Inhibition of HIV-1 replication by delivery of the SRSF1 RNA Recognition Motifs

 描述（由申请方提供）：整合的HIV-1前病毒基因组的转录受到病毒蛋白达特与几种细胞因子和RNA聚合酶II复合物相互作用的严格调控。转录的病毒前mRNA被剪接成多个mRNA以产生病毒复制所需的九种不同的基因产物。HIV还发展了许多策略来调节其转录物的剪接。病毒基因组的表达依赖于病毒启动子、RNA序列、病毒蛋白和宿主细胞因子之间的相互作用。调节病毒信使转录和剪接的机制的改变可以显著影响病毒的感染性和发病机制。利用基于细胞和生物化学方法的组合，我们已经分离出一种细胞RNA结合蛋白SRSF 1，它是病毒转录和剪接的抑制剂。SRSF 1通过与病毒转录反式激活因子达特竞争从而减少病毒转录，并通过结合病毒信使内的一系列序列来发挥其抗病毒活性，所述序列调节病毒转录物内多个剪接位点的选择。SRSF 1的过表达诱导转录和剪接机制的破坏，导致对病毒复制的强烈抑制。其抗病毒活性所需的最小SRSF 1片段由RNA识别基序（RRM）1和2，两个RNA结合结构域（RBD）组成。RRM 1和2在稳定细胞系中的表达可以使许多病毒株的复制减少多达3000倍而不改变细胞活力。我们建议评估SRSF 1 RRM结构域的治疗潜力。我们将在SRSF 1 RRM和达特细胞穿透肽（CPP）之间产生嵌合蛋白，CPP是一种短序列，其允许分子货物以高效率递送和内化至真核细胞。我们将 分析CPP-RRM嵌合体的细胞内递送效率和抗病毒活性， 白细胞来源的细胞系和从健康供体纯化并感染不同亚型（B、C和D）病毒的CD 4 + T细胞。接下来，我们将表征SRSF 1核定位信号并优化携带单个RRM 2的嵌合蛋白的核递送，所述单个RRM 2有效地结合靶RNA序列，但未能优先定位在细胞核内并以与RRM 1和2组合相当的效率下调病毒复制。最后，我们将利用一种新的内体溶解剂，命名为dfTat，其允许在与DfTat简单共孵育后大蛋白货物的有效递送和内化。 目标细胞。我们提出的方法将确定一种新的靶蛋白的治疗潜力，并建立利用动物模型的未来研究。

 DESCRIPTION (provided by applicant): Transcription of the integrated HIV-1 proviral genome is tightly regulated by the interaction of the viral protein Tat with several cellular factors and he RNA Polymerase II complex. The transcribed viral pre- mRNA is spliced in multiple mRNAs to generate the nine different gene products required for viral replication. HIV has also developed a number of strategies to regulate splicing of its transcripts. Expression of the viral genome is dependent on the interactions between the viral promoter, RNA sequences, viral proteins and host cell factors. Alteration of the mechanisms regulating transcription and splicing of the viral messenger can dramatically affect viral infectivity and pathogenesis. Utilizing a combination of cell-based and biochemical approaches we have isolated a cellular RNA binding protein, SRSF1, which is an inhibitor of both viral transcription and splicing. SRSF1 exerts its antiviral activity by competing with the viral transcriptional transactivator Tat, thus reducing viral transcription, and by binding a series of sequences within the viral messengers, which regulate the choice of multiple splicing sites within the viral transcripts. Over-expression of SRSF1 induces the disruption of both transcription and splicing mechanisms resulting in a strong inhibition of viral replication. The minimal SRSF1 fragment required for its antiviral activity is constituted by the RNA Recognition Motifs (RRMs) 1 and 2, two RNA binding domains (RBDs). Expression of RRM1 and 2, in a stable cell line, can reduce the replication of a number of viral strains up to 3000 fold without altering cell viability. We propose to evaluate the therapeutic potential of the SRSF1 RRM domains. We will create a chimeric protein between the SRSF1 RRMs and the Tat Cell Penetrating Peptide (CPP), a short sequence, which allows for the delivery and internalization of molecular cargoes to eukaryotic cells with high efficiency. We will analyze the efficiency of intracellular delivery and antiviral activity of the CPP-RRMs chimeras in a leukocyte derived cell line and in CD4+ T cells purified from healthy donors and infected with viruses from different subtypes (B, C and D). Next, we will characterize the SRSF1 nuclear localization signal and optimize the nuclear delivery of chimeric proteins carrying the single RRM2, which efficiently binds the target RNA sequences but fails to preferentially localize within the cell nucleus and down-regulate viral replication with efficiency comparable to the RRM1 and 2 combined. Finally, we will utilize a novel endosomolitic agent, named dfTat, which allows for the efficient delivery and internalization of large protein cargoes after simple co-incubation with the target cells. The approach we propose will determine the therapeutic potential of a novel target protein and set-up future studies that utilize animal models.