Cellular Factors Critical for Initiation of HIV-1 Reverse Transcription

描述：HIV-1逆转录的引物，人的tRNALys 3，与tRNALys 1，2一起被选择性地包装成病毒粒子。人赖氨酰-tRNA合成酶(LysRS)是已知的唯一与所有三种tRNALys同种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，而这两种宿主细胞因子的存在是获得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体(MSC)的一部分。近年来，LysRs被证明是从MSC动员起来的，并在多种非翻译途径中发挥作用。为了开发针对这一重要宿主细胞因子的有效治疗方法，需要阐明LysRS在HIV-1感染性中替代功能的详细分子机制。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复合体通过与GAG相互作用而改变其正常翻译功能并被招募到颗粒中的机制尚不清楚，这是目标1中需要解决的关键问题。我们最近发现，LysRS与HIV-1基因组RNA(VRNA)5‘端非编码区中的引物结合位点区域具有高亲和力。在初步研究中，我们发现基因组的这个高度结构和动态的区域包含一个tRNA样元件，负责与LysRS的相互作用。然而，LysRS结合、tRNA引物退火以及随后的逆转录酶结合和引发时发生的构象变化尚不清楚，而且到目前为止还没有报道起始复合体的高分辨结构。这些关键的悬而未决的问题将分别在目标2和目标3中解决。本工作的具体目的是：(1)阐明人LysRS被招募到HIV-1颗粒中的机制；(2)表征HIV-1 vRNA的PBS区在LysRS结合和tRNA引物退火时的整体三级结构和构象变化；(3)阐明vRNA：引物：RT起始复合体的结构。

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.