Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(Pro) of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B
Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(Pro) of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B
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DOI：
10.1074/jbc.272.49.30715
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发表时间：
1997-12-05
影响因子：
4.8
通讯作者：
Padmanabhan, R
中科院分区：
文献类型：
--
作者：
Clum, S;Ebner, KE;Padmanabhan, R
登革2型病毒是一种正链RNA病毒，其多聚蛋白的加工是由宿主的信号肽酶和一种新的丝氨酸蛋白酶家族的双组分病毒蛋白酶NS 2B/NS 3（Pro）在内质网中进行的。利用体外加工系统，我们分别检测了NS 2B/NS 3（Pro）对2B/3和4 B/5位点的顺式和反式切割，共表达NS 2B和NS 3（Pro）的BHK-21细胞裂解物在体外介导4 B/5位点的反式切割，并且蛋白酶活性与膜组分相关。使用偶联的体外转录/翻译系统（TnT）分析NS 2B-NS 3（Pro）和缺失NS 2B的功能性亲水结构域的突变体ndNS 2B-NS 3（Pro），结果表明，在TnT反应中加入微粒体膜的共翻译作用可显著增强2B/3位点的顺式切割，并呈剂量依赖性，在膜存在下合成的NS 2B也促进突变体前体中2B/3位点的反式切割，切割产物NS 2B和NS 3（Pro）是膜结合的。这种膜的要求是由NS 2B的疏水区决定的。删除NS 2B的疏水区，只留下40个氨基酸的保守亲水结构域，导致在没有微粒体膜的情况下2B-3位点的体外高效加工。
Polyprotein processing of dengue virus type 2, a positive strand RNA virus, is carried out by the host signal peptidase and a novel two-component viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum, Using an in vitro processing system, we examined the cis and trans cleavages of the 2B/3 and 4B/5 sites by NS2B/NS3(Pro), respectively, Lysates of BHK-21 cells coexpressing NS2B and NS3(Pro) mediated trans cleavage of the 4B/5 site in vitro, and the protease activity was associated with the membrane fraction, To study the role of membranes in the protease activity of NS2B/NS3(Pro), labeled precursors, NS2B-NS3(Pro) and the mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of NS2B was deleted, were analyzed using a coupled in vitro transcription/translation system (TnT), The results showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site in a dose-dependent manner, NS2B synthesized in the presence of membranes also facilitated trans cleavage of the 2B/3 site in the mutant precursor, The cleavage products, NS2B and NS3(Pro), were membrane-associated, Furthermore, this membrane requirement was dictated by the hydrophobic regions of NS2B, Deletion of hydrophobic regions of NS2B, leaving only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 site in vitro in the absence of microsomal membranes.