IMMUNOGENICITY OF DENGUE TYPE 4 VIRUS NONSTRUCTURAL PROTEIN NS1

本实验研究了登革病毒非结构蛋白NS 1的体外加工过程， 体外转录/翻译，以及重组体表达的体内 痘苗病毒在体外，制备全长NS 1-NS 2A前体， 转移到狗胰腺微粒体膜上并糖基化， 但不发生NS 1/NS 2A切割。在体内，布雷菲德菌素A阻断分泌 但不抑制NS 1/NS 2A切割。综合来看，这些结果 这表明NS 1/NS 2A切割发生在高尔基体中，或在隔室中 在ER和高尔基体之间。我们构建了牛痘重组病毒 表达嵌合preM-NS 1（3/8）-NS 2A蛋白，仅含有3或8个 NS 1的C-末端氨基酸残基。我们观察到， NS 1的残基在NS 1-NS 2A连接处被切割。那么唯一的 NS 1/NS 2A切割所需的NS 1部分是含有 8个C-末端氨基酸。类似NS 5-NS 1（3/8）-NS 2A嵌合体的结果 显示这些蛋白质被部分切割到大约相同的程度。 这些结果表明，切割效率和NS 1靶大小是 当NS 1-NS 2A连接不移位到ER中时，两者都降低。

We studied the processing of dengue virus nonstructural protein NS1 by in vitro transcription/translation, and in vivo as expressed by recombinant vaccinia virus. In vitro, the full length NS1-NS2A precursor was made, and was translocated into dog pancreas microsomal membranes and glycosylated, but NS1/NS2A cleavage did not occur. In vivo, brefeldin A blocked secretion of NS1 but did not inhibit NS1/NS2A cleavage. Taken together, these results suggest that NS1/NS2A cleavage occurs in the Golgi, or in a compartment between the ER and the Golgi. We constructed vaccinia recombinant viruses expressing chimeric preM-NS1(3/8)-NS2A proteins, containing only the 3 or 8 C-terminal amino acid residues of NS1. We observed that the chimera with 8 residues of NS1 was cleaved at the NS1-NS2A junction. Thus, the only portion of NS1 required for NS1/NS2A cleavage is the region containing the 8 C-terminal amino acids. Results with analogous NS5-NS1(3/8)-NS2A chimeras showed that these proteins were partially cleaved to about the same extent. These results suggest that the cleavage efficiency and NS1 target size are both reduced when the NS1-NS2A junction is not translocated into the ER.