IDENTIFICATION OF ATTENUATING MUTATIONS IN THE DENGUE VIRUS GENOME

该项目的目标是识别非致命性突变， 登革病毒基因组，当其被整合到感染性cDNA中时， 克隆（项目编号Z 01 BK 07001-02）将降低毒力。(1)核 登革病毒衣壳（C）蛋白的定位。 法检测 羧基端存在核定位信号（NLS） DEN4 C. 替代突变产生了 在40 aa结构域的可疑NLS内， 进行了评估。 只有一个突变体，其中所有（4）可能的NLS已被 在核运输中似乎发生了改变，这表明 黄病毒复制中的核依赖性步骤， 通过将选定的突变掺入感染性cDNA进行测试。（二） 登革病毒C. 这项工作已经完成。 膜 C的整合似乎稳定了分子对泛素的抵抗， 依赖性降解（手稿准备中）。 (3)膜集成 梭 黄病毒C含有一个保守的内部20-aa疏水区， 域 我们证明了这个域作为信号锚的功能 (SA)介导C. 此SA的功能是 对于有效识别prM信号肽至关重要， 在黄病毒多聚蛋白中紧邻C的下游。 (4)效果 DEN NS 2B蛋白突变对NS 3蛋白酶活性的影响。 到 进一步表征了NS 2B对蛋白酶活性的要求， 我们对病毒编码蛋白酶NS 3的NS 2B进行了40个氨基酸的结构域的诱变， 是蛋白酶活性不可缺少的。 分离出>60个突变体，并且 在体内和体外分析NS 2B-NS 3位点的自切割。 黄病毒NS 2B 40-aa结构域中5个保守氨基酸中的3个 是乳沟的必备条件 三个额外的半保守 还需要AAS。 一些突变导致了部分缺陷， 切割，从而确定引入 登革病毒基因组中的减毒突变（手稿在准备中）。 （五） NS 1-NS 2A位点的切割。 已知这种分裂发生在 保守基序，其是ER驻留酶切割的共有位点 蛋白酶、信号肽酶（SP）。 然而，这一主题并没有出现在 信号肽的背景，识别信号肽的要求， 切割需要NS 1的羧基末端8个氨基酸， NS 2A的氨基端70%。 今年我们展示了乳沟 在含有ER及其受体的微粒体存在下， 常驻蛋白酶。 可溶性管腔内酶的ER耗竭， 仍然具有切割活性，表明切割酶是ER 膜结合的 假设NS 1-NS 2A实体本身是一个 通过选择性诱变所有 可能需要在已知类型的活性位点， 蛋白酶 结果表明NS 1-NS 2A不是一种自身蛋白酶， SP以一种新的方式催化NS 1-NS 2A裂解。

The goal of this project is the identification of nonlethal mutations in the DEN virus genome which when incorporated into the infectious cDNA clone (project# Z01 BK 07001-02) will reduce virulence. (1) Nuclear localization of the dengue virus capsid (C) protein. We detected the presence of a nuclear localization signal (NLS) in the carboxy-terminal 40 amino acids (aa) of the DEN4 C. Substitution mutations were created within suspected NLSs in the 40 aa domain, and their nuclear transport was assessed. Only a mutant in which all (4) possible NLSs had been eliminated appeared to be altered in nuclear transport, suggesting nucleus-dependent steps in the replication of flaviviruses which can be tested by incorporation of selected mutations into infectious cDNA. (2) Ubiquitination of the dengue virus C. This work was completed. Membrane integration of C appeared to stabilize the molecule against ubiquitin- dependent degradation (manuscript in prep). (3) Membrane integration of C. The flavivirus C contains a conserved internal 20-aa hydrophobic domain. We demonstrated that this domain functions as a signal-anchor (SA) to mediate the membrane integration of C. Function of this SA is essential for efficient recognition of the prM signal peptide, which lies immediately downstream from C in the flavivirus polyprotein. (4) Effect of mutations in the DEN NS2B protein on protease activity of NS3. To further characterize the requirement of NS2B for the protease activity of the virus-coded protease, NS3, we mutagenized a 40-aa domain in NS2B indispensable for protease activity. >60 mutants were isolated, and autocleavage at the NS2B-NS3 site was analyzed in vivo and in vitro. Three of 5 aas conserved among flaviviruses in the 40-aa domain of NS2B were absolutely required for cleavage. Three additional semi-conserved aas were also required. Some mutations resulted in partial defects in cleavage, thus defining potential sites for the introduction of attenuating mutations into the dengue genome (manuscript in prep). (5) Cleavage of the NS1-NS2A site. This cleavage is known to occur after a conserved motif which is a consensus site for cleavage by the ER resident protease, signal peptidase (SP). However, the motif does not occur in the context of a signal peptide, a requirement for recognition of the motif by SP. Cleavage requires the carboxy-terminal 8 aa of NS1 and the amino-terminal 70%of NS2A. This year, we demonstrated that cleavage takes place in vitro in the presence of microsomes containing ER and its resident proteases. ER depleted of soluble intralumenal enzymes was still active for cleavage, suggesting that the cleavage enzyme is ER membrane-bound. The hypothesis that the NS1-NS2A entity is by itself an autoprotease was investigated by selectively mutagenizing all aas that could possibly be required at the active site of known types of proteases. Results suggest that NS1-NS2A is not an autoprotease and that NS1-NS2A cleavage is catalyzed by SP in a novel fashion.