Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein
Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein
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DOI：
10.1006/viro.1998.9213
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发表时间：
1998-07-05
期刊：
影响因子：
3.7
通讯作者：
Fu, JL
中科院分区：
文献类型：
--
作者：
Cui, TA;Sugrue, RJ;Fu, JL
全长登革病毒NS 3蛋白已成功表达为94-kDa GST融合蛋白在大肠杆菌中。用凝血酶处理纯化的融合蛋白释放出68-kDa的蛋白质，其是DEN 1 NS 3蛋白质的预期分子量。使用登革病毒抗血清通过蛋白质印迹法确认该蛋白的身份。重组NS 3蛋白具有与登革病毒RNA基因组3 ′-非编码区结合和NTR活性两种相关活性。我们使用带移测定证明，DEN 1 NS 3蛋白可以与3 '-非编码区（3'-NCR）中的茎环结构形成复合物，尽管茎环外的位点也可以参与结合。使用各种未标记的均聚和杂聚RNA作为结合的竞争者，进一步显示DEN 1 NS 3蛋白表现出优先结合来自登革病毒3 ′-NCR的94-nt RNA转录物。重组DEN 1 NS 3蛋白的NTR活性使用薄层层析测定来表征，我们发现DEN 1 NS 3蛋白具有NTR活性的某些方面，这与在其他黄病毒中发现的那些不同。尽管NS 3蛋白能够利用所有四种核糖核苷三磷酸作为其底物，但NS 3蛋白显示出对嘌呤三磷酸的明显偏好（即，加入poly（U）不刺激DEN 1 NS 3蛋白中的NTR活性，这与其他黄病毒NS 3蛋白的报道相反。然而，NTR的活性是特别刺激的病毒NS 5蛋白，这是表现为一个超过2倍的增加，ATP水解的速度和25%的增加，在120分钟的反应结束时，ADP的产量。这些数据表明，NS 3蛋白的NTR活性可能在病毒复制过程中受到病毒NS 5蛋白的调节。(C)北京：科学出版社.
The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay, we found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP), The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication. (C) 1998 Academic Press.