Burns C C, Lawson M A, Semler B L, Ehrenfeld E
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
J Virol. 1989 Nov;63(11):4866-74. doi: 10.1128/JVI.63.11.4866-4874.1989.
A series of short insertion mutations was introduced into the poliovirus gene for 3Dpol at a number of different locations. When substituted for wild-type sequences in a full-length, infectious cDNA and tested for infectivity, all 3D mutants were nonviable. The mutant cDNAs were introduced into a bacterial plasmid designed to direct the expression of poliovirus 3CD, a viral protein composed of contiguous protease and RNA polymerase sequences. Bacteria transformed with these plasmids all expressed similar amounts of 3CD, and all mutant proteins cleaved themselves to generate wild-type 3Cpro and mutant 3Dpol polypeptides with approximately the same efficiency as wild-type 3CD. The released mutant 3Dpol proteins were all defective in RNA-dependent RNA polymerase activity in vitro. Uncleaved 3CD is a protease required for processing the viral capsid protein precursor, P1. In an in vitro assay of P1 cleavage activity, some of the mutant 3CD proteins expressed in Escherichia coli showed normal activity, while others were clearly inactive. Thus, alterations in the sequence and/or folding of different regions of the 3D protein have differential effects on its various activities.
一系列短插入突变被引入脊髓灰质炎病毒3D聚合酶基因的多个不同位置。当在全长感染性cDNA中取代野生型序列并测试其感染性时，所有3D突变体均无活力。将突变的cDNA引入一种细菌质粒，该质粒旨在指导脊髓灰质炎病毒3CD的表达，3CD是一种由连续的蛋白酶和RNA聚合酶序列组成的病毒蛋白。用这些质粒转化的细菌均表达相似量的3CD，并且所有突变蛋白均以与野生型3CD大致相同的效率自我切割，产生野生型3C蛋白酶和突变型3D聚合酶多肽。释放的突变型3D聚合酶蛋白在体外均缺乏依赖RNA的RNA聚合酶活性。未切割的3CD是加工病毒衣壳蛋白前体P1所需的蛋白酶。在P1切割活性的体外测定中，一些在大肠杆菌中表达的突变型3CD蛋白显示出正常活性，而其他一些则明显无活性。因此，3D蛋白不同区域的序列和/或折叠改变对其各种活性具有不同的影响。
