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

登革1型和2型登革热病毒在小鼠脑内连续传代 先前被证明对人类有减弱这些病毒的作用。登革热型 4株病毒株H2 41也成功地适应在小鼠体内复制 大脑。小鼠适应的DEN4 H241N是高度神经毒力的，而 其亲本DEN4 H241P则不是。此外，DEN4 H241N的复制次数较少 在猴LLC-MK2细胞中比DEN4 H241P有效。一种非典型DEN4 含DEN4 H241N C-Prem-E结构蛋白基因的嵌合体 对LLC-MK2细胞的生长也有明显的抑制作用。分析 DEN4 H241N及其衍生产物在LLC-MK2细胞中产生的病毒蛋白 C-Prem-E嵌合体表明产生的Prem非常少，并且 检测到的迁移速度比DEN4 H241P的PreM略慢 或者它的嵌合衍生品。最近的证据表明，不成熟的 含有PreM的黄病毒的复制效率低于成熟的 含有M的病毒。进行了研究，以确定是否 Prem改变或C或E突变可能会影响正常的加工 在成熟的病毒粒子中通常存在的Prem产生M。蛋白 分析表明，在病毒粒子中检测到DEN4E、Prem、M和C DEN4 H241P或其衍生嵌合体的制备。另一方面， DEN4-H241N及其含E、Prem嵌合体的病毒粒子制备 和C，但未检测到M。这表明Prem to的卵裂 M是DEN4 H241N的缺陷，并定位了该缺陷的遗传基因座 在C-Prem-E基因中。其中有6种氨基酸的差异。 DEN4 H241P和DEN4 H241N：1之间的结构蛋白基因区域 C，Prem的2个和E的3个，以确定导致 Prem裂解缺陷，构建了8个嵌合突变 包含一个或多个氨基酸替换，这些替换存在于 突变C、Prem或E。只有突变株DEN4(H241P、S456)包含所有 E区有3个氨基酸替换出现Prem裂解缺陷。 有趣的是，在Prem中包含这两个突变的嵌合突变 正常处理Prem。这表明Prem与E在 病毒成熟。

Serial intracerebral passage of dengue type 1 and type 2 viruses in mice was shown previously to attenuate these viruses for humans. Dengue type 4 virus strain H241 was also successfully adapted to replicate in mouse brain. The mouse-adapted DEN4 H241N was highly neurovirulent, whereas its parent DEN4 H241P was not. In addition, DEN4 H241N replicated less efficiently than DEN4 H241P 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 viral proteins produced in LLC-MK2 cells by DEN4 H241N or its derived C-PreM-E chimera indicated that very little PreM was produced and that which was detected migrated slightly slower than the PreM of DEN4 H241P or its chimeric derivative. Recent evidence indicates that immature PreM-containing flaviviruses replicate less efficiently than the mature M-containing virus. Studies were performed to determine whether the altered PreM or mutations in C or E might affect the normal processing of PreM to produce M normally present in the mature virion. Protein analysis indicated that DEN4 E, PreM, M and C were detected in the virion preparation of DEN4 H241P or its derived chimera. On the other hand, the virion preparation of DEN4 H241N or its derived chimera contained E, PreM and C, but M was not detected. This suggested that cleavage of PreM to M was defective for DEN4 H241N and the genetic loci for the defect mapped within the C-PreM-E genes. There were six amino acid differences in the structural protein gene region between DEN4 H241P and DEN4 H241N: 1 in C, 2 in PreM and 3 in E. To identify mutations responsible for the defective PreM cleavage, 8 chimeric mutants were constructed that contained one or more amino acid substitutions that are present in the mutant C, PreM or E. Only mutant DEN4(H241P, S456) which contained all three amino acid substitutions in E exhibited the PreM cleavage defect. Interestingly, chimeric mutants which contained both mutations in PreM processed PreM normally. This suggests that PreM interacts with E during virus maturation.