Leblois H, Young P R
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Australia.
J Gen Virol. 1995 Apr;76 ( Pt 4):979-84. doi: 10.1099/0022-1317-76-4-979.
A series of recombinant baculoviruses was constructed in order to study the influence of downstream NS2A sequences on the processing of the dengue virus NS1 glycoprotein in insect cells. NS1 alone was expressed at a high level in its native dimeric form and processed efficiently through the Spodoptera frugiperda (Sf) cell secretory pathway. Recombinant NS1 was found associated with the plasma membrane and was also secreted into the extracellular medium. Although both intra- and extracellular NS1 were processed to an endo H-resistant form in Sf cells, Triton X-114 phase separation analysis further suggested that some modifications in addition to dimerization account for the hydrophobic properties of NS1, and that N-glycosylation was therefore not the only difference between the cell-associated and secreted forms. Cleavage at the NS1-NS2A junction of these recombinants demonstrated that as few as 26 amino acids from the N terminus of NS2A provide a sufficient, but not optimal, recognition sequence for a functional cleavage mediated by a protease present in Sf cells infected with recombinant Autographa californica nuclear polyhedrosis virus expressing NS1.
为了研究下游NS2A序列对登革病毒NS1糖蛋白在昆虫细胞中加工过程的影响，构建了一系列重组杆状病毒。单独的NS1以其天然二聚体形式高水平表达，并通过草地贪夜蛾（Sf）细胞分泌途径有效加工。发现重组NS1与质膜相关，并且也分泌到细胞外培养基中。尽管细胞内和细胞外的NS1在Sf细胞中均被加工成耐内切糖苷酶H的形式，但Triton X-114相分离分析进一步表明，除二聚化外的一些修饰导致了NS1的疏水特性，因此N-糖基化不是细胞相关形式和分泌形式之间的唯一差异。这些重组体在NS1-NS2A连接处的切割表明，来自NS2A N端的少至26个氨基酸为感染表达NS1的重组苜蓿银纹夜蛾核型多角体病毒的Sf细胞中存在的蛋白酶介导的功能性切割提供了足够但非最佳的识别序列。
