Development of the Gag/Lysyl tRNA synthetase interaction as a target for anti-HIV

描述(申请人提供)：HIV-1的基因组由RNA组成，该RNA进入细胞后必须转化为病毒DNA，然后插入细胞的DNA，并用于生产新的病毒蛋白和病毒。RNA被病毒酶逆转录酶转化为DNA。这种酶需要一种底物才能启动逆转录。HIV-1逆转录的引物是人tRNALys3，它在病毒在细胞中组装时被选择性地包装到病毒粒子中，并与病毒RNA基因组中称为引物结合位点(PBS)的区域结合(退火)。反转录正是从这个结合的tRNALys3开始的。TRNALys3的选择性包装是因为病毒蛋白GAG与细胞中主要的tRNALys3结合蛋白Lysyl-tRNA合成酶(LysRS)特异地相互作用，从而使tRNALys3和LysRS都被包装到组装病毒中。通过GAG或LysRS的突变来破坏这一反应，可以防止tRNALys3被包装到病毒中，并极大地降低反转录和病毒感染性。因此，Gag/LysRS相互作用代表了抗HIV-1治疗的新靶点。在这个项目中，我们正在寻找这种相互作用的小分子抑制剂。这将通过对化合物文库的高通量筛选(HTS)来找到抑制GAG/LysRS相互作用的分子。在LysRS/CA相互作用的HTS中，使用荧光标记的CA作为示踪剂，用荧光各向异性(FA)来测量LysRS与Gag或较小的衣壳(CA)分子的相互作用。还将研究小肽或RNA分子抑制这种相互作用的能力，使用来自GAG或LysRS的已知参与GAG/LysRS相互作用的多肽，或SELEX来分离与CA特异结合的RNA分子。项目简介许多报告现已证明，选择性地将逆转录引子tRNALys3包装到HIV-1中是病毒生命周期所必需的。TRNALys3的选择性掺入需要GAG和LysRS之间的特定相互作用，使GAG/LysRS相互作用成为抗病毒药物开发的一个有吸引力的靶点。已经证明了使用荧光各向异性分析来检测结合相互作用的可行性，并将在该项目中用于促进快速筛选技术的发展，以寻找这一新靶点的抑制剂，然后将测试它们特异性抑制HIV-1复制的能力。

DESCRIPTION (provided by applicant): The genome of HIV-1 is composed of RNA, and after this RNA enters a cell, it must be converted to viral DNA, which can then insert into the cell's DNA, and be used to produce new viral protein and viruses. The RNA is converted into DNA by the viral enzyme reverse transcriptase. This enzyme requires a primer to start reverse transcription. The primer for reverse transcription in HIV-1 is human tRNALys3, which is selectively packaged into the virion during viral assembly in the cell, and binds (anneals) to a region in the viral RNA genome termed the primer binding site (PBS). It is from this bound tRNALys3 that reverse transcription begins. The selective packaging of tRNALys3 occurs because the viral protein Gag specifically interacts with a major tRNALys3-binding protein in the cell, lysyl-tRNA synthetase (LysRS), so that both tRNALys3 and LysRS are packaged into the assembling virus. Disrupting this reaction through mutations in either Gag or LysRS prevent tRNALys3 from being packaged into the virus, and greatly reduces reverse transcription and viral infectivity. The Gag/LysRS interaction, therefore, represents a novel target for anti-HIV-1 therapy. In this project, we are seeking to identify small molecule inhibitors of this interaction. This will be done using high throughput screening (HTS) of libraries of compounds to find molecules that inhibit the Gag/LysRS interaction. The interaction of LysRS with either Gag or the smaller capsid (CA) molecule will be measured using fluorescent anisotropy (FA), using fluorescently-labeled CA as the tracer in the HTS of the LysRS/CA interaction. The ability of small peptides or RNA molecules to inhibit this interaction will also be investigated, using peptides from Gag or LysRS that are known to be involved in the Gag/LysRS interaction, or SELEX to isolate RNA molecules that specifically bind to CA. Project Narrative Many reports have now demonstrated that the selective packaging into HIV-1 of the primer for reverse transcription, tRNALys3, is essential for the viral life cycle. This selective incorporation of tRNALys3 requires a specific interaction between Gag and LysRS, making the Gag/LysRS interaction an attractive target for antiviral drug development. The feasibility of using a fluorescence anisotropy assay to detect the binding interaction has already been demonstrated, and will be used in this project facilitate the development of rapid screening techniques to search for inhibitors of this novel target, which will then be tested for their ability to specifically inhibit HIV-1 replication.