Virology DTA, Roche Palo Alto LLC, Palo Alto, California 94034, USA.
J Biol Chem. 2011 Jan 21;286(3):2067-77. doi: 10.1074/jbc.M110.162685. Epub 2010 Nov 15.
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(-1) s(-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 Mg(2+) during preincubation. The assembled elongation complex incorporates a correct nucleotide, GTP, to the primer with a K(d) of 275 ± 52 μM and k(pol) 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.
登革热病毒（DENV）每年在全球感染 5000 万至 1 亿人，造成严重的公共卫生问题。DENV RNA 依赖性 RNA 聚合酶是药物开发的一个有吸引力的靶点，它在三个阶段催化病毒基因组的从头复制：起始、转换和延伸。本工作旨在使用瞬变动力学方法表征 DENV 血清型 2 聚合酶结构域在延伸过程中催化核苷酸添加的机制。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。延伸复合物的组装是缓慢的，遵循一步结合机制，在 37°C 时，其缔合速率为 0.0016±0.0001μM(-1)s(-1)，解离速率为 0.00020±0.00005s(-1)。在 30°C 时，延伸复合物的组装速度慢 6 倍，并且在预孵育过程中需要 Mg(2+)。组装好的延伸复合物以 275±52μM 的 K(d)和 18±1s(-1)的 k(pol)将正确的核苷酸，GTP，掺入引物中。聚合酶的保真度为 1/34000、1/59000 和 1/135000，分别对应于在模板中 CMP 反位时掺入 UTP、ATP 和 CTP 的错误掺入。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。本工作首次报道了黄病毒科 RNA 依赖性 RNA 聚合酶的预稳态动力学和保真度。
