EVIDENCE THAT THE N-TERMINAL DOMAIN OF NONSTRUCTURAL PROTEIN NS3 FROM YELLOW-FEVER VIRUS IS A SERINE PROTEASE RESPONSIBLE FOR SITE-SPECIFIC CLEAVAGES IN THE VIRAL POLYPROTEIN
EVIDENCE THAT THE N-TERMINAL DOMAIN OF NONSTRUCTURAL PROTEIN NS3 FROM YELLOW-FEVER VIRUS IS A SERINE PROTEASE RESPONSIBLE FOR SITE-SPECIFIC CLEAVAGES IN THE VIRAL POLYPROTEIN
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DOI：
10.1073/pnas.87.22.8898
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发表时间：
1990-11-01
影响因子：
11.1
通讯作者：
RICE, CM
中科院分区：
文献类型：
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作者：
CHAMBERS, TJ;WEIR, RC;RICE, CM
序列同源性和分子模型研究表明，黄病毒非结构蛋白 NS3 的 N 末端三分之一充当胰蛋白酶样丝氨酸蛋白酶。为了检查 NS3 的假定蛋白水解活性，将黄热病病毒基因组片段亚克隆到质粒转录/翻译载体中，并对无细胞翻译产物进行了表征。结果表明，黄热病病毒 NS3 的 NS2B 和 N 端三分之一内编码的蛋白酶活性能够对 NS2B-NS3 切割位点进行顺式作用位点特异性蛋白酶解，并对 NS2A-NS2B 位点进行稀释不敏感切割。对组成所提出的催化三联体的 NS3 的 His-53、Asp-77、adn sSer-138 残基进行定点诱变表明该结构域是丝氨酸蛋白酶。用含有 Ser-138 突变（在体外消除或显着降低蛋白酶活性）的全长黄热病病毒 cDNA 模板转录的 RNA 转染的哺乳动物细胞中未回收感染性病毒，这表明该蛋白酶是病毒复制所必需的。
Seqence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavivirus nonstructural protein NS3 functions as a trypsin-like serine protease. To examine the putative proteolytic activity of NS3, segments of the yellow fever virus genome were subcloned into plasmid transcription/translation vectors and cell-free translation products were characterized. The results suggest that a protease activity encoded within NS2B and the N-terminal one-third of yellow fever virus NS3 is capable of cis-acting site-specific protelysis a the NS2B-NS3 cleavage site and dilution-insensitive cleavage of the NS2A-NS2B site. Site-directed mutagenesis fo the His-53, Asp-77, adn sSer-138 residues of NS3 that compose the proposed catalytic triad implicates this domain as a serine protease. Infectious virus was not recovered from mammalian cells transfected with RNAs transcribed from full-length yellow fever virus cDNA templates containing mutations at Ser-138 (which abolish or dramatically reduce protease activity in vitro), suggesting that the protease is required for viral replication.