Lama J, Paul A V, Harris K S, Wimmer E
Department of Microbiology, School of Medicine, State University of New York at Stony Brook 11794-5222.
J Biol Chem. 1994 Jan 7;269(1):66-70.
The poliovirus-specific polypeptide 3AB (B = VPg) was expressed in Escherichia coli and purified to near homogeneity. Corresponding to its known association with membranes in poliovirus-infected HeLa cells, 3AB expressed in E. coli was also membrane-associated, and it could be solubilized only in detergent-containing buffers. In soluble form, 3AB was resistant to digestion with the virus-specific proteinases 3Cpro and 3CDpro. However, it was cleaved by these enzymes to 3A and VPg when bound to the bacterial membranes, an observation suggesting that 3AB may deliver the genome-linked protein VPg to the membrane-associated poliovirus replication complex. The specific activity of 3CDpro in processing 3AB was significantly higher than that of 3Cpro. Soluble 3AB was found to stimulate nearly 100-fold poly (A)-dependent, primer-dependent poly(U) synthesis, catalyzed by purified poliovirus RNA polymerase 3Dpol. We propose that 3AB has a dual function in poliovirus genome replication: as a precursor for VPg, and as a co-factor for 3Dpol.
脊髓灰质炎病毒特异性多肽3AB（B = VPg）在大肠杆菌中表达并纯化至接近均一。与其在脊髓灰质炎病毒感染的HeLa细胞中与膜的已知关联相对应，在大肠杆菌中表达的3AB也与膜相关，并且仅在含去污剂的缓冲液中可溶解。以可溶形式存在时，3AB对病毒特异性蛋白酶3Cpro和3CDpro的消化具有抗性。然而，当与细菌膜结合时，它会被这些酶切割成3A和VPg，这一观察结果表明3AB可能将基因组连接蛋白VPg递送至与膜相关的脊髓灰质炎病毒复制复合物。3CDpro加工3AB的比活性明显高于3Cpro。发现可溶性3AB可刺激纯化的脊髓灰质炎病毒RNA聚合酶3Dpol催化的近100倍的聚（A）依赖性、引物依赖性聚（U）合成。我们提出3AB在脊髓灰质炎病毒基因组复制中具有双重功能：作为VPg的前体，以及作为3Dpol的辅助因子。
