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。____取得的成就：我们使用FLIP-SGS方法证明带有完整原病毒的感染细胞克隆在接受抗逆转录病毒治疗的患者中很常见（Halvas等，J. clint）。投资。，出版，2020年)。这项研究表明了研究潜在的治疗策略对感染细胞克隆扩增的影响的重要性。这项研究还证实，HIV的储存库是在开始抗逆转录病毒治疗之前感染的T细胞的增殖。____为了确定携带完整HIV原病毒的细胞克隆是否在抗逆转录病毒治疗抑制的儿童中持续存在，就像在成人中一样，我们分析了出生后不久开始抗逆转录病毒治疗的HIV-1感染儿童的纵向样本中的HIV种群（Katusiime等，J. Virol. 94: 01519- 190,2020）。我们在两个孩子身上发现了序列相同的完整的原病毒，这表明这种克隆可能在出生时携带艾滋病毒的孩子身上持续存在。这些数据表明，儿童体内的HIV病毒库与成人体内的HIV病毒库并没有什么不同，尽管还需要对这些位点整合到宿主基因组中的研究来证明这是真的。我们已经开始了这些研究。____为了研究HIV-1在抗逆转录病毒治疗期间在淋巴结和其他组织中复制维持HIV-1储存库的可能性，我们寻找了长达15年病毒抑制后供体中病毒复制的证据（McManus et al.， J. clinin）。投资管理学报，2019,vol . 29- vol . 42, ei。广告5:eaav2045, 2019)。我们分析了抗逆转录病毒治疗前和治疗期间组织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，并鉴定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 116: 25891-25899, 2019）。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。____与Frank Maldarelli博士合作，我们使用qPCR检测检测可能完整的前病毒和被删除的前病毒，研究了持续存在于ART上的前病毒的遗传结构。我们发现，在接受抗逆转录病毒治疗的患者中，潜在完整的原病毒数量随着时间的推移而减少（Anderson等人，J. Virol, 12: 136, 2020）。这些结果表明，尽管被完整的原病毒感染的细胞增殖，但储存库可能随着时间的推移而缩小。

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 used our FLIP-SGS method to demonstrate that infected cell clones with intact proviruses are common in patients on ART (Halvas et al., J. Clin. Invest., in press, 2020). This study demonstrates the importance of investigating potential curative strategies for their effect on clonal expansion of infected cells. This study also confirms that the reservoir for HIV is the proliferation of T cells infected prior to the initiation of ART. ____To determine if cell clones carrying intact HIV proviruses persist in children with viremia suppressed on ART, as in adults, we analyzed HIV populations in longitudinal samples from HIV-1-infected children who initiated ART shortly after birth (Katusiime et al., J. Virol. 94: e01519-19, 2020). We found sequence-identical, intact proviruses in two of the children, demonstrating that such clones likely persist in children born with HIV. These data suggest that the HIV reservoir in children is not different from the HIV reservoir in adults, although studies investigating that the sites are integrated in the host genome are needed to demonstrate it to be true. We have initiated these studies. ____To investigate the possibility that HIV-1 replication in lymph nodes and other tissues sustains the reservoir during ART, we looked for evidence of viral replication in donors after up to about 15 years of viral suppression (McManus et al., J. Clin. Invest. 129:4629-4642, 2019, Bozzi et al., Sci. Adv. 5: eaav2045, 2019). We characterized proviral populations in tissues 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 116: 25891-25899, 2019). 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. ____In collaboration with Dr. Frank Maldarelli, we investigated the genetic structure of proviruses that persist on ART using a qPCR assay that detected potentially intact proviruses and those that are deleted. We found the number of potentially intact proviruses to decrease over time in patients on ART (Anderson et al., J. Virol, 12: 136, 2020). These results suggest that the reservoir may shrink over time despite the proliferation of cells infected with intact proviruses.