Role of Lysyl-tRNA Synthetase in HIV-1 Replication

描述（由申请人提供）：HIV-1逆转录酶的引物tRNA（人tRNALys 3）与人赖氨酰-tRNA合成酶（LysRS）（使tRNALys氨酰化的酶）一起沿着选择性包装到病毒粒子中。Gag单独将LysRS包装成Gag颗粒。病毒LysRS比细胞质形式小，即使在不存在病毒蛋白酶的情况下也存在这种截短形式。我们假设LysRS是病毒蛋白识别的信号，并靶向tRNALys进行病毒包装。LysRS截短可以将其从tRNALys释放，从而促进tRNALys 3退火至病毒RNA。这些过程将在拟议的工作中进行研究，并代表抗HIV-1治疗的新靶点。本研究的具体目的是：1）研究HIV-1 Gag与人LysRS的相互作用。将在体内研究Gag/LysRS相互作用，通过Gag和LysRS突变形式的细胞表达，测量它们在细胞质中相互作用（免疫共沉淀）和将LysRS包装成Gag颗粒的能力。之间的相互作用 纯化的组分将在体外进行研究，使用定性和定量方法测量蛋白质-蛋白质相互作用。2)研究tRNA氨酰化状态对tRNALys 3包装入HIV-1和启动逆转录的作用。虽然所有可检测的细胞质tRNALys 3是氨酰化的，但tRNALys 3必须不带电荷才能作为引物。我们将通过确定能够结合tRNALys 3但不能使其氨酰化的突变型LysRS是否仍然促进tRNALys 3包装来确定tRNALys 3的氨酰化是否对于tRNALys 3包装是必需的。还将在体外检查病毒蛋白诱导tRNALys 3脱酰化的能力。3)探讨病毒LysRS的细胞来源。细胞质LysRS存在于由12种蛋白质组成的高分子量氨酰-tRNA合成酶（HMW阿尔斯）复合物中，但仅在HIV-I中发现LysRS。我们将确定不能与p38结合并因此不能作为HMW阿尔斯的组分参与的突变型LysRS是否仍然能够被包装到HIV-1中。使用RNA干扰，我们将确定病毒LysRS的来源是否是新合成的LysRS。4)表征导致HIV-1中发现的人LysRS截短形式的细胞蛋白酶。用于鉴定编码LysRS蛋白酶的cDNA的生物化学纯化方法和遗传策略将用于鉴定可以切割LysRS的细胞蛋白酶。

DESCRIPTION (provided by applicant): The primer tRNA for reverse transcriptase in HIV-1, human tRNALys3, is selectively packaged into the virion, along with human lysyl-tRNA synthetase (LysRS), the enzyme that aminoacylates tRNALys. Gag alone will package LysRS into Gag particles. Viral LysRS is smaller than the cytoplasmic form, and this truncated form is present even in the absence of viral protease. We hypothesize that LysRS is the signal recognized by viral proteins and targets tRNALys for viral packaging. LysRS truncation may release it from tRNALys, thereby facilitating tRNALys3 annealing to the viral RNA. These processes will be studied in the proposed work, and represent new targets for anti-HIV-1 therapy. The specific aims of this study are: 1) To investigate the interaction between HIV-1 Gag and human LysRS. Gag/LysRS interactions will be studied in vivo, through the cellular expression of mutant forms of Gag and LysRS, measuring their ability to interact in the cytoplasm (coimmunoprecipitation) and to package LysRS into Gag particles. Interactions between purified components will be studied in vitro, using both qualitative and quantitative methods for measuring protein-protein interactions. 2) To investigate the role of the tRNA aminoacylation state on tRNALys3 packaging into HIV-1 and on priming of reverse transcription. Although all detectable cytoplasmic tRNALys3 is aminoacylated, tRNALys3 must be uncharged to serve as a primer. We shall determine if aminoacylation of tRNALys3 is essential for tRNALys3 packaging by determining if mutant LysRS able to bind tRNALys3, but not aminoacylate it, still facilitates tRNALys3 packaging. The ability of viral proteins to induce tRNALys3 deacylation will also be examined in vitro. 3) To investigate the cellular origin of viral LysRS. Cytoplasmic LysRS is found in a high molecular weight aminoacyl-tRNA synthetase (HMW aaRS) complex composed of 12 proteins, but only LysRS is found in HIV-I. We shall determine if mutant LysRS unable to bind to p38 and therefore unable to participate as a component of the HMW aaRS, is still able to be packaged into HIV-1. Using RNA interference, we shall determine if the source of viral LysRS is newly-synthesized LysRS. 4) To characterize the cellular protease that leads to the truncated form of human LysRS found in HIV-1. Both biochemical purification methods and a genetic strategy for identifying cDNAs coding for LysRS proteases will be used to identify cellular proteases that can cleave LysRS.