Niyomrattanakit Pornwaratt, Winoyanuwattikun Pakorn, Chanprapaph Santad, Angsuthanasombat Chanan, Panyim Sakol, Katzenmeier Gerd
Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phutthamonthon 4 Rd., Nakornpathom 73170, Thailand.
J Virol. 2004 Dec;78(24):13708-16. doi: 10.1128/JVI.78.24.13708-13716.2004.
Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Phix(3)Phi motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a k(cat)/K(m) value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k(cat) values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.
登革病毒多聚蛋白的蛋白水解加工由宿主细胞蛋白酶和病毒编码的NS2B-NS3双组分蛋白酶介导。NS3蛋白酶是开发抗病毒抑制剂的一个有吸引力的靶点。NS3蛋白酶结构域的三维结构已被确定，但NS2B辅因子激活该酶所需的结构决定因素仅在有限程度上得到了表征。为了测试最近提出的Phix(3)Phi基序在NS3蛋白酶激活中可能的功能作用，我们通过位点特异性诱变靶向NS2B辅因子内的六个残基。将NS2B中的色氨酸62、丝氨酸71、亮氨酸75、异亮氨酸77、苏氨酸78和异亮氨酸79残基替换为丙氨酸，此外，还产生了L75A/I79A双突变体。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析NS2B/NS3位点的自蛋白水解切割以及用荧光肽底物GRR-AMC测定酶活性，在体外分析了这些突变对NS2B(H)-NS3pro蛋白酶活性的影响。与野生型相比，L75A、I77A和I79A突变体表现出自蛋白水解效率低下，而在W62A和L75A/I79A突变体中，自我切割似乎几乎完全被消除。除了S71A突变体对GRR-AMC肽的k(cat)/K(m)值与野生型相似外，所有其他突变体的k(cat)值都大幅降低。这些结果表明，NS2B辅因子中保守残基色氨酸62、亮氨酸75和异亮氨酸79在登革病毒NS3丝氨酸蛋白酶的结构激活中起关键作用。
