PROCESSING AND IMMUNOGENICITY OF DENGUE TYPE 4 VIRUS NONSTRUCTURAL PROTEIN NS1

我们实验室以前的结果表明， NS 1-NS 2A需要NS 2A和NS 1的C-末端8个氨基酸 乳沟 为了进一步阐明NS 1-NS 2A切割的要求，我们 构建的表达嵌合蛋白的重组牛痘病毒 由融合到NS 1+的3或8个C-末端氨基酸的PreM组成 NS2A。 结果表明，NS 1除8个外，其余均为阴性。 氨基酸切割位点，对于NS 1-NS 2A切割是不稳定的。 此外， 作为嵌合蛋白，需要信号序列来发生切割 Sig PreM-NS 1（8）-NS 2A未被切割。 这表明，针对 胞吐途径是NS 1-NS 2A切割的先决条件。 额外 进行实验以分析这种切割。 Brefeldin A，一种药物 其阻断高尔基体外的转运，但不阻断NS 1/NS 2A切割。 这表明，NS 1/NS 2A切割发生在高尔基体或在一个细胞中。 在ER和高尔基体之间的隔室。 研究了NS 1/NS 2A切割 在阻断内质网向高尔基体转运的条件下的追逐过程中： 与药物羰基氰化物间氯苯腙（CCCP）孵育， 或在14 ℃下孵育。 这两种治疗方法都完全阻断了 将未裂解的NS 1-NS 2A前体追赶成NS 1产物。 这是一致 认为NS 1/NS 2A裂解发生在从ER退出之后。 进一步阐明的裂解机制，现在可能使用在 体外ER到高尔基体转运系统最近由他人开发。

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.