Furukawa Hiroto, Inaba Hiroshi, Sasaki Yoshihiro, Akiyoshi Kazunari, Matsuura Kazunori
Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University Koyama-Minami 4-101 Tottori 680-8552 Japan
Centre for Research on Green Sustainable Chemistry, Tottori University Koyama-Minami 4-101 Tottori 680-8552 Japan.
RSC Chem Biol. 2021 Dec 21;3(2):231-241. doi: 10.1039/d1cb00166c. eCollection 2022 Feb 9.
Natural enveloped viruses, in which nucleocapsids are covered with lipid bilayers, contain membrane proteins on the outer surface that are involved in diverse functions, such as adhesion and infection of host cells. Previously, we constructed an enveloped artificial viral capsid through the complexation of cationic lipid bilayers onto an anionic artificial viral capsid self-assembled from β-annulus peptides. Here we demonstrate the embedding of the membrane protein Connexin-43 (Cx43), on the enveloped artificial viral capsid using a cell-free expression system. The expression of Cx43 in the presence of the enveloped artificial viral capsid was confirmed by western blot analysis. The embedding of Cx43 on the envelope was evaluated by detection the anti-Cx43 antibody, using fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM). Interestingly, many spherical structures connected to each other were observed in TEM images of the Cx43-embedded enveloped viral replica. In addition, it was shown that fluorescent dyes could be selectively transported from Cx43-embedded enveloped viral replicas into Cx43-expressing HepG2 cells. This study provides a proof of concept for the creation of multimolecular crowding complexes, that is, an enveloped artificial viral replica embedded with membrane proteins.
天然包膜病毒的核衣壳被脂质双层覆盖，其外表面含有参与多种功能的膜蛋白，如宿主细胞的黏附和感染。此前，我们通过将阳离子脂质双层与由β-环肽自组装而成的阴离子人工病毒衣壳复合，构建了一种包膜人工病毒衣壳。在此，我们展示了使用无细胞表达系统将膜蛋白连接蛋白43（Cx43）嵌入包膜人工病毒衣壳的过程。通过蛋白质免疫印迹分析证实了在包膜人工病毒衣壳存在的情况下Cx43的表达。使用荧光相关光谱法（FCS）和透射电子显微镜（TEM）通过检测抗Cx43抗体来评估Cx43在包膜上的嵌入情况。有趣的是，在嵌入Cx43的包膜病毒复制体的TEM图像中观察到许多相互连接的球形结构。此外，研究表明荧光染料可以从嵌入Cx43的包膜病毒复制体选择性地转运到表达Cx43的HepG2细胞中。这项研究为创建多分子拥挤复合物提供了概念验证，即一种嵌入膜蛋白的包膜人工病毒复制体。
