Ciccaglione A R, Costantino A, Marcantonio C, Equestre M, Geraci A, Rapicetta M
Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy1.
J Gen Virol. 2001 Sep;82(Pt 9):2243-2250. doi: 10.1099/0022-1317-82-9-2243.
The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331-383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced in E. coli by influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262-291) was examined by expressing different deletion mutants of E1 in an E. coli system that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg(339), Trp(368) and Lys(370) play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.
丙型肝炎病毒的E1糖蛋白是一种具有C末端锚定结构域的跨膜糖蛋白。当在大肠杆菌中表达时，E1会诱导膜通透性发生变化，这对细菌细胞有毒性。E1的C末端疏水区域（氨基酸331 - 383）主要负责膜结合以及诱导膜通透性的变化。观察到的这些变化类似于流感病毒M2、人类免疫缺陷病毒gp41和脊髓灰质炎病毒3AB蛋白在大肠杆菌中产生的变化，其疏水结构域被认为会在生物膜中形成孔道。为了在分子水平上进一步表征E1的活性，通过在一个广泛用于分析来自其他动物病毒的膜活性蛋白的大肠杆菌系统中表达E1的不同缺失突变体，来检测第二个内部疏水区域（氨基酸262 - 291）的膜通透能力。此外，对C末端疏水区域中高度保守的氨基酸进行突变，以确定对诱导膜通透性变化至关重要的残基。重组培养物的细胞生长曲线分析和膜通透性测定表明，该片段的合成增加了小分子化合物通过膜的通量并导致渐进性细胞裂解，这表明该结构域具有膜活性特性。此外，对C末端突变体的分析表明，保守氨基酸精氨酸（339）、色氨酸（368）和赖氨酸（370）在蛋白质功能中起关键作用，因为野生型克隆诱导的细胞裂解和膜通透性变化都可以被这些位置的取代所阻断。
