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; 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中的逆转录酶的引物tRNA与人赖氨酸-TRNA合成酶（LYSRS）一起选择性地包装到病毒粒子中，该酶是氨基酰基trnalys的酶。单独的插科打术会将lysr包装到插孔颗粒中。病毒lysr小于细胞质形式，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LYSR是病毒蛋白识别的信号，并靶向TRNALYS用于病毒包装。 LYSR截断可能会从Trnalys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治疗的新目标。这项研究的具体目的是：1）研究HIV-1 GAG与人类LYSR之间的相互作用。 GAG/LYSRS相互作用将在体内，通过突变形式的GAG和LYSR的细胞表达来研究它们在细胞质中相互作用的能力（共免疫沉淀），并将LYSR包装到GAG颗粒中。之间的相互作用 纯化的成分将在体外研究，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氨基酰化状态在TRNALYS3包装中的作用以及逆转录启动。尽管所有可检测到的细胞质Trnalys3都是氨基酰基3，但必须将TRNALYS3无负荷作为底漆。我们将通过确定突变体LYSR是否能够结合Trnalys3但不能氨基酰基化，但仍然促进Trnalys3包装，确定TRNALYS3的氨基酰基3对于TRNALYS3包装是否至关重要。病毒蛋白诱导Trnalys3脱酰基化的能力也将在体外检查。 3）研究病毒LYSR的细胞起源。细胞质LYSR是在高分子量氨基酰基-TRNA合成酶（HMW AARS）复合物中发现的，由12种蛋白质组成，但仅在HIV-I中发现LYSR。我们将确定突变体是否无法与p38结合，因此无法作为HMW AAR的组成部分参与，仍然可以将其包装到HIV-1中。使用RNA干扰，我们将确定病毒LYSR的来源是否是新的合成的LYSR。 4）表征导致HIV-1中发现的人类LYSR截断形式的细胞蛋白酶。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LYSR的细胞蛋白酶。