GENETIC LOCI RESPONSIBLE FOR GROWTH RESTRICTION OF MOUSE-ADAPTED DENGUE VIRUSES

负责小鼠适应登革热病毒生长限制的基因位点

• 批准号: 2566881
• 负责人: C J LAI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/2566881
• 关键词: dengue virus; gene mutation; laboratory mouse; microorganism culture; virus genetics; virus replication

Serial intracerebral passage of dengue type 1 or type 2 virus in mice was shown previously to select for mouse neurovirulent mutants which also proved to be attenuated for humans. In a similar manner a neurovirulent mutant of DEN4 (strain H241) was selected by serial intracerebral passage in mice. DEN4 H241 neurovirulent (N) replicated less efficiently than its DEN4 H241 parent (P) in simian LLC-MK2 cells. An intratypic DEN4 chimera containing the C-PreM-E structural protein genes from DEN4 H241N also exhibited marked restriction of growth in LLC-MK2 cells. Analysis of virion proteins of DEN4 H241N or its derived C-PreM-E chimera grown in mosquito C6/36 cells indicated that very little M was produced. There is considerable evidence that immature PreM-containing flavivirus virions are less infectious than the mature M-containing virus. For this reason, we constructed a panel of chimeras that contained one or more of the variant amino acids of the mutant C-PreM-E sequence substituting for the corresponding sequence of the parental virus. These chimeras were analyzed to determine if any of these mutations was responsible for inefficient PreM cleavage. Mutants which contained two substitutions in PreM, i. e., Lys165-Glu and Val188-Ala, with or without the Thr81-Ile substitution in C, exhibited reduced PreM cleavage. Reduction of PreM cleavage was also observed for chimeras which contain multiple mutations in E, i.e., Thr434-Ile, Ser435-Pro, and Phe680-Leu, with the exception of the chimera containing Thr434-Ile and Ser435-Pro. Efficient PreM cleavage was required for virus to achieve high level of replication in simian LLC-MK2 cells. Similarly, the genetic basis for the reduced replicative efficiency of the mouse-passaged MD-1 vaccine strain in LLC-MK2 cells compared to its parental DEN1 Hawaii strain was investigated. A combination of three substitutions in E independently caused restriction of replication in simian cells. A single Val207-Ala substitution in PreM was also independently responsible for restriction of replication of MD-1 or its derived chimera in LLC-MK2 cells. This PreM mutation is located immediately downstream of the N-terminus of M, which is cleaved from PreM by the host cell furin enzyme. The Val207-Ala mutation did not affect PreM cleavage in mosquito cells, but this mutation might possibly restrict PreM cleavage in simian cells and thus, be responsible for growth restriction.

登革热 1 型或 2 型病毒在小鼠脑内连续传代 先前显示用于选择小鼠神经毒性突变体，该突变体也 已被证明对人类有减弱作用。 以类似的方式，神经毒力 通过连续脑内传代选择 DEN4 突变体（H241 株） 在小鼠中。 DEN4 H241 神经毒力 (N) 的复制效率低于 其 DEN4 H241 亲本 (P) 在猿猴 LLC-MK2 细胞中。 型内 DEN4 含有来自 DEN4 H241N 的 C-PreM-E 结构蛋白基因的嵌合体 LLC-MK2 细胞的生长也表现出明显的限制。 分析 DEN4 H241N 或其衍生的 C-PreM-E 嵌合体的病毒体蛋白的数量 在蚊子 C6/36 细胞中，表明产生的 M 非常少。那里 大量证据表明，未成熟的含有 PreM 的黄病毒颗粒 与成熟的含 M 病毒相比，其传染性较低。 为此原因， 我们构建了一组嵌合体，其中包含一个或多个 突变体C-PreM-E序列的变体氨基酸取代 亲代病毒的相应序列。 这些嵌合体是 分析以确定是否有任何这些突变导致 PreM 裂解效率低下。 含有两个取代的突变体 PreM，i。例如，Lys165-Glu 和 Val188-Ala，有或没有 Thr81-Ile C 中的取代，表现出 PreM 裂解减少。 减少PreM 还观察到包含多个突变的嵌合体的裂解 在 E 中，即 Thr434-Ile、Ser435-Pro 和 Phe680-Leu，但例外 含有 Thr434-Ile 和 Ser435-Pro 的嵌合体。 高效学前班 病毒需要进行切割才能实现高水平的复制 猿猴 LLC-MK2 细胞。 同样，减少的遗传基础 小鼠传代 MD-1 疫苗株的复制效率 LLC-MK2 细胞与其亲本 DEN1 Hawaii 株相比 调查了。 E 中三个独立取代的组合 导致猿猴细胞的复制受到限制。 单个 Val207-Ala PreM 中的替代也独立地负责限制 MD-1 或​​其衍生嵌合体在 LLC-MK2 细胞中的复制。 这 PreM 突变位于 M N 末端的紧下游， 它被宿主细胞弗林蛋白酶从 PreM 上切割下来。 Val207-丙氨酸 突变并不影响蚊子细胞中的 PreM 裂解，但这 突变可能会限制猿猴细胞中的 PreM 裂解，因此， 负责生长限制。