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Effect of transcription inhibition and generation of suppressive viral non-coding RNAs
Retrovirology ( IF 2.7 ) Pub Date : 2019-04-29 , DOI: 10.1186/s12977-019-0475-0
Daniel O Pinto 1 , Tristan A Scott 2 , Catherine DeMarino 1 , Michelle L Pleet 1 , Thy T Vo 1 , Mohammed Saifuddin 1 , Dmytro Kovalskyy 3 , James Erickson 1 , Maria Cowen 1 , Robert A Barclay 1 , Chen Zeng 4 , Marc S Weinberg 5, 6 , Fatah Kashanchi 1, 7
Affiliation  

BackgroundHIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo.ResultsHere, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state.ConclusionsCollectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of “RNA Machines” that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.

中文翻译:

转录抑制的影响和抑制性病毒非编码 RNA 的产生

背景接受联合抗逆转录病毒疗法 (cART) 的 HIV-1 患者在感染后存活下来,但需要以高昂的费用终生坚持。在无法检测到病毒滴度的慢性 cART 治疗患者中,仍可检测到细胞相关病毒 RNA,这表明病毒转录泄漏水平较低。迄今为止,还没有 FDA 批准的抗 HIV-1 转录的药物。我们之前已经证明 F07#13,一种对 Cdk9/T1-Tat 结合位点具有竞争活性的第三代 Tat 肽模拟物,在体外和体内抑制 HIV-1 转录。结果在这里,我们证明了 F07#13 浓度的增加( 0.01、0.1、1 µM) 通过抑制 Cdk9/T1-Tat 复合物的形成和随后的泛素介导的 Tat 螯合和降解,以剂量依赖性方式导致 Tat 水平降低。我们的数据表明复合物 I 和 IV 包含不同的泛素化 Tat 模式,并且 F07#13 诱导的转录抑制导致 Tat 水平的整体降低。这种减少可能由 F07#13 触发,但最终由 TAR-gag 病毒 RNA 介导,这些 RNA 结合抑制性转录因子(类似于 7SK、NRON、HOTAIR 和 Xist lncRNA)以增强转录基因沉默和潜伏期。这些 RNA 与 PRC2、Sin3A 和 Cul4B 复合,导致表观遗传修饰。最后,我们通过靶向长末端重复序列(HIV-1 启动子区域,LTR)的 R 区域,促进暂停的聚合酶和提高受感染细胞中 CRISPR/Cas9 编辑的效率,观察到 F07#13 介导的病毒负荷降低。这意味着基因编辑可能最好在受抑制的转录状态下进行。结论总的来说,我们的结果表明 F07#13 可以在不同位点终止 RNA 聚合酶 II,可以生成支架 RNA,这些 RNA 可以组装成特定的“RNA 机器”组,有助于基因调控。在具有不同启动子强度、突变 LTR 和患者样本的各种进化枝中是否也能看到这些影响还有待观察。
更新日期:2019-04-29
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