Echeverri A C, Dasgupta A
Department of Microbiology and Immunology, UCLA School of Medicine 90024, USA.
Virology. 1995 Apr 20;208(2):540-53. doi: 10.1006/viro.1995.1185.
The poliovirus-encoded, membrane-associated polypeptide 2C is required for viral replication. We have expressed 2C, its precursor 2BC, and a number of 2C deletion mutants in eukaryotic (HeLa) cells and examined their localization using indirect immunofluorescence. Results presented here demonstrate that proteins 2C and 2BC are capable of localizing to the endoplasmic reticulum area in transfected cells in the absence of other poliovirus proteins. Additionally, 2C binds tightly to microsomal membranes in a direct in vitro membrane binding assay. Although the 2C protein lacks a defined membrane binding domain, we demonstrate that the N-terminal region encompassing amino acids 21-54 and containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In contrast, most of the C-terminus portion of the protein appears to be unnecessary for membrane association. The susceptibility of membrane-associated 2C to proteinase K digestion in vitro suggests that all or most of 2C is exposed to the cytoplasmic face of the membrane. Furthermore, unlike most proteins targeted to the endoplasmic reticulum, 2C does not appear to be glycosylated or cleaved by signal peptidases. The implication of the membrane binding domain of 2C in viral RNA replication is discussed.
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。我们在真核（HeLa）细胞中表达了2C、其前体2BC以及多个2C缺失突变体，并使用间接免疫荧光法检测了它们的定位。此处呈现的结果表明，在没有其他脊髓灰质炎病毒蛋白的情况下，2C和2BC蛋白能够定位于转染细胞的内质网区域。此外，在直接的体外膜结合试验中，2C与微粒体膜紧密结合。尽管2C蛋白缺乏明确的膜结合结构域，但我们证明，包含氨基酸21 - 54且含有一个假定的两亲性螺旋的N端区域在体内和体外的膜结合中都起着重要作用。相比之下，该蛋白的大部分C端部分似乎对于膜结合并非必需。体外膜相关的2C对蛋白酶K消化的敏感性表明，2C的全部或大部分暴露于膜的细胞质面。此外，与大多数靶向内质网的蛋白不同，2C似乎未被糖基化或被信号肽酶切割。本文讨论了2C的膜结合结构域在病毒RNA复制中的意义。
