vmiR-TAR与Tat相互作用在HIV-1病毒潜伏感染中的作用机制研究

HIV-1 infection and AIDS imperil a big amount of people worldwide. The current HAART treatment can restrict viral replication to a very low level, but the virus re-activate in some treated patients. Transcription of HIV-1 in infected cells depends on Tat binding of transactivation responsive element(TAR). Recently, TAR was reported to encode viral miRNAs (vmiR-TAR) to restrict viral replication, while Tat has evolved a suppressor of RNA silencing (SRS) function to facilitate viral replication. The interactions of vmiR-TAR and Tat, as a mouse-cat game, thus contribute to viral latent infection, but the mechanism remains dramatically unclear. Based on our previours work, we plan to uncover the biogenesis of vmiR-TAR though our Drosha and Dicer in vitro systems. To investigate the viral replication suppress function of vmiR-TAR, we use monocyte/macrophage and resting/activate CD4+T cells as infection models, as they are the major viral pools in latent infection. HIV-1 wild type strains Bal、Jago and JRFL infected macrophges and infectious clone pNL4-3 transfected resting/active CD4+T cells are detected by northern blot. Also, small RNA fragments under 30 nt are extracted for deep sequencing. Deep sequencing data will not only provide us actual vmiR-TAR sequences and copy numbers in each different infection condition, but also show cellular miRNA profile changes related to HIV-1 infection. To test inhibition of HIV-1 infection and replication by vmiR-TAR, chemical synthesized vmiR-TAR (5' and 3') are transfected into cell models (transfection for pNL4-3 in T cells), P24 of HIV-1 in the supernatant of cell culture can reflect HIV-1 infection and replication under suppressure by miR-TAR. What's more, our previous work had identified structure determinants of pre-miRNAs cleaved by human Dicer and interactions of Dicer and pre-miRNAs, including binding and catalysis process. Here, we presume that, based on high affinity binding of Tat and TAR, Tat probably bind a big range of pre-miNRA hairpins in the cytoplasm,including TAR precusor hairpin, thus module miRNA biogenesis by block Dicer recognition and binding of pre-miRNAs competitively. This study might give evidence for vmiR-TAR biogenesis, supressing function on HIV-1 transcription, infection and replication. The discovering of mechanism of vmiR-TAR and Tat interactions might also provide novel insights into viral encoded miRNA and viral suppressor of RNA silencing (SRS) function, which may suggest new techniques for antiviral strategy.

艾滋病的治疗手段HAART只能控制病毒复制，而防止病毒复制再激活为目前治疗方法的瓶颈。最新研究发现，HIV-1 编码的TAR因子可产生抑制病毒复制的miRNA（vmiR-TAR），而病毒Tat蛋白能阻断vmiR-TAR生成途径，利于病毒潜伏感染和再复制，但其机制未明。本项目拟利用Drosha体外实验平台研究vmiR-TAR生成途径；在HIV-1潜伏感染模型上研究miR-TAR抑制病毒转录复制效应，并通过深度测序确定vmiR-TAR序列与拷贝数；利用Dicer体外实验平台研究Tat与包括TAR发夹环在内的pre-miRNA结合对Dicer酶切割底物活性的影响。本研究将探索HIV-1病毒编码的vmiR-TAR的发生途径、揭示抑制病毒转录复制的作用机理，提出Tat蛋白抑制调节Dicer酶活性新机制，并探索利用病毒自身编码miRNA抑制病毒复制的可能性，为控制HIV-1复制提供新思路。

HIV-1病毒编码的TAR序列可产生小分子RNA（miR-TAR）已获得认同，但其产生及表达规律以及效应并不十分清楚。我们证实了在AIDS病人临床样本、感染HIV-1的巨噬细胞内miR-TAR-3p有表达，且其表达与HIV-1复制水平相关；同时，表达的miR-TAR-3p反过来具有抑制病毒复制的效应，整体上可理解为病毒编码TAR来源的小分子RNA抑制自身的过度复制，对病毒建立缓慢的潜伏感染具有重要意义。我们的研究成果已发表SCI论文一篇；另一篇正在准备完善数据尽快投稿；利用表达载体设计具有调节病毒复制水平的小分子RNA部分计划申报专利一项。