Abstract
Accumulating evidence demonstrates that autophagy and microRNAs (miRNAs) play key roles in regulating virus-host interactions and can restrict or facilitate viral replication. In the present study we examined whether a functional relationship exists between autophagy, miRNA and porcine circovirus type 2 (PCV2) infection, using several approaches. We demonstrated that there was a positive correlation between PCV2 infection and autophagy in 3D4/21 cells and autophagy induced by PCV2 infection triggered PCV2 replication. Four miRNA were selected by real-time PCR and further studied, but only miR-30a-5p mimic had a significant effect on PCV2 replication. Overexpression of miR-30a-5p significantly enhanced PCV2 infection and autophagy in a dose-dependent manner. Blockage of miR-30a-5p significantly decreased PCV2 replication. We provided further evidence that miR-30a-5p regulate the link between PCV2 infection and host immune system. Furthermore, miR-30a-5p targeted and regulated 14-3-3 gene, which is a regulator of autophagy. Flow cytometry data demonstrated that miR-30a-5p promotes cell cycle arrest at the G2 phase to regulate PCV2 replication and autophagy by interacting directly with 14-3-3, but not with the PCV2 genome. These data not only provide new insights into virus-host interactions during PCV2 infection but also suggest a potential new antiviral therapeutic strategy against PCV2 infection.
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References
Mizushima N (2007) Autophagy: process and function. Genes Dev 21(22):2861–2873
Sato K, Tsuchihara K, Fujii S, Sugiyama M, Goya T, Atomi Y, Ueno T, Ochiai A, Esumi H (2007) Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation. Cancer Res 67(20):9677–9684
Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147(4):728–741
Dumit VI, Dengjel J (2012) Autophagosomal protein dynamics and influenza virus infection. Front Immunol 3:1–10
Rabinowitz JD, White E (2010) Autophagy and metabolism. Science 330(6009):1344–1348
Azad MB, Chen Y, Henson ES, Cizeau J, Mcmillanward E (2008) Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3. Autophagy 4(2):195–204
Chen Y, Klionsky DJ (2011) The regulation of autophagy—unanswered questions. J Cell Sci 124(Pt 2):161–170
Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451(7182):1069–1075
Ravikumar B, Sarkar SDavies JE, Futter M, Garcia AM, Green Thompson ZW (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90(4):1383–1435
Dales S, Eggers HJ, Tamm I, Palade GE (1965) Electron microscopic study of the formation of poliovirus. Virology 26(26):379–389
Chen Q, Fang L, Wang D, Wang S, Li P, Li M, Luo R, Chen R, Xiao S (2012) Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication. Virus Res 163(2):650–655
Lee YR, Lei HY, Liu MT, Wang JR, Chen SH, Jiang-Shieh YF, Lin YS, Yeh TM, Liu CC, Liu HS (2008) Autophagic machinery activated by dengue virus enhances virus replication. Virology 374(2):240–248
Zhu B, Zhou Y, Xu F, Shuai J, Li X, Fang W (2012) Porcine circovirus type 2 induces autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in PK-15 cells. J Virol 86(22):12003–12012
Zhu BL, Xu F, Li J, Shuai JB, Li XL, Fang WH (2012) Porcine circovirus type 2 explores the autophagic machinery for replication in PK-15 cells. Virus Res 163(2):476–485
Fu YX, Xu WT, Chen DY, Feng CH, Zhang L, Wang XH, Lv XW, Zheng N, Jin Y, Wu ZW (2015) Enterovirus 71 induces autophagy by regulating has-miR-30a expression to promote viral replication. Antivir Res 124:43–53
Richards AL, Jackson WT (2013) How positive-strand RNA viruses benefit from autophagosome maturation. J Virol 87:9966–9972
Jheng JR, Ho JY, Horng JT (2014) ER stress, autophagy, and RNA viruses. Front Microbiol 5:388
Kudchodkar SB, Levine B (2009) Viruses and autophagy. Rev Med Virol 19(6):359–378
Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V (2004) Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119:753–766
Schmid D, Munz C (2007) Innate and adaptive immunity through autophagy. Immunity 27(1):11–21
Shelly S, Lukinova N, Bambina S, Berman A, Cherry S (2009) Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus. Immunity 30(4):588–598
Huang SC, Chang CL, Wang PS, Tsai Y, Liu HS (2009) Enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication. J Med Virol 81:1241–1252
O‘Donnell V, Pacheco JM, LaRocco M, Burrage T, Jackson W, Rodriguez LL, Borca MV, Baxt B (2011) Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication. Virology 410(1):142–150
Kirkegaard K (2009) Subversion of the cellular autophagy pathway by viruses. Curr Top Microbiol Immunol 335:323–333
Ramamoorthy S, Opriessnig T, Pal N, Huang FF, Meng XJ (2011) Effect of an interferon-stimulated response element (ISRE) mutant of porcine circovirus type 2 (PCV2) on PCV2-induced pathological lesions in a porcine reproductive and respiratory syndrome virus (PRRSV) co-infection model. Vet Microbiol 147:49–58
Allan GM, Mcneilly F, Kenedy S, Daft B, Clarke EG, Ellis JA, Haines Dm, Meehan BM, Adair BM (1998) Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. Vet Diagn Invest 10:3–10
Krakowka S, Ellis J, McNeilly F, Waldner C, Allan G (2005) Features of porcine circovirus-2 disease: correlations between lesions, amount and distribution of virus, and clinical outcome. J Vet Diagn Invest 17(3):213–222
Ellis J, Hassard L, Clark E, Harding J, Allan G, Willson P, Strokappe J, Martin K, McNeilly F, Meehan B, Todd D, Haines D (1998) Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J 39(1):44–51
Tischer I, Rasch R, Tochtermann G (1974) Characterization of papovavirus and picornavirus-like particles in permanent pig kidney cell lines. Zentralbl Bakteriol Hyg A 226:153–167
Guo J, Lu YH, Wei W, Huang LP, Liu M (2010) Porcine circovirus type 2 (PCV2): genetic variation and newly emerging genotypes in China. Virol J 7:273
Zhai SL, Chen SN, Xu ZH, Tang MH, Wang FG, Li XJ, Sun BB, Deng SF, Hu J, Lv DH, Wen XH, Yuan J, Luo ML, Wei WK (2014) Porcine circovirus type 2 in China: an update on and insights to its prevalence and control. Virol J 11:88
Liu J, Zhang X, Ma C, Jiang P, Yun S (2017) Hsp90 inhibitor reduces porcine circovirus 2 replication in the porcine monocytic line 3D4/31. Virus Genes 53(1):95–99
Liu J, Bai J, Zhang LL, Jiang P (2013) Hsp70 positively regulates porcine circovirus type 2 replication in vitro. Virology 447(1–2):52–62
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233
Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4):445–544
Fu LL, Wen X, Bao JK, Liu B (2012) MicroRNA-modulated autophagic signaling networks in cancer. Int J Biochem Cell Biol 44(5):733–736
Gottwein E, Cullen BR (2008) Viral and cellular microRNAs as determinants of viral pathogenesis and immunity. Cell Host Microbe 3(6):375–387
Skalsky RL, Cullen BR (2010) Viruses, microRNAs, and host interactions. Annu Rev Microbiol 64(1):123–141
Schligel A, Ms L, Th KKG (1996) Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol 70(10):6576–6588
Hong JS, Kim NH, Choi CY, Lee JS, Na D, Chun T, Lee YS (2015) Changes in cellular microRNA expression induced by porcine circovirus type 2-encoded proteins. Vet Res 46(1):39
Nunez-Hernandez F, Perez LJ, Munoz M, Vera G, Tomas A, Egea R, Cordoba S, Segales J, Sanchez A, Nunez JI (2015) Identification of microRNAs in PCV2 subclinically infected pigs by high throughput sequencing. Vet Res 46:18
Nunez-Hernandez F, Perez LJ, Vera G, Cordoba S, Segales J, Sanchez A, Nunez JI (2015) Evaluation of the capability of the PCV2 genome to encode miRNAs: lack of viral miRNA expression in an experimental infection. Vet Res 46(1):48
Bookout AL, Cummins CL, Mangelsdorf DJ, Pesola JM, Kramer MF (2006) High-throughput real-time quantitative reverse transcription PCR. Curr Protoc Mol Biol 73:1581–15828
Noda T, Ohsumi Y (1998) Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J Biol Chem 273(7):3963–3966
Meng C, Zhou Z, Jiang K, Yu SQ, Jia LJ, Wu YT, Liu YQ, Meng SS, Ding C (2012) Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication. Arch Virol 157(6):1011–1018
Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelarova H, Meijer AJ (1997) The phosphatidylinositol 3-kinase inhibitors Wortmannin and LY294002 inhibit in isolated rat hepatocytes. Eur J Biochem 243(1–2):240–246
Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M, Baehrecke EH, Bahr BA, Ballabio A et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4(2):151–175
Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326
Zhou A, Li SF, Zhang SJ (2014) miRNAs and genes expression in MARC-145 cell in response to PRRSV infection. Infect Genet Evol 27:173–180
Jopling CL (2010) Targeting microRNA-122 to Treat Hepatitis C Virus Infection. Viruses 2(7):1382–1393
Zhang Q, Guo XK, Gao L, Huang C, Li N, Jia XJ, Liu WJ, Feng WH (2014) MicroRNA-23 inhibits PRRSV replication by directly targeting PRRSV RNA and possibly by upregulating type I interferons. Virology 450–451:182–195
Paludan C, Schmid D, Landthaler M, Vockerodt M, Kube D, Tuschl T, Munz C (2005) Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 307:593–596
Pei Y, Chen ZP, Ju HQ, Komatsum M, Ji YH, Liu G, Guo CW, Zhang YJ, Yang CR, Wang YF, Kitazato K (2011) Autophagy is involved in anti-viral activity of pentagalloylglucose (PGG) against Herpes simplex virus type 1 infection in vitro. Biochem Biophys Res Commun 405:186–191
Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, Wu L, Kominami E, Ueno T, Yamamoto A, Federico M, Panganiban A, Vergne I, Deretic V (2009) Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. J Cell Biol 186:255–268
Xin L, Xiao ZH, Ma XL, He F, Yao HL, Liu ZW (2014) Coxsackievirus B3 induces crosstalk between autophagy and apoptosis to benefit its release after replicating in autophagosomes through a mechanism involving caspase cleavage of autophagy-related proteins. Infect Genet Evol 26:95–102
Liu B, Fang M, Hu Y, Huang B, Li N, Chang C, Huang R, Xu X, Yang Z, Chen Z, Liu W (2014) Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation. Autophagy 10:416–430
Jackson WT, Giddings JrTH, Taylor MP, Mulinyawe S, Rabinovitch M, Kopito RR, Kirkegaard K (2005) Subversion of cellular autophagosomal machinery by RNA viruses. PLos Biol 3:e156
Belov GA, Altan-Bonnet N, Kovtunovych G, Jackson CL, Lippincott-Schwartz J, Ehrenfeld E (2007) Hijacking components of the cellular secretory pathway for replication of poliovirus RNA. J Viral 81:558–567
Gu YX, Qi BZ, Zhou YS, Jiang XW, Zhang X, Li XL, Fang WH (2016) Porcine circovirus type 2 activates CaMMKβ to initiate autophagy in PK-15 cells by increasing cytosolic calcium. Viruses 8:135
Kumar S, Gupta P, Khanal S, Shahi A, Kumar P, Sarin SK, Venugopal SK (2015) Overexpression of microRNA-30a inhibits hepatitis B virus X protein-induced autophagosome formation in hepatic cells. The FEBS Journal 282(6):1152–1163
Wang P, Liang J, Li Y, Li J, Yang X, Zhang X, Han S, Li S, Li J (2014) Down-regulation of miRNA-30a alleviates cerebral ischemic injury through enhancing Beclin 1-mediated autophagy. Neurochem Res 39:1279–1291
Pan W, Zhong Y, Cheng C, Liu B, Wang L, Li A, Xiong L, Liu S (2013) MiR-30-regulated autophagy mediates angiotensin II-induced myocardial hypertrophy. PLoS One 8:e53950
Shin HR, Kim H, Oh S, Lee J, Kee M, Ko H, Kweon M, Won K, Baek SH (2016) AMPK–SKP2–CARM1 signalling cascade in transcriptional regulation of autophagy. Nature 534:553–557
Pozuelo-Rubio M (2012) 14-3-3 proteins are regulators of autophagy. Cells 1:754–773
Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol 40:617–647
Wang RC, Wei Y, An Z, Zou Z, Xiao G, Bhagat G, White M, Reichelt J, Levine B (2012) Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation. Science 338:956–959
Quan R, Wei L, Zhu SS, Wang J, Cao YC, Xue CY, Yan X, Liu J (2016) Phase cell cycle arrest induced by porcine circovirus type 2 replication. Sci Rep 6:27917. doi:10.1038/srep27917
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This study was funded by the National Natural Science Foundation of China (31302071), Independent Innovation of Agricultural Sciences in Jiangsu province (CX (14)5041) and Special Fund for Public Welfare Industry of Chinese Ministry of Agriculture (201303046).
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Wang, X., Xu, X., Wang, W. et al. MicroRNA-30a-5p promotes replication of porcine circovirus type 2 through enhancing autophagy by targeting 14-3-3. Arch Virol 162, 2643–2654 (2017). https://doi.org/10.1007/s00705-017-3400-7
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DOI: https://doi.org/10.1007/s00705-017-3400-7