Role of Lysyl-tRNA Synthetase in HIV-1 Replication

赖氨酰-tRNA 合成酶在 HIV-1 复制中的作用

• 批准号: 6785379
• 负责人: LAWRENCE KLEIMAN
• 金额: $ 25.92万
• 依托单位: SIR MORTIMER B. DAVIS JEWISH GEN HOSP
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6785379
• 关键词: acylation; aminoacid tRNA ligase; calorimetry; complementary DNA; fluorescence polarization; gag protein; gel filtration chromatography; genetic screening; human immunodeficiency virus 1; immunoprecipitation; point mutation; protein binding; protein protein interaction; protein purification; protein sequence; virus replication; yeast two hybrid system

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.

描述（由申请人提供）：HIV-1 中逆转录酶的引物 tRNA（人 tRNALys3）与人赖氨酰-tRNA 合成酶（LysRS）（氨酰化 tRNALys 的酶）一起被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，即使在没有病毒蛋白酶的情况下，这种截短的形式也存在。我们假设 LysRS 是病毒蛋白识别的信号，并以 tRNALys 为目标进行病毒包装。 LysRS 截短可能会将其从 tRNALys 中释放出来，从而促进 tRNALys3 与病毒 RNA 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和人类LysRS之间的相互作用。 Gag/LysRS 相互作用将在体内研究，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）以及将 LysRS 包装到 Gag 颗粒中的能力。之间的相互作用 纯化的成分将在体外进行研究，使用定性和定量方法来测量蛋白质-蛋白质相互作用。 2) 研究tRNA氨酰化状态对tRNALys3包装成HIV-1和启动逆转录的作用。尽管所有可检测的细胞质 tRNALys3 均被氨酰化，但 tRNALys3 必须不带电荷才能用作引物。我们将通过确定突变型 LysRS 是否能够结合 tRNALys3 但不能对其进行氨酰化，从而仍然促进 tRNALys3 包装，从而确定 tRNALys3 的氨酰化是否对于 tRNALys3 包装至关重要。病毒蛋白诱导 tRNALys3 脱酰化的能力也将在体外进行检测。 3) 研究病毒LysRS的细胞起源。细胞质 LysRS 存在于由 12 种蛋白质组成的高分子量氨酰基-tRNA 合成酶 (HMW aaRS) 复合物中，但在 HIV-1 中仅发现 LysRS。我们将确定无法与 p38 结合并因此无法作为 HMW aaRS 的组成部分参与的突变型 LysRS 是否仍然能够被包装到 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) 表征导致 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编码 LysRS 蛋白酶的 cDNA 的遗传策略都将用于鉴定可以裂解 LysRS 的细胞蛋白酶。