Université Paris Cité, Institut Cochin, INSERM U1016, CNRS, UMR8104, 75014 Paris, France.
Université Paris Cité, Paris, France.
Cell Rep Methods. 2023 Apr 5;3(4):100443. doi: 10.1016/j.crmeth.2023.100443. eCollection 2023 Apr 24.
Unintegrated HIV DNA represents between 20% and 35% of the total viral DNA in infected patients. Only the linear forms (unintegrated linear DNAs [ULDs]) can be substrates for integration and for the completion of a full viral cycle. In quiescent cells, these ULDs may be responsible for pre-integrative latency. However, their detection remains difficult due to the lack of specificity and sensitivity of existing techniques. We developed an ultra-sensitive, specific, and high-throughput technology for ULD quantification called DUSQ (DNA ultra-sensitive quantification) combining linker-mediated PCR and next-generation sequencing (NGS) using molecular barcodes. Studying cells with different activity levels, we determined that the ULD half-life goes up to 11 days in resting CD4 T cells. Finally, we were able to quantify ULDs in samples from patients infected with HIV-1, providing a proof of concept for the use of DUSQ to track pre-integrative latency. DUSQ can be adapted to the detection of other rare DNA molecules.
未整合的 HIV 病毒 DNA 占感染患者体内总病毒 DNA 的 20%至 35%。只有线性形式（未整合的线性 DNA[ULDs]）可以作为整合和完成完整病毒周期的底物。在静止细胞中，这些 ULDs 可能是导致预整合潜伏的原因。然而，由于现有技术缺乏特异性和灵敏度，它们的检测仍然很困难。我们开发了一种超灵敏、特异和高通量的 ULD 定量技术，称为 DUSQ（DNA 超灵敏定量），该技术结合了连接介导的 PCR 和使用分子条码的下一代测序（NGS）。通过研究具有不同活性水平的细胞，我们确定 ULD 的半衰期在静止的 CD4 T 细胞中最长可达 11 天。最后，我们能够定量检测 HIV-1 感染患者样本中的 ULDs，为使用 DUSQ 跟踪预整合潜伏提供了概念验证。DUSQ 可以适应于其他稀有 DNA 分子的检测。
