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， human tRNALys3，与human lysyl-tRNA合成酶（LysRS）一起被选择性地包装到病毒粒子中，LysRS是使tRNALys氨基化的酶。Gag单独将LysRS包装成Gag颗粒。病毒的LysRS比细胞质形式小，即使在没有病毒蛋白酶的情况下也存在这种截短的形式。我们假设LysRS是病毒蛋白识别的信号，并针对tRNALys进行病毒包装。截断LysRS可以将其从tRNALys中释放出来，从而促进tRNALys3退火到病毒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（共免疫沉淀）和将LysRS包装成Gag颗粒的能力。之间的相互作用

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.