FUNCTIONAL ANALYSIS OF DENGUE VIRUS ANTIGENS NS3 AND NS5

描述（改编自申请人的摘要）：本研究的总体目标 建议是阐明登革病毒抗原NS 3和 NS 5蛋白质加工和基因组复制。 登革热是人类 由蚊子传播的登革热病毒引起的疾病。除了登革热 发烧是一种更严重和致命的疾病，发生在儿童身上。 称为登革出血热或登革休克综合征。一 与发展战略有关的潜在问题 单价登革热疫苗是指感染一种血清型的个体 很容易被其他人感染这种继发性感染 可能导致严重和致命的疾病。的一种方法 控制病毒感染的关键是了解病毒的复制策略， 这样就能研制出抗病毒药物。病毒 抗原NS 3和NS 5是这类研究的有用靶标，因为它们 是高度保守的黄病毒非结构蛋白， 参与蛋白质加工和病毒复制。 NS 3显示 在前180个氨基酸内含有丝氨酸蛋白酶结构域。 与这个结构域相邻的区域， 解旋酶和复制蛋白。 NS 5含有基序Gly-Asp- Asp，其通常存在于几种RNA依赖性RNA聚合酶中。 NS 3 RNA解旋酶是否在起始中起作用的问题 病毒复制的高度保守和稳定的二级 在DEN-2 RNA的3 '-末端发现的结构将被解决。体外 转录本含有相同的序列和极性的DEN-2 RNA， 以及模型双链RNA底物将用于 表征NS 3解旋酶，然后进行NS 3解旋酶的定点诱变。 推定的解旋酶结构域。 NS 5 RNA聚合酶可能 在病毒负链合成中发挥作用。 模型RNA 作为模板/引物的底物将用于表征 NS 5的合成活性。Gly-Asp-Asp的定点突变 NS 5的结构域将进行检查其在RNA中的功能作用 合成.登革热病毒的所有非结构蛋白都已经 使用重组牛痘病毒系统表达。同样地， 所有的结构蛋白都将被表达。 为了分解蛋白质 以及起始和延伸所需的核苷酸序列 复制步骤，缺陷干扰（DI）构建体， 5 '-和3'-非编码RNA基因组将在存在下使用， 结构和非结构蛋白质。 了解多聚蛋白前体的加工机制， 产生成熟蛋白质如NS 3和NS 5， 基因组复制所需的蛋白质。证据 我们的实验室表明，NS 3需要NS 2B才能发挥作用， 蛋白酶在涉及顺式和反式的NS 3-> NS 5前体的加工中的应用 反式裂解NS 2B和NS 3将使用 常规以及亲和柱色谱法。几 含有二元残基的底物类似物，以及蛋白酶 抑制剂将用于表征纯化的NS 2B/NS 3蛋白酶。

DESCRIPTION (Adapted from applicant's abstract): The overall aim of this proposal is to elucidate the role of the dengue viral antigens NS3 and NS5 in protein processing and genome replication. Dengue is a human disease caused by the mosquito-borne dengue virus.Apart from dengue fever, a more severe and fatal form of the disease occurs in children called dengue hemorrhagic fever, or the dengue shock syndrome. A potential problem associated with the strategy for the development of monovalent dengue vaccines is that individuals infected with one serotype are fully susceptible to infection by others. This secondary infection may result in the severe and fatal form of the disease. One approach to control the virus infection is to understand the replication strategy of the virus so that an antiviral agent can be developed. The viral antigens, NS3 and NS5, are useful targets for such studies, because they are highly conserved flavivirus nonstructural proteins, which have been implicated in protein processing and viral replication. NS3 was shown to contain a serine protease domain within the first 180 amino acids. Adjacent to this domain, there are regions of similarity to several RNA helicases, and to replication proteins. NS5 contains the motif Gly-Asp- Asp, which is often found in several RNA-dependent RNA polymerases. The question of whether the NS3 RNA helicase plays a role in initiation of viral replication by melting the highly conserved and stable secondary structure found at the 3'-end of DEN-2 RNA will be addressed. In vitro transcripts containing the same sequence and polarity of DEN-2 RNA, as well as model double-stranded RNA substrates will be used to characterize the NS3 helicase, followed by site-directed mutagenesis of the putative helicase domains. The NS5 RNA polymerase could potentially play a role in the viral negative strand synthesis. Model RNA substrates as template/ primers will be used to characterize the synthetic activity of NS5. Site-directed mutagenesis of the Gly-Asp-Asp domain of NS5 will be carried out to examine its functional role in RNA synthesis. All the nonstructural proteins of dengue virus have already been expressed using the recombinant vaccinia virus system. Similarly, all the structural proteins will be expressed. To dissect the protein and the nucleotide sequence requirements for initiation and elongation steps in replication, defective-interfering (DI) constructs containing the 5'-and 3'-noncoding RNA genome will be used in the presence of structural and nonstructural proteins. Understanding the mechanism of processing of the polyprotein precursors, which generates mature proteins such as NS3 and NS5, is critical to shed light on the protein requirements for genome replication. Evidence from our laboratory suggests that NS2B is required for NS3 to function as a protease in the processing of NS3->NS5 precursor involving both cis and trans cleavages. NS2B and NS3 will be purified to homogeneity using conventional as well as affinity column chromatographic methods. Several substrate analogues containing dibasic residues, as well as protease inhibitors will be used to characterize the purified NS2B/NS3 protease.