IDENTIFICATION OF ATTENUATING MUTATIONS IN THE DENGUE VIRUS GENOME

登革热病毒基因组减毒突变的鉴定

• 批准号: 3748171
• 负责人: B FALGOUT
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3748171
• 关键词: attenuated microorganism; complementary DNA; dengue virus; endopeptidases; gene mutation; genome; intracellular transport; membrane structure; nucleocapsid; protein sequence; protein signal sequence; protein transport; ubiquitin; virulence; virus RNA; virus genetics; virus infection mechanism; virus protein; virus replication

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.

该项目的目标是鉴定非致命突变 DEN 病毒基因组，当掺入感染性 cDNA 时 克隆（项目# Z01 BK 07001-02）将降低毒力。 (1) 核电 登革热病毒衣壳 (C) 蛋白的定位。 我们检测到 羧基末端存在核定位信号 (NLS) DEN4 C 的 40 个氨基酸 (aa)。创建了替代突变 40 个氨基酸域中可疑的 NLS 及其核运输 被评估。 只有一个突变体，其中所有（4）个可能的 NLS 都已被 消除的核运输似乎发生了改变，表明 黄病毒复制过程中的核依赖性步骤 通过将选定的突变掺入感染性 cDNA 中进行测试。 (2) 登革热病毒C的泛素化。这项工作已完成。 膜 C的整合似乎可以稳定该分子，对抗泛素- 依赖性降解（准备中的手稿）。 (3)膜整合 黄病毒 C 含有保守的内部 20-aa 疏水性 领域。 我们证明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 信号肽至关重要，该信号肽位于 紧邻黄病毒多蛋白中C的下游。 (4)效果 DEN NS2B 蛋白突变对 NS3 蛋白酶活性的影响。 到 进一步表征 NS2B 对蛋白酶活性的要求 对于病毒编码的蛋白酶 NS3，我们诱变了 NS2B 中的 40 个氨基酸结构域 蛋白酶活性所必需的。 分离出 >60 个突变体，并且 在体内和体外分析了 NS2B-NS3 位点的自动切割。 NS2B 40-aa 结构域中 5 个氨基酸中的 3 个在黄病毒中保守 绝对需要裂解。 另外三个半保守 还需要 aas。 一些突变导致部分缺陷 裂解，从而确定引入的潜在位点 减弱登革热基因组突变（准备中的手稿）。 (5) NS1-NS2A 位点的裂解。 已知这种裂解发生在 保守基序，是 ER 驻留者切割的共有位点 蛋白酶、信号肽酶(SP)。 然而，该主题并不出现在 信号肽的背景，识别的要求 SP 的主题。 切割需要 NS1 的羧基末端 8 个氨基酸和 NS2A 的氨基末端 70%。 今年，我们证明了分裂 在含有 ER 的微粒体存在的情况下在体外发生 常驻蛋白酶。 耗尽可溶性腔内酶的 ER 仍然具有裂解活性，表明裂解酶是 ER 膜结合。 NS1-NS2A 实体本身就是一个假设 通过选择性诱变所有氨基酸来研究自体蛋白酶 可能需要在已知类型的活性位点 蛋白酶。 结果表明 NS1-NS2A 不是自体蛋白酶并且 NS1-NS2A 裂解由 SP 以一种新颖的方式催化。