Cellular Factors Critical for Initiation of HIV-1 Reverse Transcription

对 HIV-1 逆转录启动至关重要的细胞因素

基本信息

批准号：
8803615
负责人：
Karin M Musier-Forsyth
金额：
$ 29.21万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8803615
关键词：
5&apos Untranslated Regions Acquired Immunodeficiency Syndrome Address Affect Affinity Amino Acyl-tRNA Synthetases Base Pairing Beryllium Binding Binding Sites Cell Fractionation Cell Nucleus Cell membrane Cells Complex Confocal Microscopy Crystallization DNA Development Dimerization Energy Transfer Enzymes Event Extracellular Space Gagging Genome Genomics HIV-1 Human Human Genome IgE Immunofluorescence Immunologic Infection Integration Host Factors Interferons Lead Lysine-Specific tRNA Lysine-tRNA Ligase Mass Spectrum Analysis Modification Molecular Molecular Conformation Mutagenesis Outcome Pathway interactions Phosphorylation Phosphotransferases Play Positioning Attribute Post-Translational Protein Processing RNA RNA Folding RNA-Directed DNA Polymerase Recruitment Activity Reporting Resolution Reverse Transcription Roentgen Rays Role Site Solutions Stimulus Structure TNF gene Therapeutic Transcription Initiation Transfer RNA Translations Untranslated Regions Variant Viral Virion Work X-Ray Crystallography combat design design and construction new therapeutic target novel particle public health relevance three dimensional structure trafficking viral RNA

项目摘要

DESCRIPTION: The primer for reverse transcription in HIV-1, human tRNALys3, is selectively packaged into virions along with tRNALys1,2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNALys depends on the ability of the tRNA to bind to LysRS and the presence of both host cell factors is required for optimal viral infectivity. LysR is normally part of a dynamic mammalian multisynthetase complex (MSC). In recent years, LysRS has been shown to be mobilized from the MSC and to function in a wide variety of non-translational pathways. Elucidating the detailed molecular mechanism for the alternative function of LysRS in HIV-1 infectivity is needed in order to develop effective therapeutics aimed at targeting this essential host cell factor. While some aspects of tRNA primer packaging into HIV-1 particles are now understood, the mechanism by which the LysRS/tRNA complex is diverted from its normal function in translation and recruited into particles through interaction with Gag is unclear, and this is the key question to be addressed in Aim 1. We have recently shown that LysRS binds with high affinity to the primer binding site region in the 5'UTR of HIV-1 genomic RNA (vRNA). In preliminary studies, we found that this highly structured and dynamic region of the genome contains a tRNA-like element that is responsible for the interaction with LysRS. However, the conformational changes that occur upon LysRS binding, tRNA primer annealing, and subsequent binding and initiation by reverse transcriptase are unknown, and no high-resolution structure of the initiation complex has been reported to date. These key open questions will be addressed in Aims 2 and 3, respectively. The specific aims of the proposed work are: (1) To elucidate the mechanism by which human LysRS is recruited into HIV-1 particles, (2) To characterize the global tertiary structure and conformational changes of the PBS region of HIV-1 vRNA upon LysRS binding and tRNA primer annealing, and (3) To elucidate the structure of the vRNA:primer:RT initiation complex.

描述：HIV-1 逆转录引物，人 tRNALys3，与 tRNALys1,2 一起被选择性包装到病毒体中。人类赖氨酰-tRNA 合成酶 (LysRS) 是唯一已知与所有三种 tRNALys 同工受体特异性相互作用的细胞因子，也被包装到 HIV-1 中。 tRNALys 的选择性包装取决于 tRNA 与 LysRS 结合的能力，并且两种宿主细胞因子的存在都是最佳病毒感染性所必需的。 LysR 通常是动态哺乳动物多合成酶复合物 (MSC) 的一部分。近年来，LysRS 已被证明是从 MSC 中动员出来的，并在多种非翻译途径中发挥作用。需要阐明 LysRS 在 HIV-1 感染中替代功能的详细分子机制，以便开发针对这一重要宿主细胞因子的有效疗法。虽然现在已经了解了 tRNA 引物包装到 HIV-1 颗粒中的某些方面，但 LysRS/tRNA 复合物在翻译中偏离其正常功能并通过与 Gag 相互作用招募到颗粒中的机制尚不清楚，这是关键问题目标 1 中要解决的问题。我们最近表明，LysRS 以高亲和力与 HIV-1 基因组 RNA (vRNA) 5'UTR 中的引物结合位点区域结合。在初步研究中，我们发现基因组的这个高度结构化和动态的区域包含一个类似 tRNA 的元件，负责与 LysRS 相互作用。然而，LysRS 结合、tRNA 引物退火以及随后的结合和逆转录酶启动时发生的构象变化尚不清楚，并且迄今为止尚未报道起始复合物的高分辨率结构。这些关键的开放性问题将分别在目标 2 和 3 中得到解决。拟议工作的具体目标是：（1）阐明人类 LysRS 被招募到 HIV-1 颗粒中的机制，（2）表征 HIV-1 vRNA PBS 区域的整体三级结构和构象变化LysRS 结合和 tRNA 引物退火，以及 (3) 阐明 vRNA:引物:RT 起始复合物的结构。