Inhibition of Autophagy by 3-Methyladenine restricts Murine Cytomegalo virus Replication

Abstract Background:Cytomegalovirus (CMV) could induce autophagy early upon infection, which might have an impact on virus replication and the survival of host cells. The purpose ofthe present study was to determine how autophagy effects virus replication and whether it is associated with caspase-3 dependent apoptosis duringmurine cytomegalovirus (MCMV) infection. Methods:The eyecup isolated from adultC57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were cultured and infected with MCMV K181 strain, following by treated with 3-methyladenine (3-MA)or rapamycin to block or activate autophagy.Immunofluorescence staining and western blot were used to detect the expression of early antigen (EA) of MCMV, autophagy and cell death related factors. Plaque assay was performed to detect the virus titer in different groups. TUNEL assay was used to measure the percentage of cell death.Results: Results showed that autophagy was induced at 24,72 and 96hours post infection (hpi)with MCMV in MEFs. In the eyecup culture, it also showed that autophagy was induced at 4 and 7days post infection (dpi).In addition, caspase-3 dependent apoptosis and receptor-interacting kinase 1/ receptor-interacting kinase 3/mixed lineage kinase domain-like protein (RIPK1/RIPK3/MLKL) dependent necroptosis were induced by MCMV infection in eyecup.In MEFs, caspase-3 dependent apoptosis was inhibited, whileRIPK1/RIPK3/MLKL dependent necroptosis was activated with MCMV infection. Once treatment with 3-MA, there were significantly less active virus particles released in MEFs and eyecup, also EA expression was significantly inhibited in the eyecup. However, treatment with rapamycin have no such significant influence on either virus titer or EA expression in MEFs and eyecup. Furthermore, cleaved caspase-3 was elevated, while RIPK1/RIPK3/MLKL pathway was inhibited with treatment of 3-MA both in MEFs and eyecup. Conclusion:Inhibition of autophagy by 3-MA could both restrict virus replication and promote caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection.It can be explained that on the early periodof MCMV infection, suppressed autophagy process directly reduced virus release.Thereafter, caspase-3 dependent apoptosis was activated and resulted in decreased virus replication.