PURIFICATION AND CHARACTERIZATION OF POLIOVIRUS POLYPEPTIDE 3CD, A PROTEINASE AND A PRECURSOR FOR RNA-POLYMERASE
PURIFICATION AND CHARACTERIZATION OF POLIOVIRUS POLYPEPTIDE 3CD, A PROTEINASE AND A PRECURSOR FOR RNA-POLYMERASE
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DOI：
10.1128/jvi.66.12.7481-7489.1992
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发表时间：
1992-12-01
影响因子：
5.4
通讯作者：
WIMMER, E
中科院分区：
文献类型：
--
作者：
HARRIS, KS;REDDIGARI, SR;WIMMER, E
利用T7表达系统在细菌中表达了一个编码脊髓灰质炎病毒2型（萨宾株）3CD蛋白酶（3CD(pro)）的cDNA克隆，3CD(pro)是蛋白酶3C(pro)和RNA聚合酶3D(pol)的前体。对3C/3D切割位点进行了定点诱变，以产生自身加工为3C(pro)和3D(pol)能力受损的有活性的蛋白水解前体。在这些突变中，将3C/3D切割位点P4位置的苏氨酸残基替换为赖氨酸残基（3CD(pro) T181K），产生的突变多肽自加工量最少。该突变体被纯化至86%的纯度，并用于后续的蛋白水解研究。纯化的3CD(proM)（M表示切割位点突变体3CD(pro) T181K）能够切割P1衣壳前体、一个代表2BC切割位点的肽段以及2BC前体多肽。在衣壳前体加工实验中，纯化的3CD(proM)表现出与使用P1和脊髓灰质炎病毒感染的HeLa细胞裂解物粗提物所观察到的相同的去污剂敏感性。纯化的3CD(proM)没有可检测到的RNA聚合酶活性，而在纯化的最后一步通过凝胶过滤从3CD(proM)中分离出的3D(pol)则有活性。我们得出结论，3CD(proM)能够加工脊髓灰质炎病毒多聚蛋白的结构和非结构前体，并且对合成肽底物有活性。此外，3CD切割为3D(pol)是激活3D RNA聚合酶所必需的。
A cDNA clone encoding the 3CD proteinase (3CD(pro)) of poliovirus type 2 (Sabin), the precursor to proteinase 3C(pro) and RNA polymerase 3D(pol), was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3C(pro) and 3D(pol). Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CD(pro) T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CD(proM) (M designates the cleavage site mutant 3CD(pro) T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CD(proM) demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CD(proM) did not have any detectable RNA polymerase activity, whereas 3D(pol), separated from 3CD(proM) by gel filtration in the last step of purification, did. We conclude that 3CD(proM) can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3D(pol) is needed to activate the 3D RNA polymerase.