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首页> 外文期刊>Journal of Molecular Biology >Nucleocapsid protein annealing of a primer-template enhances (+)-strand DNA synthesis and fidelity by HIV-1 reverse transcriptase
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Nucleocapsid protein annealing of a primer-template enhances (+)-strand DNA synthesis and fidelity by HIV-1 reverse transcriptase

机译：引物模板的核衣壳蛋白退火可通过HIV-1逆转录酶增强（+）链DNA合成和保真度

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摘要

Human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer, which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer-template. Through the use of a pre-steady-state kinetics approach, the NCp7-annealed primer-template has a substantial increase (3- to 7-fold) in the rate of incorporation (k pol) by RT as compared to heat-annealed primer-template with single-nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (K d) of the nucleotide. These differences in k pol and K d were not through direct interactions between HIV-1 RT and NCp7. When extension by RT was examined, the data suggest that the NCp7-annealed primer-template facilitates the formation of a longer product more quickly compared to the heat-annealed primer-template. This enhancement in rate is mediated through interactions with NCp7's zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer-template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV.
机译：人类1型免疫缺陷病毒（HIV-1）需要逆转录酶（RT）和HIV-1核衣壳蛋白（NCp7）才能正确复制病毒。 HIV-1 NCp7已显示可增强逆转录的各个步骤，包括tRNA起始和链转移，这可以通过与RT以及RNA和DNA寡核苷酸的相互作用来介导。通过使用DNA寡核苷酸，我们已经检查了NCp7与RT的相互作用，以及在（+）链合成过程中使用NCp7促进退火的引物模板进行逆转录的动力学。通过使用稳态前动力学方法，与热退火的引物相比，NCp7退火的引物模板的RT掺入率（k pol）显着增加（3至7倍） -具有单核苷酸掺入的模板。核苷酸的结合亲和常数（K d）也增加了2倍。这些k pol和K d差异不是通过HIV-1 RT和NCp7之间的直接相互作用来实现的。当检查通过RT的延伸时，数据表明NCp7退火的引物模板与热退火的引物模板相比，可更快地促进更长产物的形成。速率的提高是通过与NCp7的锌指，N末端结构域和核酸的相互作用介导的。 NCp7退火的引物模板还通过减缓错误核苷酸的掺入速率来增强RT的保真度（3倍）。两者合计，这项研究阐明了通过促进HIV-1 RT逆转录过程中DNA定向DNA合成的NCp7的新作用，这可能转化为增强的病毒适应性，并为针对HIV的靶向治疗干预提供了途径。

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