathog. 13:e1006230, 2016; Patro et al., PNAS, accepted upon revision). Our current studies aim to determine if expanded clones sustain the HIV-1 reservoir in all infected individuals. ____To identify the possible sources of persistent viremia and to investigate the effect of ART on viral replication, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low (van Zyl et al., J. Clin. Invest. 127:3827-3834, 2017). Eight children started ART at 10 months of age and had plasma viremia suppressed for 7-9 years. Two children had uncontrolled viremia for 15 and 30 months, respectively, before viremia suppression. The latter two children showed clear evidence of virus evolution, whereas none of the eight children suppressed on ART showed virus evolution by multiple methods of analysis. These suggest that ongoing HIV replication in the blood is not common on ART and is, therefore, not a significant barrier to curing HIV-1 infection. ____To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in five donors after up to 13 years of viral suppression (McManus et al., J. Clin. Invest. epub before print). We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was detected in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART. ____We applied the FLIP-SGS method to lymph node and PBMCs from five ART-treated donors to determine if groups of identical sub-genomic sequences in the two compartments are the result of clonal expansion of infected cells or a genetic bottleneck on the virus (Patro et al., PNAS, accepted upon revision). We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T-cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found two clones from one donor that carried identical proviruses except for nonoverlapping deletions from which we could infer the sequence of the intact parental provirus. Thus, FLIP-SGS can be used for "viral paleontology" to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrated that identical sequences observed by standard SGS are not always sufficient to established proviral clonality.