Role of Lysyl-tRNA Synthetase in HIV-1 Replication

描述(申请人提供)：HIV-1中逆转录酶的引物tRNA，人tRNALys3，与人赖氨酰-tRNA合成酶(LysRS)一起选择性地包装到病毒粒子中，LysRS是氨酰化tRNALys的酶。GAG单独会将LysRS打包成GAG颗粒。病毒LysRS比细胞质形式小，即使在没有病毒蛋白水解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，并针对tRNALys进行病毒包装。LysRS截断可能会将其从tRNALys中释放出来，从而促进tRNALys3对病毒RNA的退火。这些过程将在拟议的工作中进行研究，并代表抗HIV-1治疗的新目标。本研究的具体目的是：1)研究HIV-1 Gag与人LysRS的相互作用。GAG/LysRS的相互作用将通过突变形式的GAG和LysRS的细胞表达在体内进行研究，测量它们在细胞质中相互作用的能力(免疫共沉淀)，并将LysRS包装到GAG颗粒中。之间的相互作用 纯化的成分将在体外进行研究，使用定性和定量的方法来测量蛋白质之间的相互作用。2)探讨tRNA氨酰化状态在tRNALys3包装到HIV-1中的作用及其在逆转录启动中的作用。尽管所有可检测到的细胞质tRNALys3都是氨基酰化的，但tRNALys3必须不带电才能作为引物。我们将通过确定突变体LysRS是否能够结合tRNALys3，但不能氨基酰化tRNALys3，来确定tRNALys3的氨酰化是否对tRNALys3包装是必要的，这仍然有助于tRNALys3包装。病毒蛋白诱导tRNALys3脱酰化的能力也将在体外进行测试。3)探讨病毒LysRS的细胞来源。在由12种蛋白质组成的高分子量氨基酰-tRNA合成酶(HMW AARs)复合体中发现细胞质LysRS，而在HIV-I中仅发现LysRS。我们将确定突变体LysRS是否无法与p38结合，因此无法作为HMW Aars的一个组成部分参与，是否仍能包装到HIV-1中。利用RNA干扰，我们将确定病毒LysRS的来源是否是新合成的LysRS。4)鉴定导致在HIV-1中发现的人类LysRS截短形式的细胞蛋白酶。生化纯化方法和鉴定LysRS蛋白酶编码基因的遗传策略都将被用来鉴定能够裂解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.