Cellular Factors Critical for Initiation of HIV-1 Reverse Transcriptase

项目摘要 HIV-1逆转录的引物，人的tRNALys 3，被选择性地包装成病毒粒子 人赖氨酰-tRNA合成酶(LysRS)，唯一与两者特异相互作用的细胞因子 TRNALys等受体，也被包装到HIV-1中。TRNALys的选择性包装取决于 LysRS与tRNALys结合，两种宿主细胞因子的存在是最佳病毒感染性所必需的。LysRS 通常是动态哺乳动物多合成酶复合体的一部分。在之前的授权期内，我们做出了 在阐明人类LysRS被招募到HIV-1颗粒中的机制方面取得了实质性进展。 我们报道，HIV-1感染导致了一个部分重新激活的磷酸化LysRS(pS207-LysRS)游离池。 定位于靶细胞的核。在HIV-1产生细胞中阻断这一途径可消除LysRS 包装，并导致更少的传染性后代病毒粒子。我们还扩展了早期的工作，表明LysRS 与HIV-1基因组RNA(GRNA)引物结合位点(PBS)区域的tRNA样元件结合，该元件 促进tRNA从LysRS释放，并将引物放置到PBS上。在下一个授权期，我们将建立 通过检验磷酸化LysRS促进不带电tRNA的总体假设，对这些结果进行了分析 引物包装和tRNALys3对PBS退火改变了gRNA的构象和动力学，从而 影响对HIV-1生命周期至关重要的下游事件。这项工作将改变我们的理解 两种宿主细胞因子在HIV-1复制中的作用。目的1将探讨LysRS磷酸化在 包装一个不带电荷的tRNALys3，并验证这个亚型的LysRS作为治疗靶点。目标2将 建立tRNA退火和pS207-LysRS结合对gRNA结构和动力学的影响，并提供 对病毒生命周期中这一关键步骤的时机的新见解，可能揭示一种新的治疗策略。

Project Summary 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 that interacts specifically with both tRNALys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNALys depends on the ability of LysRS to bind to tRNALys and the presence of both host cell factors is required for optimal viral infectivity. LysRS is normally part of a dynamic mammalian multi-synthetase complex. In the previous grant period, we made substantial progress towards elucidating the mechanism by which human LysRS is recruited into HIV-1 particles. We reported that HIV-1 infection results in a free pool of phosphorylated LysRS (pS207-LysRS) that is partially re- localized to the nucleus of target cells. Blocking this pathway in HIV-1 producing cells abolished LysRS packaging and resulted in less infectious progeny virions. We also extended earlier work showing that LysRS binds to a tRNA-like element in the primer-binding site (PBS) region of HIV-1 genomic RNA (gRNA), which facilitates tRNA release from LysRS and primer placement onto the PBS. In the next grant period, we will build on these results by examining the overall hypotheses that phosphorylated LysRS facilitates uncharged tRNA primer packaging and that tRNALys3 annealing to the PBS alters gRNA conformation and dynamics, thereby impacting downstream events that are critical for the HIV-1 lifecycle. This work will transform our understanding of the role of two host cell factors in HIV-1 replication. Aim 1 will explore the role of LysRS phosphorylation in packaging an uncharged form of tRNALys3 and validate this isoform of LysRS as a therapeutic target. Aim 2 will establish the effect of tRNA annealing and pS207-LysRS binding on gRNA structure and dynamics and provide new insights into the timing of this key step in the viral lifecyle, which may reveal a novel therapeutic strategy.