Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.
Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.
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登革热病毒蛋白酶表达质粒的构建和体外蛋白酶试验筛选抗病毒抑制剂。
DOI：
10.1007/978-1-4939-0348-1_21
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Padmanabhan,Radhakrishnan
中科院分区：
文献类型：
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作者：
Lai,Huiguo;Teramoto,Tadahisa;Padmanabhan,Radhakrishnan
登革病毒血清型1-4型(DENV1-4)是具有全球意义的蚊媒人类病原体，每年在全世界造成约3.9亿例病例。病毒感染一般会引起一种自限性疾病，称为登革热，但有时也会导致更严重的形式，特别是在二次感染、登革出血热和登革休克综合征期间，每年造成约25,000人死亡。DENV基因组含有单链正义RNA，全长约11kb。5‘端具有I型帽状结构。3‘端没有Poly(A)尾巴。病毒RNA有一个长的开放阅读框架，由宿主翻译机制翻译产生多蛋白前体。多蛋白前体的加工由细胞蛋白酶共翻译，由内质网(ER)中的病毒丝氨酸蛋白酶翻译后产生3种结构蛋白(衣壳(C)、前体膜(PRM)和包膜(E))和7种非结构(NS)蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)。NS2B是内质网中的一个完整的膜蛋白，NS3的N端部分(180个氨基酸残基)含有类胰蛋白酶丝氨酸蛋白酶结构域，具有H51、D75和S135催化三联体。NS3的C-末端有~170-618个氨基酸残基，编码一个NTPase/RNA解旋酶和5‘-RNA三磷酸酶活性，后者是5’-封顶的第一步所必需的。病毒蛋白的切割位点由两个碱性氨基酸残基KR、RR或QR组成，其次是短链氨基酸残基G、S或T。由于病毒复制酶对多蛋白的切割是病毒复制酶组装必不可少的，因此阻断NS2B/NS3Pro的活性为设计登革病毒小分子药物提供了一种有效的手段。本文介绍了DENV2蛋白水解酶小分子抑制剂的筛选。
Dengue virus serotypes 1–4 (DENV1–4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5′-end has a type I cap structure. The 3′-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170–618 amino acid residues, encodes an NTPase/RNA helicase and 5′-RNA triphosphatase activities; the latter enzyme is required for the first step in 5′-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.