Dewalt P G, Blair W S, Semler B L
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Virology. 1990 Feb;174(2):504-14. doi: 10.1016/0042-6822(90)90104-y.
A mutagenic oligonucleotide cassette was used to introduce single and tandem amino acid substitutions into the proteinase 3C coding region of an infectious poliovirus type 1 cDNA. The sites targeted for mutagenesis, residues 60, 61, and 66, are located within a putative helical loop structure which may be involved in substrate recognition by the enzyme. Fourteen viable 3C proteinase mutants were isolated. A Lys----Arg substitution at position 60 resulted in cold sensitivity for growth at 33 degrees. Replacement of Lys 60 with Ile, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 3D RNA polymerase than wild-type poliovirus. 3C-mediated processing of the remaining sites within the polyprotein was not noticeably affected. The overproduction of 3D is a consequence of more efficient processing of the carboxy-terminal Gln-Gly amino acid pair of 3C. Together with a previous report in which substitution of Val 54 with an Ala residue results in a poliovirus that produces decreased levels of 3D, these observations provide evidence that the putative loop region (residues 51-66) may be a functional domain involved in recognition of the carboxy-terminal Gln-Gly cleavage site of 3C.
使用诱变寡核苷酸盒将单个和串联氨基酸取代引入到1型感染性脊髓灰质炎病毒cDNA的蛋白酶3C编码区。诱变的靶向位点，即第60、61和66位残基，位于一个假定的螺旋环结构内，该结构可能参与该酶对底物的识别。分离出了14个有活力的3C蛋白酶突变体。第60位的赖氨酸突变为精氨酸导致在33℃下生长时对温度敏感。将第60位的赖氨酸单独或与第61位的取代组合替换为异亮氨酸，产生的病毒产生的3D RNA聚合酶比野生型脊髓灰质炎病毒多三到五倍。多聚蛋白中其余位点的3C介导的加工没有受到明显影响。3D的过量产生是3C的羧基末端谷氨酰胺-甘氨酸氨基酸对加工更有效的结果。连同之前一份报告中用丙氨酸残基取代第54位缬氨酸导致脊髓灰质炎病毒产生的3D水平降低，这些观察结果提供了证据，表明假定的环区域（第51-66位残基）可能是参与识别3C羧基末端谷氨酰胺-甘氨酸切割位点的功能域。
