Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis. The eyecup isolated from adult C57BL/6J mice (6–8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine, or rapamycin to block or stimulate autophagy. In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (hpi), but was increased at 48 hpi In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition, caspase-3 cleavage was increased at 48 hpi in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10, and 14 dpi. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups. Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication.

中文翻译：

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3-甲基腺嘌呤抑制自噬限制鼠巨细胞病毒复制

巨细胞病毒 (CMV) 诱导的自噬影响病毒复制和受感染细胞的存活。本研究的目的是研究 3-甲基腺嘌呤 (3-MA) 抑制自噬对鼠巨细胞病毒 (MCMV) 复制的作用，以及它是否与 caspase-3 依赖性细胞凋亡有关。从成年 C57BL/6J 小鼠（6-8 周大）和小鼠胚胎成纤维细胞 (MEF) 分离的眼罩用 MCMV K181 株感染，然后用 3-甲基腺嘌呤 (3-MA)、氯喹或雷帕霉素处理阻断或刺激自噬。在培养的 MEF 中，LC3I/II 的比率在感染后 24 小时 (hpi) 降低，但在 48 hpi 时增加。在眼罩培养中，LC3I/II 比率在感染后 (dpi) 4 天和 7 天也降低。此外，在 MEF 中，caspase-3 切割在 48 hpi 时增加，在 MCMV 感染的眼罩中也在 4、7、10 和 14 dpi 时升高。3-MA 处理显着抑制了 MEF 和眼罩中的病毒复制。MCMV 早期抗原 (EA) 在 MEF 和眼罩中的表达也降低。同时，MCMV感染的MEF和眼罩中3-MA的存在促进了裂解的caspase-3依赖性细胞死亡，而眼罩中的3-MA抑制了RIPK1/RIPK3/MLKL通路。3-MA 对自噬的抑制限制了病毒复制并促进了具有 MCMV 感染的眼罩和 MEF 中的 caspase-3 依赖性细胞凋亡。可以解释为，MCMV感染早期，自噬过程的抑制直接减少了病毒的释放，但后期依赖caspase-3的细胞凋亡占主导地位，导致病毒复制减少。