Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.
PLoS Pathog. 2012;8(4):e1002615. doi: 10.1371/journal.ppat.1002615. Epub 2012 Apr 5.
Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an "escape clock" approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication.
HIV DNA 的持续存在是当前治疗方法完全控制 HIV 感染的主要障碍。大多数研究表明，在治疗下潜伏整合的 HIV 细胞的衰减非常缓慢。然而，在活跃感染期间研究 HIV DNA 的更替和持续存在要困难得多。我们开发了一种“逃逸时钟”方法来测量静止 CD4+T 细胞中 HIV DNA 的更替。该方法研究了在早期感染中存在的野生型（WT）SIV DNA 在随后感染中被 CTL 逃逸突变（EM）株取代的情况。我们使用一种特定于株的实时 PCR 检测法，定量了血浆 SIV RNA 和细胞 SIV DNA 中 WT 和 EM 株的相对量。因此，我们可以跟踪分选的静止 CD4+T 细胞中 SIV DNA 的形成和更替。我们研究了 20 只感染 SIV 的 Mane-A*10 阳性长尾猕猴的连续血浆和 PBMC 样本，这些猕猴具有特征性的 Gag CTL 逃逸突变。在病毒载量较低的动物中，早期感染中形成的 WT 病毒极其稳定，这种 WT 种的衰减非常缓慢，与接受抗逆转录病毒药物治疗的受试者的发现一致。然而，在活跃的高病毒载量感染期间，静止细胞中的大多数 SIV DNA 正在快速更替，这表明存在大量由近期感染事件产生的短寿命 DNA。我们的研究结果表明，为了减少 SIV DNA 稳定种群的形成，早期干预或在活跃复制期间进行干预非常重要。
