Department of Immunovirology and Pathogenesis, Level 5, Wallace Wurth Building, The Kirby Institute for Infection and Immunity, UNSW Sydney, Kensington, Sydney, NSW, 2052, Australia.
Retrovirology. 2018 Oct 4;15(1):67. doi: 10.1186/s12977-018-0451-0.
Current antiretroviral therapy is effective in controlling HIV-1 infection. However, cessation of therapy is associated with rapid return of viremia from the viral reservoir. Eradicating the HIV-1 reservoir has proven difficult with the limited success of latency reactivation strategies and reflects the complexity of HIV-1 latency. Consequently, there is a growing need for alternate strategies. Here we explore a "block and lock" approach for enforcing latency to render the provirus unable to restart transcription despite exposure to reactivation stimuli. Reactivation of transcription from latent HIV-1 proviruses can be epigenetically blocked using promoter-targeted shRNAs to prevent productive infection. We aimed to determine if independent and combined expression of shRNAs, PromA and 143, induce a repressive epigenetic profile that is sufficiently stable to protect latently infected cells from HIV-1 reactivation when treated with a range of latency reversing agents (LRAs).
J-Lat 9.2 cells, a model of HIV-1 latency, expressing shRNAs PromA, 143, PromA/143 or controls were treated with LRAs to evaluate protection from HIV-1 reactivation as determined by levels of GFP expression. Cells expressing shRNA PromA, 143, or both, showed robust resistance to viral reactivation by: TNF, SAHA, SAHA/TNF, Bryostatin/TNF, DZNep, and Chaetocin. Given the physiological importance of TNF, HIV-1 reactivation was induced by TNF (5 ng/mL) and ChIP assays were performed to detect changes in expression of epigenetic markers within chromatin in both sorted GFP and GFP cell populations, harboring latent or reactivated proviruses, respectively. Ordinary two-way ANOVA analysis used to identify interactions between shRNAs and chromatin marks associated with repressive or active chromatin in the integrated provirus revealed significant changes in the levels of H3K27me3, AGO1 and HDAC1 in the LTR, which correlated with the extent of reduced proviral reactivation. The cell line co-expressing shPromA and sh143 consistently showed the least reactivation and greatest enrichment of chromatin compaction indicators.
The active maintenance of epigenetic silencing by shRNAs acting on the HIV-1 LTR impedes HIV-1 reactivation from latency. Our "block and lock" approach constitutes a novel way of enforcing HIV-1 "super latency" through a closed chromatin architecture that renders the virus resistant to a range of latency reversing agents.
目前的抗逆转录病毒疗法在控制 HIV-1 感染方面非常有效。然而，一旦停止治疗，病毒库中的病毒载量就会迅速回升。由于潜伏激活策略的成功有限，以及 HIV-1 潜伏的复杂性，根除 HIV-1 病毒库一直具有挑战性。因此，人们越来越需要替代策略。在这里，我们探索了一种“阻断和锁定”的方法，用于强制潜伏，使前病毒无法在暴露于激活刺激时重新启动转录。使用靶向启动子的 shRNA 可以阻断潜伏 HIV-1 前病毒的转录激活，从而防止产生感染。我们的目的是确定独立和联合表达 shRNA、PromA 和 143 是否能诱导一种足够稳定的抑制性表观遗传特征，以保护潜伏感染的细胞免受各种潜伏逆转剂 (LRAs) 的 HIV-1 再激活。
J-Lat 9.2 细胞是 HIV-1 潜伏的模型，表达 shRNA PromA、143、PromA/143 或对照，用 LRAs 处理，以 GFP 表达水平评估对 HIV-1 再激活的保护。表达 shRNA PromA、143 或两者的细胞对 TNF、SAHA、SAHA/TNF、Bryostatin/TNF、DZNep 和 Chaetocin 的病毒再激活表现出强大的抗性。鉴于 TNF 的生理重要性，用 TNF（5ng/ml）诱导 HIV-1 再激活，并在分别含有潜伏或激活前病毒的分选 GFP 和 GFP 细胞群中进行 ChIP 分析，以检测染色质中表观遗传标记的表达变化。普通的双因素方差分析用于识别 shRNA 与与整合前病毒中抑制性或活性染色质相关的染色质标记之间的相互作用，结果显示在 LTR 中，H3K27me3、AGO1 和 HDAC1 的水平发生了显著变化，这与前病毒再激活的程度降低相关。共表达 shPromA 和 sh143 的细胞系表现出最低的再激活和最丰富的染色质紧缩指标。
shRNA 对 HIV-1 LTR 的作用主动维持表观遗传沉默，阻止 HIV-1 从潜伏中重新激活。我们的“阻断和锁定”方法通过封闭的染色质结构构成了一种强制 HIV-1“超级潜伏”的新方法，使病毒能够抵抗一系列潜伏逆转剂。
