Inhibition of autophagy by 3‐methyladenine restricts murine cytomegalovirus replication. Issue 8 (9th February 2021)

• Record Type: Journal Article
• Authors: Zhang, Xinyan; Zhang, Linlin; Bi, Yidan; Xi, Ting; Zhang, Zhan; Huang, Yuan; Lu, Yuan Yuan; Liu, Xinglou; Shu, Sainan; Fang, Feng
• Is Part Of: Journal of medical virology. Volume 93:Issue 8(2021)
• Page Start: 5001
• Page End: 5016
• Publication Date: 2021-02-09
• Subjects: 3‐MA -- apoptosis -- autophagy -- murine cytomegalovirus; Virology -- Periodicals; 616
• DOI: 10.1002/jmv.26787
• Languages: English
• ISSNs: 0146-6615

Abstract

Abstract: 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. Highlights: Firstly, we found that autophagy inhibition by 3‐MA could both inhibit virus replication and promote caspase‐3 dependent apoptosis in the eyecup culture following murine cytomegalovirus (MCMV) infection, while autophagy inducer rapamycin had no such effects. Secondly, as MCMV replication had been controlled by 3‐MA even when caspase‐3 was not activated at early time of MCMV‐infected eyecups, it indicated that during this period of MCMV infection it was autophagy inhibition that resulted in the reduced virus growth. However, when caspase‐3 dependent apoptosis was significantly activated later, it was more likely that the increased caspased‐3 dependent apoptosis caused by autophagy inhibition led to the restriction of virus replication. Last but not least, another autophagy inhibitor CQ also could limit MCMV replication and promote caspase‐3 dependent apoptosis in mouse embryo fibroblast cells (MEFs) and eyecups, which further confirmed the role of autophagy and caspase‐3 dependent apoptosis in MCMV replication.