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。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）。在一些病例中，扩增的原病毒与抗逆转录病毒治疗期间持续病毒血症中可检测水平的病毒变异相匹配；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; Halvas et al.， J. clinin .）。中国农业科学，2013,(3):557 - 557。在这些例子中，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 et al., Nat. Med. 22:1043-1049, 2016）。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多位移扩增单基因组测序（MDA-SGS）测定的新方法，该方法使我们能够分析高度扩增克隆中的原病毒（Patro等人，PNAS 116:25891-25899, 2019）。MDA-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前病毒是否具有完整的序列，还是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：我们使用MDA-SGS方法证明，具有复制能力的原病毒感染的细胞克隆在抗逆转录病毒治疗患者中很常见，并且是治疗期间持续低水平病毒血症的来源（Halvas等人，J. clint）。中国农业科学，2013,(3):557 - 557。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病毒库并无不同。____为了进一步研究儿童中的HIV病毒库，我们进行了研究，以确定是否在出生时感染HIV病毒的儿童中存在扩增的受感染细胞克隆，以及这些细胞克隆是否在抗逆转录病毒治疗中持续存在（Bale， Katusiime等人，mBio 12:e00568- 21,2021）。我们发现，感染的细胞克隆出现在儿童早期（2个月大），并在治疗期间持续至少9年。这些发现表明，儿童体内的艾滋病毒库是通过治疗前感染细胞的克隆扩增来维持的。____我们建立了一个新的公共数据库来存储和注释ART供者体内持续存在的HIV前病毒序列（Shao等人，AIDS Res. Hum）。逆转录病毒36:1-3,2020)。该数据库（PSD; https://psd.cancer.gov）可用于对抗逆转录病毒治疗中持续存在的原病毒进行荟萃分析，以更好地了解艾滋病毒库并为治疗策略提供信息。____我们开发了一种新的生物信息学管道来注释和检测抗逆转录病毒治疗期间持续存在的完整HIV原病毒（Wright等人，Retrovirology 18:16, 2021）。我们的管道是独一无二的，因为它可以整合到现有的管道中，从下一代测序实验中提取和校准HIV序列。____与Frank Maldarelli博士（HIV动力学和复制计划）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会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. 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 several cases, expanded proviruses were shown to match viral variants present at detectable levels in 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; Halvas et al., J. Clin. Invest. 130:5847-5857, 2020). These are examples where the source of infectious virus in blood has been traced to clones 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., Nat. Med. 22:1043-1049, 2016). 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 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 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 MDA-SGS method to demonstrate that infected cell clones with replication-competent proviruses are common in patients on ART and a source of persistent, low-level viremia during treatment (Halvas et al., J. Clin. Invest. 130:5847-5857, 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. ____To further investigate the HIV reservoir in children, we conducted studies to determine if expanded, infected cell clones are present in children born with HIV infection and if these cell clones persist on ART (Bale, Katusiime et al., mBio 12:e00568-21, 2021). We found that infected cell clones arise early in children (2 months old) and persist for at least 9 years during treatment. These findings demonstrate that the HIV reservoir in children is maintained by clonal expansion of cells that were infected prior to initiating treatment. ____We established a new public database to store and annotate HIV proviral sequences that persist in donors on ART (Shao et al., AIDS Res. Hum. Retroviruses 36:1-3, 2020). This database (PSD; https://psd.cancer.gov) is available for meta-analyses of proviruses that persist on ART to better understand the HIV reservoir and to inform strategies towards a cure. ____We developed a new bioinformatic pipeline to annotate and detect intact HIV proviruses that persist during ART (Wright et al., Retrovirology 18:16, 2021). Our pipeline is unique because it can be incorporated into existing pipelines to extract and align HIV sequence reads from next-generation sequencing experiments. ____In collaboration with Dr. Frank Maldarelli (HIV Dynamics and Replication Program), 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.