Echeverri A, Banerjee R, Dasgupta A
Department of Microbiology and Immunology, The University of California, Los Angeles, School of Medicine, 90095, USA.
Virus Res. 1998 Apr;54(2):217-23. doi: 10.1016/s0168-1702(98)00016-1.
The poliovirus-encoded, membrane associated polypeptide 2C is required for viral replication. We have previously established that, while the 2C protein lacks a defined membrane binding domain, the N-terminal region containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In order to determine whether the N-terminal region is sufficient for membrane binding, we have made fusion constructs between this region of 2C (amino acids 1-72 and 1-88) and a soluble protein, chloramphenicol acetyltransferase (CAT). The ability of CAT and the fusion polypeptides to bind to membranes was examined by in vitro translation in the presence of microsomal membrane. While CAT was found in the soluble fraction, both 2C/CAT fusion constructs (1-72/CAT and 1-88/CAT) were membrane associated, suggesting that the N-terminal region of 2C was sufficient to impart membrane binding. To confirm these results in vivo, CAT, 1-72/CAT, and 1-88/CAT were expressed in HeLa cells and their localization was examined using indirect immunofluorescence. Results presented here demonstrate that, while CAT is expressed throughout the cell, 1-72/CAT and 1-88/CAT constructs are capable of localizing to the endoplasmic reticulum (ER) area in transfected cells in the absence of other poliovirus proteins. These results suggest that the first 72 amino acids of 2C contain a membrane binding domain that is capable of targeting soluble proteins to the ER region of the cell.
脊髓灰质炎病毒编码的膜相关多肽2C是病毒复制所必需的。我们之前已经确定，虽然2C蛋白缺乏明确的膜结合结构域，但包含推定两亲性螺旋的N端区域在体内和体外的膜结合中都起着重要作用。为了确定N端区域是否足以实现膜结合，我们构建了2C的该区域（氨基酸1-72和1-88）与可溶性蛋白氯霉素乙酰转移酶（CAT）之间的融合构建体。通过在微粒体膜存在下进行体外翻译，检测了CAT和融合多肽与膜结合的能力。虽然在可溶部分中发现了CAT，但两种2C/CAT融合构建体（1-72/CAT和1-88/CAT）都与膜相关，这表明2C的N端区域足以赋予膜结合能力。为了在体内证实这些结果，在HeLa细胞中表达了CAT、1-72/CAT和1-88/CAT，并使用间接免疫荧光检测它们的定位。此处呈现的结果表明，虽然CAT在整个细胞中表达，但在没有其他脊髓灰质炎病毒蛋白的情况下，1-72/CAT和1-88/CAT构建体能够在转染细胞中定位于内质网（ER）区域。这些结果表明，2C的前72个氨基酸包含一个膜结合结构域，该结构域能够将可溶性蛋白靶向到细胞的内质网区域。
