McBride A E, Schlegel A, Kirkegaard K
Department of Molecular, Cellular, and Developmental Biology and Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0347, USA.
Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2296-301. doi: 10.1073/pnas.93.6.2296.
A HeLa cDNA expression library was screened for human polypeptides that interacted with the poliovirus RNA-dependent RNA polymerase, 3D, using the two-hybrid system in the yeast Saccharomyces cerevisiae. Sam68 (Src-associated in mitosis, 68 kDa) emerged as the human cDNA that, when fused to a transcriptional activation domain, gave the strongest 3D interaction signal with a LexA-3D hybrid protein. 3D polymerase and Sam68 coimmunoprecipitated from infected human cell lysates with antibodies that recognized either protein. Upon poliovirus infection, Sam68 relocalized from the nucleus to the cytoplasm, where poliovirus replication occurs. Sam68 was isolated from infected cell lysates with an antibody that recognizes poliovirus protein 2C, suggesting that it is found on poliovirus-induced membranes upon which viral RNA synthesis occurs. These data, in combination with the known RNA- and protein-binding properties of Sam68, make Sam68 a strong candidate for a host protein with a functional role in poliovirus replication.
利用酿酒酵母中的双杂交系统，对一个HeLa细胞cDNA表达文库进行筛选，以寻找与脊髓灰质炎病毒RNA依赖性RNA聚合酶3D相互作用的人类多肽。Sam68（有丝分裂中与Src相关的68 kDa蛋白）作为一种人类cDNA出现，当它与转录激活结构域融合时，与LexA-3D融合蛋白产生最强的3D相互作用信号。3D聚合酶和Sam68可从感染的人类细胞裂解物中用识别任一蛋白的抗体进行共免疫沉淀。脊髓灰质炎病毒感染后，Sam68从细胞核重新定位到细胞质，脊髓灰质炎病毒在细胞质中进行复制。用识别脊髓灰质炎病毒蛋白2C的抗体从感染细胞裂解物中分离出Sam68，这表明它存在于脊髓灰质炎病毒诱导的膜上，病毒RNA合成在该膜上发生。这些数据，结合Sam68已知的RNA和蛋白质结合特性，使Sam68成为在脊髓灰质炎病毒复制中起功能作用的宿主蛋白的有力候选者。
