J Virol 2011 Nov;85 (21): 11183-95. [IF:5.189]
Inhibition of dengue virus by targeting viral NS4B protein.
Xie X , Wang QY , Xu HY , Qing M , Kramer L , Yuan Z , Shi PY .
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan, China.
武汉大学，中国科学院武汉病毒研究所，病毒学国家重点实验室
Abstract
We report a novel inhibitor that selectively suppresses dengue virus (DENV) by targeting viral NS4B protein. The inhibitor was identified by screening a 1.8-million-compound library using a luciferase replicon of DENV serotype 2 (DENV-2). The compound specifically inhibits all four serotypes of DENV (50% effective concentration [EC(50)], 1 to 4 μM; and 50% cytotoxic concentration [CC(50)], >40 μM), but it does not inhibit closely related flaviviruses (West Nile virus and yellow fever virus) or nonflaviviruses (Western equine encephalomyelitis virus, Chikungunya virus, and vesicular stomatitis virus). A mode-of-action study suggested that the compound inhibits viral RNA synthesis. Replicons resistant to the inhibitor were selected in cell culture. Sequencing of the resistant replicons revealed two mutations (P104L and A119T) in the viral NS4B protein. Genetic analysis, using DENV-2 replicon and recombinant viruses, demonstrated that each of the two NS4B mutations alone confers partial resistance and double mutations confer additive resistance to the inhibitor in mammalian cells. In addition, we found that a replication defect caused by a lethal NS4B mutation could be partially rescued through trans complementation. The ability to complement NS4B in trans affected drug sensitivity when a single cell was coinfected with drug-sensitive and drug-resistant viruses. Mechanistically, NS4B was previously shown to interact with the viral NS3 helicase domain; one of the two NS4B mutations recovered in our resistance analysis-P104L-abolished the NS3-NS4B interaction (I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan, J. Gen. Virol. 87:2605-2614, 2006). Collectively, the results suggest that the identified inhibitor targets the DENV NS4B protein, leading to a defect in viral RNA synthesis.
摘要
我们报告了一种新型抑制剂，它能通过靶向病毒NS4B蛋白选择性地抑制登革热病毒（DENV）。抑制剂的鉴定，我们使用登革2型（DENV- 2）的荧光素酶的复制子筛选了1.8万化合物库鉴定出这个抑制剂。这个化合物能特异性抑制登革所有四个血清型（50％有效浓度[EC（50）]，1至4微米;和50％细胞毒性浓度[CC（50）]，>40微米），但不抑制相似度高的虫媒病毒（西尼罗河病毒和黄热病病毒）或非虫媒病毒（西部马脑脊髓炎病毒，基孔肯雅病毒，水疱性口炎病毒）。一个行为模式的研究表明，该化合物抑制病毒RNA合成。在细胞培养中选用了耐受抑制剂的复制子。复制子测序发现病毒NS4B蛋白存在两个突变（P104L和A119T）。对ENV- 2复制子和重组病毒进行遗传分析表明两个NS4B突变单独具有部分抵抗作用，双突变在哺乳动物细胞中的会具有更强的抵抗作用。此外，我们发现由NS4B突变引起的复制缺陷可以部分通过反式互补方式补救。当一个细胞同时被药敏和抗药病毒感染时，这种互补NS4B的能力可以影响药物的敏感性。机理研究发现，NS4B可以和病毒的NS3解旋酶域相互租用;在抵抗分析中P104L消除了NS3 - NS4B相互作用后两个NS4B中的一个复原了（I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan, J. Gen. Virol. 87:2605-2614, 2006)）。总的来说，这些结果表明所鉴定出的抑制剂能靶向登革病毒NS4B蛋白，并导致病毒RNA合成的缺陷。
