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 前面显示的是为小鼠神经毒性突变体选择的 对人类来说是衰减的。后来，以类似的方式，一种神经毒性突变体 经连续脑内传代筛选出DEN4菌株(菌株H241)。 老鼠。此外，DEN4 H241神经毒力株(N)复制较少 在猴LLC-MK2细胞中比DEN4-H_241亲本(P)有效。一个 含C-Prem-E结构蛋白基因的异型DEN4嵌合体 来自DEN4的H241N对LLC-MK2细胞的生长也有明显的抑制作用 细胞。DEN4在LLC-MK2细胞中产生的病毒蛋白分析 H241N或其衍生的C-Prem-E嵌合体表明很少的Prem是 产生的和被检测到的迁移速度略慢于 DEN4 H241P或其嵌合衍生物的前缀。最近的证据表明 未成熟的含有Prem的黄病毒粒子的传染性比 成熟的含M病毒。进行了研究以确定 Prem的改变或C或E的突变是否会影响正常 产生通常占主导地位的M的PREM的处理 产物中的成熟病毒粒子。蛋白质分析表明，DEN4E， 在DEN4 H241P或其病毒粒子制剂中检测到Prem、M和C 衍生嵌合体。另一方面，DEN4 H241N的病毒粒子制备 或其衍生的嵌合体包含E、Prem和C，但不包含M。 提示DEN4 H241N的Prem到M的切割是有缺陷的， 该缺陷的遗传位点定位在C-Prem-E基因内。确实有 日本血吸虫结构蛋白基因区6个氨基酸的差异 DEN4 H241P和DEN4 H241N：一个C调、两个PREM调和三个E调至 确定导致Prem裂解缺陷的突变，八个突变体 是由非典型DEN4P嵌合病毒构建而成的 突变体C中存在的一个或多个氨基酸取代， Prem或E.只有突变体DEN4(H241P，S456)，三个氨基酸都变成了 介绍了DEN4N的E中的酸取代反应，并展示了 Prem卵裂缺陷。有趣的是，嵌合突变体 在正常处理的Prem中包含这两种突变。这表明 Prem在病毒成熟过程中与E相互作用，并在 后者可以对前者的蛋白质加工产生深远的影响。

Serial intracerebral passage of dengue type 1 or type 2 virus in mice was shown previously to select for mouse neurovirulent mutants which were attenuated for humans. Later, in a similar manner a neurovirulent mutant of DEN4 (strain H241) was selected by serial intracerebral passage in mice. In addition, DEN4 H241 neurovirulent (N) replicated less efficiently than 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 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 flavivirus virions are less infectious than the mature M-containing virus. Studies were performed to determine whether altered PreM or mutations in C or E might affect the normal processing of PreM that yields M which is normally the predominant product in the mature virion. Protein analysis indicated that DEN4 E, PreM, M and C were detected in a virion preparation of DEN4 H241P or its derived chimera. On the other hand, a virion preparation of DEN4 H241N or its derived chimera contained E, PreM and C, but not M. 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 are six amino acid differences in the structural protein gene region between DEN4 H241P and DEN4 H241N: one in C, two in PreM and three in E. To identify mutations responsible for defective PreM cleavage, eight mutants were constructed from the intratypic DEN4 P chimeric virus that contained one or more amino acid substitutions that are present in the mutant C, PreM or E. Only mutant DEN4(H241P, S456), into which all three amino acid substitutions present in the E of DEN4 N were introduced, 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 and changes in the latter can have a profound effect on processing of the former protein.