Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA
Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA
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DOI：
10.1074/jbc.ra119.007679
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发表时间：
2019-07-26
影响因子：
4.8
通讯作者：
Heng, Xiao
中科院分区：
文献类型：
--
作者：
Brady, Samantha;Singh, Gatikrushna;Heng, Xiao
DHX9/RNA解旋酶A(RHA)是一种宿主RNA解旋酶，参与HIV-1生命周期的许多关键步骤。它与病毒RNA基因组共同组装成衣壳核心。由于逆转录效率降低，缺乏RHA的病毒粒子的传染性较低，这表明与病毒粒子相关的RHA在病毒粒子获得新宿主的RHA之前促进了逆转录。在这里，我们量化了HIV-1感染的T细胞中的逆转录中间产物，以阐明RHA提高HIV-1逆转录效率的机制。一贯地，纯化的重组人RHA在体外条件下促进了逆转录效率，这种条件模仿了衣壳核心去涂层之前的早期逆转录步骤。我们没有观察到RHA介导的tRNA(Lys3)-病毒RNA-退火物复合体的结构重构。RHA直到掺入前几个核苷酸才能提高DNA合成速度，这表明RHA主要参与反转录的延伸阶段。稳态前和稳态动力学研究表明，RHA对单核苷酸掺入动力学几乎没有影响。在TRAP dsDNA存在下进行的引物延伸分析表明，RHA增强了HIV-1逆转录酶(RT)的处理能力。这里使用的生化分析有效地反映和解释了从RHA耗尽的细胞产生的HIV-1病毒粒子的低RT活性。此外，我们检测的RT活性表明，在病毒感染过程中，在去衣壳之前，HIV-1病毒粒子中的RHA是有效催化(-)cDNA合成所必需的。我们的研究确定RHA是HIV-1RT的一个进程因素。
DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNA(Lys3)-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.