IDENTIFICATION OF ATTENUATING MUTATIONS IN THE DENGUE VIRUS GENOME

该项目的目标是识别非致命性突变 DEN病毒基因组，当被整合到有感染性的cDNA中时 克隆(项目#Z01 BK 07001-02)将降低毒力。(1)核能 登革病毒衣壳(C)蛋白的定位。我们检测到了 在羧基末端存在核定位信号(NLS) 产生了40个氨基酸(AA)的DEN4 C替换突变 在40AA结构域的可疑NLSS内，以及它们的核运输 进行了评估。只有一个突变体，其中所有(4)个可能的NLS都是 消除的似乎在核运输中发生了变化，这表明 黄病毒复制中依赖于核的步骤，这可能是 通过将选定的突变掺入到有感染性的cDNA中进行测试。(2) 登革热病毒C的泛素化这项工作已经完成。膜 C的整合似乎稳定了分子对抗泛素- 依赖降级(准备中的手稿)。(3)膜集成 黄病毒C含有保守的内部20-aa疏水性。 域。我们演示了该域作为信号锚的功能 (SA)介导C的膜整合。该SA的功能是 对于有效识别PRM信号肽是必不可少的，它位于 紧接在C下游的黄病毒多蛋白。(4)效果 DEN NS2B蛋白突变对NS3酶活性的影响。至 进一步表征NS2B对蛋白酶活性的要求 在病毒编码的蛋白酶NS3中，我们突变了NS2B中的一个40-AA结构域 蛋白水解酶活性不可缺少的物质。&gt；60个突变体，并 在体内和体外分析了NS2B-NS3位点的自切作用。 在NS2B的40-AA结构域中，5个氨基酸中有3个在黄病毒中保守 对于乳沟来说是绝对必要的。三个额外的半保守 AAS也是必需的。一些突变导致了部分缺陷 切割，从而定义了引入的潜在位置 减弱登革热基因组中的突变(手稿在准备中)。(5) NS1-NS2a位点的裂解。已知这种卵裂发生在 保守的基序，是ER居民进行切割的共识位置 蛋白水解酶、信号肽(SP)。然而，主题不会出现在 信号肽的上下文，识别信号肽的要求 主题由SP提供。切割需要NS1的羧基末端8个氨基酸和 NS2A70%的氨基末端。今年，我们展示了乳沟 在含有ER及其受体的微粒体存在的情况下，体外发生 居民的抗议活动。ER耗尽了可溶的腔内酶 仍然具有切割活性，表明切割酶是内质网 膜结合的。假设NS1-NS2A实体本身是一个 通过选择性地诱变所有符合以下条件的原子来研究自身蛋白酶 可能需要在已知类型的活动站点 蛋白酶。结果表明，NS1-NS2a不是一种自身蛋白水解酶 SP以一种新的方式催化NS1-NS2A的切割。

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.