PROCESSING AND IMMUNOGENICITY OF DENGUE TYPE 4 VIRUS NONSTRUCTURAL PROTEIN NS1

我们实验室以前的结果证明，70%的N-末端 NS1-NS2A需要NS2A和NS1的C-末端8个氨基酸 乳沟。为进一步说明NS1-NS2A型切割的要求，我们 构建表达嵌合蛋白的重组痘苗病毒 由融合到NS1+的3或8个C-末端氨基酸的Prem组成 NS2A。结果显示，除8个外，所有的NS1都是 氨基酸裂解位点是NS1-NS2a裂解所必需的。此外，还有一个 信号序列是以嵌合蛋白的形式发生切割所必需的 SigPrem-NS1(8)-NS2a未被切割。这表明，针对 胞外途径是NS1-NS2a裂解的先决条件。其他内容 对这种分裂进行了实验分析。Brefeldin A，一种药物 它阻止了高尔基体的运输，并没有阻止NS1/NS2A的切割。 这表明NS1/NS2a裂解发生在高尔基体或 急诊室和高尔基人之间的隔间。对NS1/NS2A的切割进行了研究 在阻止内质网到高尔基体运输的条件下进行追逐： 与药物间氯苯肼(CCCP)孵育， 或在14oC下孵化。这两种治疗方法都完全阻断了 将未裂解的NS1-NS2a前体追逐成NS1产物。这是一致的 认为NS1/NS2A切割发生在退出ER后。 现在可以使用In来进一步阐明切割机制 最近由其他人开发的体外内质网到高尔基体转运系统。

Previous results from our laboratory established that the N-terminal 70% of NS2A and the C-terminal 8 amino acids of NS1 are required for NS1-NS2A cleavage. To further delineate the requirements for NS1-NS2A cleavage, we constructed recombinant vaccinia viruses expressing chimeric proteins consisting of PreM fused to the 3 or 8 C-terminal amino acids of NS1 plus NS2A. The results showed that all of NS1, with the exception of the 8 amino acid cleavage site, is dispensable for NS1-NS2A cleavage. Also, a signal sequence is required for cleavage to occur as the chimeric protein Sig PreM-NS1(8)-NS2A was not cleaved. This suggests that targeting to the exocytic pathway is a prerequisite for NS1-NS2A cleavage. Additional experiments were performed to analyze this cleavage. Brefeldin A, a drug which blocks transport out of the Golgi, did not block NS1/NS2A cleavage. This suggests that NS1/NS2A cleavage occurs in the Golgi or in a compartment between the ER and the Golgi. NS1/NS2A cleavage was studied during a chase under conditions that block ER-to-Golgi transport: incubation with the drug carbonyl cyanide m-chlorophenylhydrazone (CCCP), or incubation at 14oC. Both of these treatments completely blocked the chase of uncleaved NS1-NS2A precursor into NS1 product. This is consistent with the notion that NS1/NS2A cleavage occurs after exit from the ER. Further elucidation of the cleavage mechanism is now possible using an in vitro ER-to-Golgi transport system recently developed by others.