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 后来，以类似的方式， DEN 4（菌株H241）通过连续脑内传代选择， 小鼠 此外，DEN 4 H241神经毒力（N）复制较少 在猿猴LLC-MK2细胞中，比DEN 4 H241亲本（P）更有效。 一个 含有C-PreM-E结构蛋白基因的胞内DEN 4嵌合体 来自DEN 4 H241 N的菌株也表现出对LLC-MK2生长的显著限制 细胞 通过DEN 4分析LLC-MK2细胞中产生的病毒蛋白 H241 N或其衍生的C-PreM-E嵌合体表明， 生产和检测迁移略慢于 DEN 4 H241 P或其嵌合衍生物的PreM。 最近的证据表明 未成熟的含PreM的黄病毒病毒粒子的感染性低于 成熟的含M病毒 进行研究以确定 PreM的改变或C或E的突变是否会影响正常 处理PreM，产生通常占主导地位的M 在成熟病毒体中的产物。 蛋白质分析表明，DEN 4 E， 在DEN 4 H241 P或其衍生物的病毒体制备物中检测到PreM、M和C。 衍生嵌合体。 另一方面，DEN 4 H241 N的病毒体制备物 或其衍生的嵌合体含有E、PreM和C，但不含M。 这 提示，对于DEN 4 H241 N，PreM至M的裂解是缺陷的， 在C-PreM-E基因内映射的缺陷的遗传基因座。 有 在结构蛋白基因区域的六个氨基酸的差异， DEN 4 H241 P和DEN 4 H241 N：C中一个，PreM中两个，E中三个。 到 鉴定导致缺陷性PreM切割的突变，8个突变体 构建自胞内DEN 4 P嵌合病毒， 存在于突变体C中的一个或多个氨基酸取代， PreM或E. 只有突变体DEN 4（H241 P，S456），所有三个氨基酸都被转化为DEN 4（H241 P，S456）。 在DEN 4 N的E中存在的酸取代被引入， PreM分裂缺陷。 有趣的是， 在PreM正常加工的PreM中含有这两种突变。 这表明 PreM在病毒成熟过程中与E相互作用， 后者可以对前者蛋白质的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.