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

登革1型或2型病毒在小鼠脑内连续传代， 先前显示选择小鼠神经毒性突变体， 对人类来说是减毒的 以类似的方式， 通过连续脑内传代，选择DEN 4突变株（H241株 对小鼠 DEN 4 H241神经毒性（N）复制效率低于 其DEN 4 H241亲本（P）在猿猴LLC-MK2细胞中。 一种内源性DEN 4 含有来自DEN 4 H241 N的C-PreM-E结构蛋白基因的嵌合体 在LLC-MK2细胞中也表现出明显的生长限制。 分析 DEN 4 H241 N或其衍生的C-PreM-E嵌合体的病毒体蛋白的生长 在蚊子C6/36细胞中，表明产生非常少的M。那里 有相当多的证据表明，不成熟的含PreM的黄病毒病毒粒子 比成熟的含M病毒传染性小 基于这个原因， 我们构建了一组嵌合体， 突变体C-PreM-E序列的变体氨基酸取代 亲本病毒的相应序列。 这些嵌合体 分析以确定这些突变中的任何一个是否与 低效的PreM切割。 突变体中含有两个取代， 普雷姆岛例如，Lys 165-Glu和Val 188-Ala，有或没有Thr 81-Ile C中的取代，表现出减少的PreM切割。 PreM的减少 在含有多个突变的嵌合体中也观察到切割 在E中，即，Thr 434-Ile、Ser 435-Pro和Phe 680-Leu，但 含Thr 434-Ile和Ser 435-Pro的嵌合体。 高效PreM 病毒需要切割才能实现高水平的复制， 猴LLC-MK2细胞。 同样，减少的遗传基础 小鼠传代MD-1疫苗株在 LLC-MK 2细胞与其亲本DEN 1夏威夷株相比， 研究了 E中三个取代的组合独立地 导致猿猴细胞的复制受到限制。 单个Val 207-Ala PreM中的取代也独立地负责限制 MD-1或其衍生的嵌合体在LLC-MK2细胞中复制。 这 PreM突变位于M的N-末端的紧下游， 其被宿主细胞弗林蛋白酶从PreM上切割下来。 Val207-Ala 突变并不影响蚊子细胞中的PreM切割，但这一突变并不影响蚊子细胞中的PreM切割。 突变可能会限制猴细胞中的PreM切割， 负责限制增长。

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.