Characterization of the elongation complex of dengue virus RNA polymerase: assembly, kinetics of nucleotide incorporation, and fidelity.
Characterization of the elongation complex of dengue virus RNA polymerase: assembly, kinetics of nucleotide incorporation, and fidelity.
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DOI：
10.1074/jbc.m110.162685
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发表时间：
2011-01-21
期刊：
影响因子：
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通讯作者：
Swinney DC
中科院分区：
文献类型：
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作者：
Jin Z;Deval J;Johnson KA;Swinney DC
登革病毒（DENV）每年感染全球5000万至1亿人，造成严重的公共卫生问题。登革病毒RNA依赖性RNA聚合酶是药物开发的一个有吸引力的靶标，它在三个阶段催化病毒基因组的从头复制：起始、过渡和延伸。本工作的目的是使用瞬时动力学方法表征在延伸期间由DENV血清型2的聚合酶结构域催化的核苷酸添加的机制。我们通过预孵育实验测量了含有聚合酶和双链RNA的延伸复合物形成的动力学。在37 °C下，延伸复合物的组装是缓慢的，遵循一步结合机制，缔合速率为0.0016 ± 0.0001 μm− 1 s −1，解离速率为0.00020 ± 0.00005 s−1。延伸复合物组装在30 °C下慢6倍，并且在预孵育期间需要Mg 2+。组装的延伸复合物将正确的核苷酸GTP掺入引物，Kd为275 ± 52 μm，kpol为18 ± 1 s−1。对于模板中UTP、ATP和与CMP相反的CTP的错误掺入，聚合酶的保真度分别为1/34，000、1/59，000、1/135，000。DENV聚合酶的保真度与HIV逆转录酶和脊髓灰质炎病毒聚合酶相当。这项工作报告的前稳态动力学和保真度的RNA依赖的RNA聚合酶的黄病毒科的第一个描述。
Dengue virus (DENV) infects 50–100 million people worldwide per year, causing severe public health problems. DENV RNA-dependent RNA polymerase, an attractive target for drug development, catalyzes de novo replication of the viral genome in three phases: initiation, transition, and elongation. The aim of this work was to characterize the mechanism of nucleotide addition catalyzed by the polymerase domain of DENV serotype 2 during elongation using transient kinetic methods. We measured the kinetics of formation of the elongation complex containing the polymerase and a double-stranded RNA by preincubation experiments. The elongation complex assembly is slow, following a one-step binding mechanism with an association rate of 0.0016 ± 0.0001 μm−1s−1 and a dissociation rate of 0.00020 ± 0.00005 s−1 at 37 °C. The elongation complex assembly is 6 times slower at 30 °C and requires Mg2+ during preincubation. The assembled elongation complex incorporates a correct nucleotide, GTP, to the primer with a Kd of 275 ± 52 μm and kpol of 18 ± 1 s−1. The fidelity of the polymerase is 1/34,000, 1/59,000, 1/135,000 for misincorporation of UTP, ATP, and CTP opposite CMP in the template, respectively. The fidelity of DENV polymerase is comparable with HIV reverse transcriptase and the poliovirus polymerase. This work reports the first description of presteady-state kinetics and fidelity for an RNA-dependent RNA polymerase from the Flaviviridae family.