Division of Immunology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences-SGGW, Ciszewskiego 8, 02-786, Warsaw, Poland.
Arch Immunol Ther Exp (Warsz). 2011 Dec;59(6):463-71. doi: 10.1007/s00005-011-0149-7. Epub 2011 Oct 5.
Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.
已有数项研究报告揭示了自噬和细胞凋亡在病毒和细菌疾病发病机制以及外来抗原呈现中的新作用。我们的模型研究集中在高度毒性的莫斯科型疱疹病毒（ECTV-MOS）复制过程中细胞凋亡的参与。在这里，我们展示了自噬在鼠痘复制过程中被诱导的证据，该研究使用了细胞系。荧光显微镜显示，与未感染的对照 L929 细胞相比，感染细胞中的 LC3（微管相关蛋白 1 轻链 3）聚集增加。此外，Western blot 分析显示，ECTV-MOS 在 L929 细胞中的复制导致 LC3-II（自噬活性标志物）的表达增加。Beclin 1 在感染细胞中与核外病毒复制中心强烈共定位，而荧光显微镜显示，在这些中心处 Bcl-2 的表达减少。Beclin 1-Bcl-2 相互作用的丧失可能导致病毒感染的 L929 细胞发生自噬。为了评估 Beclin 1 在 ECTV-MOS 感染期间调节细胞凋亡中的作用，我们在感染后使用针对 beclin 1 的小干扰 RNA。通过 AnnexinV 和碘化丙啶染色后，通过流式细胞术分析早期和晚期凋亡细胞。Beclin 1 的沉默导致 ECTV-MOS 在 L929 细胞中的感染后期早期和晚期凋亡细胞的百分比降低。我们得出结论，Beclin 1 在调节 ECTV-MOS 在 L929 允许细胞中的复制过程中的自噬和凋亡方面发挥重要作用。
