细胞自噬促进柯萨奇病毒B3复制的研究
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摘要
本研究探索柯萨奇病毒B3(Coxsackievirus B3,CVB3)感染引起的自噬与病毒复制之间的关系。CVB3感染HeLa细胞,并在病毒感染后6 h、8 h和10 h时检测LC3-Ⅰ蛋白、LC3-Ⅱ蛋白和p62蛋白的表达水平。结果显示CVB3病毒感染促使LC3-Ⅱ/LC3-Ⅰ比值升高,同时降低p62蛋白的表达。分别将自噬诱导剂雷帕霉素(Rapamy-cin)、自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3MA)或溶酶体抑制剂阿洛司他丁(Aloxistatin,E46D)预处理HeLa细胞2 h,CVB3感染药物处理细胞并在病毒感染6 h后收集细胞、检测CVB3病毒VP1蛋白的表达。结果显示雷帕霉素和E64D促使CVB3病毒VP1蛋白表达增加,而3MA降低CVB3病毒VP1蛋白的表达。本研究得出结论 CVB3病毒感染诱导自噬进而促进病毒复制。
To explore the relationship between autophagy and viral replication in HeLa cells infected with coxsackievirus B3(CVB3), the expression levels of LC3-Ⅰ, LC-Ⅱ and p62 were detected at 6 hrs, 8 hrs and 10 hrs post-infection. We found that CVB3 infection increased the ratio of LC3-Ⅱ/LC3-Ⅰ and decreased the expression of p62 protein. After HeLa cells were exposed to autophagy inducer Rapamycin, autophagy inhibitor 3-Methyladenine(3 MA) or lysosomal inhibitor Aloxistatin(E64 D) for 2 hrs, respectively, HeLa cells were infected with CVB3, then the expression of CVB3 VP1 was detected at 6 hrs post-infection. The results showed that both Rapamycin treatment and E64 D treatment increased the expression of VP1 protein in the HeLa cells infected with CVB3, while 3 MA treatment decreased the expression of VP1 protein.CVB3 infection activated autophagy to facilitate viral replication.
To explore the relationship between autophagy and viral replication in HeLa cells infected with coxsackievirus B3(CVB3), the expression levels of LC3-Ⅰ, LC-Ⅱ and p62 were detected at 6 hrs, 8 hrs and 10 hrs post-infection. We found that CVB3 infection increased the ratio of LC3-Ⅱ/LC3-Ⅰ and decreased the expression of p62 protein. After HeLa cells were exposed to autophagy inducer Rapamycin, autophagy inhibitor 3-Methyladenine(3 MA) or lysosomal inhibitor Aloxistatin(E64 D) for 2 hrs, respectively, HeLa cells were infected with CVB3, then the expression of CVB3 VP1 was detected at 6 hrs post-infection. The results showed that both Rapamycin treatment and E64 D treatment increased the expression of VP1 protein in the HeLa cells infected with CVB3, while 3 MA treatment decreased the expression of VP1 protein.CVB3 infection activated autophagy to facilitate viral replication.
引文
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[2] Van Kuppeveld F J, Hoenderop J G, Smeets R L, Willems P H, Dijkman H B, Galama J M, Melchers W J.Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release[J]. Embo j, 1997,16(12):3519-3532.
[3] Harvala H,Kalimo H,Bergelson J,Stanway G,Hyypia T. Tissue tropism of recombinant coxsackieviruses in an adult mouse model[J]. J Gen Virol, 2005, 86(Pt 7):1897-1907.
[4] Qian M,Fang X,Wang X.Autophagy and inflammation[J]. Clin TranslMed,2017, 6(1):24.
[5] Wong J,Zhang J,Si X,Gao G, Mao I, Mcmanus B M,Luo H.Autophagosome supports coxsackievirus B3replication in host cells[J]. J Virol, 2008, 82(18):9143-9153.
[6] Liu W J,Ye L,Huang W F,Guo L J,Xu Z G,Wu H L,Yang C,Liu H F. p62 links the autophagy pathwayand the ubiqutin-proteasome system upon ubiquitinated protein degradation[J]. Cell Mol Biol Lett, 2016,21(29.
[7] Choi Y, Bowman J W, Jung J U. Autophagy during viral infection-a double-edged sword[J]. Nat Rev Microbiol, 2018, 16(6):341-354.
[8] Wang X,Qi X,Yang B,Chen S,Wang J. Autophagy benefits the replication of egg drop syndrome virus in duck embryo fibroblasts[J]. Front Microbiol, 2018, 9:1091.
[9] Ravikumar B,Sarkar S,Davies J E,Futter M,GarciaArencibia M,Green-Thompson Z W,Jimenez-Sanchez M,Korolchuk V I,Lichtenberg M,Luo S,Massey DC, Menzies F M, Moreau K, Narayanan U, Renna M,Siddiqi F H,Underwood B R,Winslow A R,Rubinsztein D C. Regulation of mammalian autophagy in physiology and pathophysiology[J]. Physiol Rev, 2010, 90(4):1383-1435.
[10] Jordan T X, Randall G. Manipulation or capitulation:virus interactions with autophagy[J]. Microbes Infect,2012, 14(2):126-139.
[11] Orvedahl A, Levine B. Viral evasion of autophagy[J].Autophagy,2008,4(3):280-285.
[12] Kim Y C,Guan K L. mTOR:a pharmacologic target for autophagy regulation[J]. J Clin Invest, 2015,125(1):25-32.