Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir, but limited data exist on its use in vitro Moreover, most NGS studies do not separate integrated from unintegrated DNA, even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare the infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions, we obtained HIV sequences 2, 4, and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/ml interleukin-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection, the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo Massively deleted viral sequences formed more frequently in resting cells, likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures.IMPORTANCE The major implication of our work is that the decay of intact proviruses in vitro is extremely rapid, perhaps as a result of enhanced expression. Gaining a better understanding of why intact proviruses decay faster in vitro might help the field identify strategies to purge the reservoir in vivo When used wisely, in vitro models are a powerful tool to study the selective pressures shaping the viral landscape. Our finding that massively deleted sequences rarely succeed in integrating has several ramifications. It demonstrates that the total HIV DNA can differ substantially in character from the integrated HIV DNA under certain circumstances. The presence of unintegrated HIV DNA has the potential to obscure selection pressures and confound the interpretation of clinical studies, especially in the case of trials involving treatment interruptions.

中文翻译：

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HIV 感染直接模型中的下一代测序揭示了与体内水库动力学的重要相似之处和不同之处。

下一代测序 (NGS) 是揭示 HIV 宿主基因组成的强大工具，但其在体外使用的数据有限这两种形式应该是不同的。我们推断我们可以使用 NGS 在体外比较静息和激活的 CD4 T 细胞的感染，以解决代谢状态如何影响水库形成和动态。为了解决这些问题，我们在 NL4-3 感染代谢激活和静止的 CD4 T 细胞（用 2 ng/ml 白细胞介素 7 培养）后 2、4 和 8 天获得了 HIV 序列。我们通过在进行测序之前使用脉冲场电泳分离整合的 HIV DNA 来比较整合和总 HIV DNA 的组成。单轮感染后，大多数整合的 HIV DNA 在静止和活化的 T 细胞中都是完整的。在静止和活化的 T 细胞中，整合的完整原病毒的衰变是快速和相似的。缺陷形式相对于完整形式积累，类似于在体内观察到的情况 大量缺失的病毒序列在静息细胞中更频繁地形成，可能是由于脱氧核苷三磷酸 (dNTP) 水平较低和存在多种限制因子。令我们惊讶的是，这些删除的序列中的大多数都没有整合到人类基因组中。使用 NGS 在体外研究储层动力学提供了一个模型，该模型概括了储层动力学的重要方面。此外，在某些临床环境中，将整合的 HIV DNA 与未整合的 HIV DNA 分开对于正确研究选择压力非常重要。重要性我们工作的主要含义是完整的前病毒在体外的衰变非常迅速，可能是由于表达增强。更好地理解为什么完整的前病毒在体外衰减得更快，可能有助于该领域确定清除体内储存库的策略。如果使用得当，体外模型是研究塑造病毒景观的选择压力的有力工具。我们发现大量删除的序列很少成功整合有几个后果。它表明，在某些情况下，总 HIV DNA 在特性上可能与整合的 HIV DNA 有很大不同。未整合的 HIV DNA 的存在有可能掩盖选择压力并混淆临床研究的解释，