Dipartimento di Bioscienze, Università degli Studi di Milano e Istituto di Biofisica, CNR, Milano, Italy.
Membrane Biophysics Group, Department of Biology, Technical University Darmstadt, Germany.
J Gen Virol. 2013 Nov;94(Pt 11):2549-2556. doi: 10.1099/vir.0.055251-0. Epub 2013 Aug 5.
Most chloroviruses encode small K(+) channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K(+) channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K(+) channel from chlorovirus MA-1D to search for the viral K(+) channel in the virus particle. The results showed that the antibody was specific and bound to the tetrameric channel on the extracellular side. The antibody reacted in a virus-specific manner with protein extracts from chloroviruses that encoded channels similar to that from MA-1D. There was no cross-reactivity with chloroviruses that encoded more diverse channels or with a chlorovirus that lacked a K(+) channel gene. Together with electron microscopic imaging, which revealed labelling of individual virus particles with the channel antibody, these results establish that the viral particles contain an active K(+) channel, presumably located in the lipid membrane that surrounds the DNA in the mature virions.
大多数噬藻体病毒编码小的 K(+) 通道,这些通道在电生理测定中具有功能。实验发现,病毒感染的初始步骤对通道抑制剂的敏感性与病毒的 K(+) 通道相同,这导致了一个假说,即这些通道是位于病毒粒子内部膜中的结构蛋白。最近,这个假说受到了质疑,因为蛋白质组学研究未能在原型噬藻体病毒 Paramecium bursaria chlorella virus 1 (PBCV-1) 的病毒粒子中检测到通道蛋白。在这里,我们使用针对噬藻体病毒 MA-1D 的功能性 K(+) 通道的 mAb 来搜索病毒粒子中的病毒 K(+) 通道。结果表明,该抗体是特异性的,与细胞外的四聚体通道结合。该抗体以病毒特异性的方式与编码与 MA-1D 相似通道的噬藻体病毒的蛋白提取物发生反应。与编码更多样化通道的噬藻体病毒或缺乏 K(+) 通道基因的噬藻体病毒没有交叉反应。与电子显微镜成像结合,该成像显示了通道抗体对单个病毒粒子的标记,这些结果确立了病毒粒子中含有活性的 K(+) 通道,该通道可能位于成熟病毒粒子中 DNA 周围的脂质膜中。