lncRNA MALAT1影响HIV-1感染与复制的分子机制研究

As human immunodeficiency virus (HIV) could be persistent and latent infection in CD4 T cells and form viral reservior, highly active antiretroviral therapy didn’t effectively remove viruses. Therefore it is most important for AIDS treatment to understand the molecular mechanism on regulating transcription and latent infected by HIV-1. The detection and identification and function of new long non-coding RNA (lncRNA) is kept the focus on the cell development and tumorigenesis. It is largely unknown that the function and mechanism of lncRNAs acts on HIV-1 infection. Previously, we identified 72 novel lncRNAs in HIV-1 infection CD4+T cells by RNA-Seq. Interestingly, a novel lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was expressed significantly increased in HIV-1 infected CD4+T cells compared to mock infected cells by qRT-PCR method. Moreover, we confirmed that over-expression of MALAT1 evidently resulted in increased HIV-1 replication by qRT-PCR and western blotting methods. In this study, the interaction and biding site/domain between HIV-1 and lncRNA MALAT1 will be further confirmed by the methods of northern blotting and sequencing mutational analysis. Then we will investigate how the interaction on HIV-1 and lncRNA MALAT1 effects viral infection and viral RNA stabilization and replication by the analysis of transcriptional activity, flow cytometry, western blotting and RT-PCR. It is great important significance that these findings will elucidate the new regulatory function of lncRNA MALAT1 and revel the molecular mechanism on HIV-1 infection, and will provide the helpful clue for anti-viral therapy.

由于人类免疫缺陷病毒（HIV）能在CD4 T细胞中潜伏并形成病毒库，而高效抗逆转录疗法并不能有效清除病毒，因此了解调控HIV-1转录和潜伏分子机制对于艾滋病治疗尤为重要。长非编码RNA（lncRNA）的发现、鉴定及功能研究目前较集中在细胞发育、肿瘤发生等，而关于lncRNA与HIV-1功能及机制尚不清楚。前期利用RNA-Seq技术在HIV-1感染人CD4+T细胞中发现72个候选新lncRNA，qRT-PCR验证得到一个显著差异表达lncRNA MALAT1，且过表达MALAT1显著促进HIV-1感染。本项目拟进一步利用序列突变、流式细胞术、转录活性分析、RNA分析、蛋白印迹和RT-PCR等探讨MALAT1与HIV-1相互作用对病毒感染、病毒RNA稳定、复制等功能影响。本项目的完成将对探索lncRNA MALAT1功能、揭示新的HIV-1感染分子机制、为抗病毒治疗提供理论依据等具有重要意义。

长链非编码RNA (lncRNAs)已被证明是整个细胞生物学中RNA和蛋白质功能的有效调节因子，包括病毒复制。最近研究发现lncRNAs通过调节不同的细胞机制激活或抑制HIV-1的复制和潜伏感染。转移相关肺腺癌转录本1 (MALAT1)是一种可致癌lncRNA，并已被证明与病毒感染有关。然而，它影响HIV-1感染靶细胞的潜在机制尚不清楚。本研究通过RNA深度测序，比较了感染HIV-1或不感染HIV-1的巨噬细胞中lncRNAs表达谱，发现MALAT1在感染HIV-1巨噬细胞中显著上调。进一步利用蛋白质组学分析确定了HIV-1感染巨噬细胞中MALAT1调控的蛋白表达谱，基于CHCHD2下调表达最低，我们选择CHCHD2作为后续研究对象。研究发现MALAT1和CHCHD2均可以促进HIV-1复制，并且MALAT1作为ceRNA可通过竞争性结合miR-145-5p调节CHCHD2表达。一方面，CHCHD2通过抑制STAT2信号通路，引起MxB表达下降，进而促进HIV-1入核；另一方面，CHCHD2又可以抑制IRF7表达，降低IFN-α和IFN-β启动子的活性，减少IFN-α和IFN-β的产生，最终促进HIV-1感染与复制，阐释了CHCHD2作为先天免疫应答负调控因子在病毒感染调控中的作用；同时也增加了lncRNA MALAT1调控HIV-1复制机制的理解，为潜在的靶向免疫治疗干预AIDS提供了新的见解。