Ypma-Wong M F, Semler B L
Nucleic Acids Res. 1987 Mar 11;15(5):2069-88. doi: 10.1093/nar/15.5.2069.
We describe a completely in vitro system for generating defined poliovirus proteinase mutations and subsequently assaying the phenotypic expression of such mutations. A complete cDNA copy of the entire poliovirus genome has been inserted into a bacteriophage T7 transcription vector. We have introduced proteinase and/or cleavage site mutations into this cDNA. Mutant RNA is transcribed from the altered cDNA template and is subsequently translated in vitro. Employing such a system, we provide direct evidence for the bimolecular cleavage events carried out by the 3C proteinase. We show that specific genetically-altered precursor polypeptides containing authentic Q-G cleavage sites will not act as substrates for 3C either in cis or in trans. We also provide evidence that almost the entire P3 region is required to generate 3C proteinase activity capable of cleaving the P1 precursor to capsid proteins. However, only the 3C portion of P3 is required to generate 3C proteinase activity capable of cleaving P2 and its processing products.
我们描述了一种完全体外系统，用于产生特定的脊髓灰质炎病毒蛋白酶突变，随后分析此类突变的表型表达。脊髓灰质炎病毒全基因组的完整cDNA拷贝已被插入噬菌体T7转录载体。我们已将蛋白酶和/或切割位点突变引入该cDNA。突变RNA从改变的cDNA模板转录而来，随后在体外进行翻译。利用这样的系统，我们为3C蛋白酶进行的双分子切割事件提供了直接证据。我们表明，含有真实Q-G切割位点的特定基因改变的前体多肽，无论是顺式还是反式，都不会作为3C的底物。我们还提供证据表明，几乎整个P3区域是产生能够切割衣壳蛋白P1前体的3C蛋白酶活性所必需的。然而，产生能够切割P2及其加工产物的3C蛋白酶活性仅需要P3的3C部分。
