Abstract
Autophagy is a conserved lysosomal degradation process, and abnormal autophagy has been associated with various pathological processes, e.g., neurodegeneration, cancer, and pathogen infection. Small chemical modulators of autophagy show the potential to treat autophagy-associated diseases. Diterpenoids, nature products found in various plants, exhibit a wide range of bioactivity, and we have recently isolated and characterized over 150 diterpenoids from Isodon species distributed in China. Here, we applied a high-content fluorescence imaging-based assay to assess these diterpenoids’ ability to affect autophagic flux in HeLa cells. We found that enanderinanin J, an ent-kauranoid dimer, is an autophagy inhibitor, manifested by its ability to increase lysosomal pH and inhibit the fusion between autophagosomes and lysosomes. Autophagy has been shown to be either positively or negatively involved in the life cycle of Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), and enterovirus-A71 (EV-A71). We found that enanderinanin J significantly inhibited the infection of ZIKV, DENV, JEV, or EV-A71. Interestingly, although ATG5 knockdown inhibited ZIKV or JEV infection, enanderinanin J further inhibited the infection of ZIKV or JEV in ATG5-knockdown cells. Taken together, our data indicate that enanderinanin J inhibits autophagosome-lysosome fusion and is a potential antiviral agent.
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Abbreviations
- ARJ:
-
acetonide of Rubescensin J
- BAF:
-
bafilomycin A1
- LC3:
-
microtubule-associated protein 1 light chain 3
- GFP:
-
green fluorescence protein
- RFP:
-
red fluorescence protein
- JEV:
-
Japanese encephalitis virus
- ZIKV:
-
Zika virus
- EV-A17:
-
enterovirus A17
- DENV:
-
Dengue virus
- (+)ss RNA viruses:
-
positive-sense single-stranded RNA viruses
References
Acharya B, Gyeltshen S, Chaijaroenkul W, Na-Bangchang K. Significance of autophagy in dengue virus infection: a brief review. Am J Trop Med Hyg. 2019;100:783–90.
Ahmad L, Mostowy S, Sancho-Shimizu V. Autophagy-virus interplay: from cell biology to human disease. Front Cell Dev Biol. 2018;6:155.
Cao B, Parnell LA, Diamond MS, Mysorekar IU. Inhibition of autophagy limits vertical transmission of Zika virus in pregnant mice. J Exp Med. 2017;214:2303–13.
Daep CA, Munoz-Jordan JL, Eugenin EA. Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus. J Neuro-Oncol. 2014;20:539–60.
Dash S, Aydin Y, Wu T. Integrated stress response in hepatitis C promotes Nrf2-related chaperone-mediated autophagy: a novel mechanism for host-microbe survival and HCC development in liver cirrhosis[C]//Seminars in cell & developmental biology. Academic Press. 2020;101:20–35.
Davis-Kaplan SR, Ward DM, Shiflett SL, Kaplan J. Genome-wide analysis of iron-dependent growth reveals a novel yeast gene required for vacuolar acidification. J Biol Chem. 2004;279:4322–9.
de Souza NJ, Dohadwalla AN, Reden J. Forskolin: a labdane diterpenoid with antihypertensive, positive inotropic, platelet aggregation inhibitory, and adenylate cyclase activating properties. Med Res Rev. 1983;3:201–19.
Echavarria-Consuegra L, Smit JM, Reggiori F. Role of autophagy during the replication and pathogenesis of common mosquito-borne flavi- and alphaviruses. Open Biol. 2019;9:190009.
Eskelinen EL. Maturation of autophagic vacuoles in Mammalian cells. Autophagy. 2005;1:1–10.
Fu Y, Xu W, Chen D, Feng C, Zhang L, Wang X, et al. Enterovirus 71 induces autophagy by regulating has-miR-30a expression to promote viral replication. Antivir Res. 2015;124:43–53.
Fujita E, Node M. Diterpenoids of Rabdosia species[M]//Fortschritte der Chemie organischer Naturstoffe/Progress in the Chemistry of Organic Natural Products. Vienna: Springer; 1984. p. 77–157.
Gratton R, Agrelli A, Tricarico PM, et al. Autophagy in Zika virus infection: a possible therapeutic target to counteract viral replication[J]. International journal of molecular sciences. 2019;20(5):1048.
Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N, et al. Biology of Zika virus infection in human skin cells. J Virol. 2015;89:8880–96.
Huang L, Yue J. The interplay of autophagy and enterovirus[C]//Seminars in Cell & Developmental Biology. Academic Press, 2019.
Janku F, McConkey DJ, Hong DS, Kurzrock R. Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol. 2011;8:528–39.
Jin R, Zhu W, Cao S, Chen R, Jin H, Liu Y, et al. Japanese encephalitis virus activates autophagy as a viral immune evasion strategy. PLoS One. 2013;8:e52909.
Jones-Jamtgaard KN, Wozniak AL, Koga H, Ralston R, Weinman SA. Hepatitis C virus infection increases autophagosome stability by suppressing lysosomal fusion through an Arl8b-dependent mechanism. J Biol Chem. 2019;294:14257–66.
Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, et al. An autophagic flux probe that releases an internal control. Mol Cell. 2016;64:835–49.
Kawai A, Uchiyama H, Takano S, Nakamura N, Ohkuma S. Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy. 2007;3:154–7.
Ke PY. The multifaceted roles of autophagy in flavivirus-host interactions. Int J Mol Sci. 2018;19(12):3940.
Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nat Rev Cancer. 2005;5:886–97.
Lai JKF, Sam IC, Verlhac P, Baguet J, Eskelinen EL, Faure M, et al. 2BC non-structural protein of enterovirus A71 interacts with SNARE proteins to trigger autolysosome formation. Viruses. 2017;9:169.
Lee YR, Lei HY, Liu MT, Wang JR, Chen SH, Jiang-Shieh YF, et al. Autophagic machinery activated by dengue virus enhances virus replication. Virology. 2008;374:240–8.
Lee YR, Wang PS, Wang JR, Liu HS. Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model. J Biomed Sci. 2014;21:80.
Leung CH, Grill SP, Lam W, Han QB, Sun HD, Cheng YC. Novel mechanism of inhibition of NF-κB DNA-binding activity by diterpenoids isolated from Isodon rubescens. Mol Pharmacol. 2005;68:286–97.
Li JK, Liang JJ, Liao CL, Lin YL. Autophagy is involved in the early step of Japanese encephalitis virus infection. Microbes Infect. 2012;14:159–68.
Li C, Huang L, Sun W, Chen Y, He ML, Yue J, et al. Saikosaponin D suppresses enterovirus A71 infection by inhibiting autophagy. Signal Transduct Target Ther. 2019;4:4.
Liang Q, Luo Z, Zeng J, Chen W, Foo SS, Lee SA, et al. Zika virus NS4A and NS4B proteins deregulate Akt-mTOR signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy. Cell Stem Cell. 2016;19:663–71.
Liu M, Wang WG, Sun HD, Pu JX. Diterpenoids from Isodon species: an update. Nat Prod Rep. 2017;34:1090–140.
Liu Y, Gordesky-Gold B, Leney-Greene M, Weinbren NL, Tudor M, Cherry S. Inflammation-induced, STING-dependent autophagy restricts Zika virus infection in the Drosophila brain. Cell Host Microbe. 2018;24:57–68 e53.
Lu Y, Dong S, Hao B, Li C, Zhu K, Guo W, et al. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A. Autophagy. 2014;10:1895–905.
Manders E, Verbeek F, Aten J. Measurement of co-localization of objects in dual-colour confocal images. J Microsc. 1993;169:375–82.
Manfredi JJ, Horwitz SB. Taxol: an antimitotic agent with a new mechanism of action. Pharmacol Ther. 1984;25:83–125.
Marwaha R, Sharma M. DQ-Red BSA trafficking assay in cultured cells to assess cargo delivery to lysosomes. Bio Protoc. 2017;7:e2571.
Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev. 2010;90:1383–435.
Sampath A, Padmanabhan R. Molecular targets for flavivirus drug discovery. Antivir Res. 2009;81:6–15.
Sharma M, Bhattacharyya S, Nain M, Kaur M, Sood V, Gupta V, et al. Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes. Autophagy. 2014;10:1637–51.
Sharma M, Bhattacharyya S, Sharma KB, Chauhan S, Asthana S, Abdin MZ, et al. Japanese encephalitis virus activates autophagy through XBP1 and ATF6 ER stress sensors in neuronal cells. J Gen Virol. 2017;98:1027–39.
Sharma M, Sharma KB, Chauhan S, Bhattacharyya S, Vrati S, Kalia M. Diphenyleneiodonium enhances oxidative stress and inhibits Japanese encephalitis virus induced autophagy and ER stress pathways. Biochem Biophys Res Commun. 2018;502:232–7.
Shen Y, Liang WJ, Shi YN, Kennelly EJ, Zhao DK. Structural diversity, bioactivities, and biosynthesis of natural diterpenoid alkaloids. Nat Prod Rep. 2020;37:763–96.
Solomon T, Mallewa M. Dengue and other emerging flaviviruses. J Inf Secur. 2001;42:104–15.
Song Z, Xu Y, Bao L, Zhang L, Yu P, Qu Y, et al. From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses. 2019;11:59.
Su W, Huang S, Zhu H, Zhang B, Wu X. Interaction between PHB2 and Enterovirus A71 VP1 induces autophagy and affects EV-A71 infection. Viruses. 2020;12:414.
Sun HDX, Y L, Jiang B. Diterpenoids from Isodon Species; 2001, P 3.
Sun H-D, Huang S-X, Han Q-B. Diterpenoids from Isodon species and their biological activities. Nat Prod Rep. 2006;23:673–98.
Tasaki T, Nukuzuma S, Takegami T. Impaired Japanese encephalitis virus replication in p62/SQSTM1 deficient mouse embryonic fibroblasts. Microbiol Immunol. 2016;60:708–11.
Tong J, Yan X, Yu L. The late stage of autophagy: cellular events and molecular regulation. Protein Cell. 2010;1:907–15.
White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 2012;12:401–10.
Wong HH, Sanyal S. Manipulation of autophagy by (+) RNA viruses. Semin Cell Dev Biol. 2019;8(7):674.
Wong HH, Sanyal S. Manipulation of autophagy by (+) RNA viruses. Semin Cell Dev Biol. 2020;101:3–11.
Wu WKK, Yue J. Autophagy in host-microbe interactions. Semin Cell Dev Biol. 2020;101:1–2.
Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998;23:33–42.
Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010;12:814–22.
Yao R, Chen ZL, Zhou CC, Luo M, Shi XJ, Li JG, et al. Xerophilusin B induces cell cycle arrest and apoptosis in esophageal squamous cell carcinoma cells and does not cause toxicity in nude mice. J Nat Prod. 2015;78:10–6.
Zhang H, Baehrecke EH. Eaten alive: novel insights into autophagy from multicellular model systems. Trends Cell Biol. 2015;25:376–87.
Zhang S, Yi C, Li C, Zhang F, Peng J, Wang Q, et al. Chloroquine inhibits endosomal viral RNA release and autophagy-dependent viral replication and effectively prevents maternal to fetal transmission of Zika virus. Antivir Res. 2019;169:104547.
Funding
This work was supported by the CAS-Croucher Funding Scheme (JY), Hong Kong Research Grant Council (RGC) grants (11101717 and 11103620 to JY), NSFC (21778045 and 32070702 to JY and 81673329 to TP), Shenzhen government research grant (JCYJ20160229165235739 and JCYJ20170413141331470 to JY), Sichuan Science and Technology Program (# 2019YJ063 to JY), and the Second Tibetan Plateau Scientific Expedition and Research (STEP) program (2019QZKK0502 to TP).
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L.H., Q.F., J-M.D., D.Y., and D.W. performed the experiments; T.P. and J.Y. wrote the manuscript; and J.Y. conceived the study and designed the experiments.
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Huang, L., Fu, Q., Dai, JM. et al. High-content screening of diterpenoids from Isodon species as autophagy modulators and the functional study of their antiviral activities. Cell Biol Toxicol 37, 695–713 (2021). https://doi.org/10.1007/s10565-021-09580-6
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DOI: https://doi.org/10.1007/s10565-021-09580-6