Abstract
Viruses utilize cellular lipids and manipulate host lipid metabolism to ensure their replication and spread. Therefore, the identification of lipids and metabolic pathways that are suitable targets for antiviral development is crucial. Using a library of compounds targeting host lipid metabolic factors and testing them for their ability to block pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) infection, we found that U18666A, a specific inhibitor of Niemann-Pick C1 (NPC1), is highly potent in suppressing the entry of diverse viruses including pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NPC1 deficiency markedly attenuates viral growth by decreasing cholesterol abundance in the plasma membrane, thereby inhibiting the dynamics of clathrin-coated pits (CCPs), which are indispensable for clathrin-mediated endocytosis. Significantly, exogenous cholesterol can complement the dynamics of CCPs, leading to efficient viral entry and infectivity. Administration of U18666A improves the survival and pathology of PRV- and influenza A virus-infected mice. Thus, our studies demonstrate a unique mechanism by which NPC1 inhibition achieves broad antiviral activity, indicating a potential new therapeutic strategy against SARS-CoV-2, as well as other emerging viruses.
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Altan-Bonnet, N. (2017). Lipid tales of viral replication and transmission. Trends Cell Biol 27, 201–213.
Barrows, N.J., Campos, R.K., Liao, K.C., Prasanth, K.R., Soto-Acosta, R., Yeh, S.C., Schott-Lerner, G., Pompon, J., Sessions, O.M., Bradrick, S. S., et al. (2018). Biochemistry and molecular biology of flaviviruses. Chem Rev 118, 4448–4482.
Bender, F.C., Whitbeck, J.C., Ponce de Leon, M., Lou, H., Eisenberg, R.J., and Cohen, G.H. (2003). Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. J Virol 77, 9542–9552.
Bhattacharyya, S., Warfield, K.L., Ruthel, G., Bavari, S., Aman, M.J., and Hope, T.J. (2010). Ebola virus uses clathrin-mediated endocytosis as an entry pathway. Virology 401, 18–28.
Blanchard, E., Belouzard, S., Goueslain, L., Wakita, T., Dubuisson, J., Wychowski, C., and Rouille, Y. (2006). Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80, 6964–6972.
Bokelmann, M., Edenborough, K., Hetzelt, N., Kreher, P., Lander, A., Nitsche, A., Vogel, U., Feldmann, H., Couacy-Hymann, E., and Kurth, A. (2020). Utility of primary cells to examine NPC1 receptor expression in Mops condylurus, a potential Ebola virus reservoir. PLoS Negl Trop Dis 14, e0007952.
Bourke, E., Cassetti, A., Villa, A., Fadlon, E., Colotta, F., and Mantovani, A. (2003). IL-1β scavenging by the type II IL-1 decoy receptor in human neutrophils. J Immunol 170, 5999–6005.
Carneiro, F.A., Lapido-Loureiro, P.A., Cordo, S.M., Stauffer, F., Weissmüller, G., Bianconi, M.L., Juliano, M.A., Juliano, L., Bisch, P. M., Da Poian, A.T., et al. (2006). Probing the interaction between vesicular stomatitis virus and phosphatidylserine. Eur Biophys J 35, 145–154.
Cenedella, R.J., Sarkar, C.P., and Towns, L. (1982). Studies on the mechanism of the epileptiform activity induced by U18666A. II. Concentration, half-life and distribution of radiolabeled U18666A in the brain. Epilepsia 23, 257–268.
Chu, B.B., Liao, Y.C., Qi, W., Xie, C., Du, X., Wang, J., Yang, H., Miao, H. H., Li, B.L., and Song, B.L. (2015). Cholesterol transport through lysosome-peroxisome membrane contacts. Cell 161, 291–306.
Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. (2020). The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5, 536–544.
Cruz, J.C., Sugii, S., Yu, C., and Chang, T.Y. (2000). Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol. J Biol Chem 275, 4013–4021.
Daecke, J., Fackler, O.T., Dittmar, M.T., and Krausslich, H.G. (2005). Involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry. J Virol 79, 1581–1594.
Dai, J., Ting-Beall, H.P., and Sheetz, M.P. (1997). The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells. J Gen Physiol 110, 1–10.
Desplanques, A.S., Nauwynck, H.J., Vercauteren, D., Geens, T., and Favoreel, H.W. (2008). Plasma membrane cholesterol is required for efficient pseudorabies virus entry. Virology 376, 339–345.
Ding, Z., Fang, L., Yuan, S., Zhao, L., Wang, X., Long, S., Wang, M., Wang, D., Foda, M.F., and Xiao, S. (2017). The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/MDA5 activation. Oncotarget 8, 49655–49670.
Doki, T., Tarusawa, T., Hohdatsu, T., and Takano, T. (2020). In vivo antiviral effects of U18666A against type I feline infectious peritonitis virus. Pathogens 9, 67.
Edeling, M.A., Smith, C., and Owen, D. (2006). Life of a clathrin coat: insights from clathrin and AP structures. Nat Rev Mol Cell Biol 7, 32–44.
Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M.L., and Kirchhausen, T. (2004). Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118, 591–605.
Elgner, F., Ren, H., Medvedev, R., Ploen, D., Himmelsbach, K., Boller, K., and Hildt, E. (2016). The intracellular cholesterol transport inhibitor U18666A inhibits the exosome-dependent release of mature hepatitis C virus. J Virol 90, 11181–11196.
Ferner, R.E., and Aronson, J.K. (2020). Chloroquine and hydroxychloroquine in COVID-19. BMJ 369, m1432.
Grove, J., and Marsh, M. (2011). The cell biology of receptor-mediated virus entry. J Cell Biol 195, 1071–1082.
Heaton, N.S., and Randall, G. (2011). Multifaceted roles for lipids in viral infection. Trends Microbiol 19, 368–375.
Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., Schiergens, T.S., Herrler, G., Wu, N.H., Nitsche, A., et al. (2020). SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280.e8.
Jiang, J., Kolpak, A.L., and Bao, Z.Z. (2010). Myosin IIB isoform plays an essential role in the formation of two distinct types of macropinosomes. Cytoskeleton 67, 32–42.
Kiss, T., Fenyvesi, F., Bácskay, I., Váradi, J., Fenyvesi, E., Iványi, R., Szente, L., Tósaki, A., and Vecsernyés, M. (2010). Evaluation of the cytotoxicity of β-cyclodextrin derivatives: Evidence for the role of cholesterol extraction. Eur J Pharm Sci 40, 376–380.
Li, M., Yang, C., Tong, S., Weidmann, A., and Compans, R.W. (2002). Palmitoylation of the murine leukemia virus envelope protein is critical for lipid raft association and surface expression. J Virol 76, 11845–11852.
Li, Z., Guo, D., Qin, Y., and Chen, M. (2019). PI4KB on inclusion bodies formed by ER membrane remodeling facilitates replication of human parainfluenza virus type 3. Cell Rep 29, 2229–2242.e4.
Lingwood, D., and Simons, K. (2010). Lipid rafts as a membrane-organizing principle. Science 327, 46–50.
Liscum, L., and Faust, J.R. (1989). The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one. J Biol Chem 264, 11796–11806.
Lorizate, M., and Kräusslich, H.G. (2011). Role of lipids in virus replication. Cold Spring Harbor Perspect Biol 3, a004820.
Lozano, R., Naghavi, M., Foreman, K., Lim, S., Shibuya, K., Aboyans, V., Abraham, J., Adair, T., Aggarwal, R., Ahn, S.Y., et al. (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2095–2128.
Lu, F., Liang, Q., Abi-Mosleh, L., Das, A., De Brabander, J.K., Goldstein, J.L., and Brown, M.S. (2015). Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection. eLife 4, e12177.
Lu, R., Zhao, X., Li, J., Niu, P., Yang, B., Wu, H., Wang, W., Song, H., Huang, B., Zhu, N., et al. (2020). Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565–574.
Martín-Acebes, M.A., González-Magaldi, M., Sandvig, K., Sobrino, F., and Armas-Portela, R. (2007). Productive entry of type C foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol. Virology 369, 105–118.
Menendez-Arias, L., Alvarez, M., and Pacheco, B. (2014). Nucleoside/ nucleotide analog inhibitors of hepatitis B virus polymerase: mechanism of action and resistance. Curr Opin Virol 8, 1–9.
Mercer, J., and Helenius, A. (2009). Virus entry by macropinocytosis. Nat Cell Biol 11, 510–520.
Mettlen, M., Chen, P.H., Srinivasan, S., Danuser, G., and Schmid, S.L. (2018). Regulation of clathrin-mediated endocytosis. Annu Rev Biochem 87, 871–896.
Miller, E.H., Obernosterer, G., Raaben, M., Herbert, A.S., Deffieu, M.S., Krishnan, A., Ndungo, E., Sandesara, R.G., Carette, J.E., Kuehne, A.I., et al. (2012). Ebola virus entry requires the host-programmed recognition of an intracellular receptor. EMBO J 31, 1947–1960.
Miller, S., and Krijnse-Locker, J. (2008). Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6, 363–374.
Monnerie, H., Romer, M., Jensen, B.K., Millar, J.S., Jordan-Sciutto, K.L., Kim, S.F., and Grinspan, J.B. (2017). Reduced sterol regulatory element-binding protein (SREBP) processing through site-1 protease (S1P) inhibition alters oligodendrocyte differentiation in vitro. J Neurochem 140, 53–67.
Moriishi, K., and Matsuura, Y. (2012). Exploitation of lipid components by viral and host proteins for hepatitis C virus infection. Front Microbio 3, 54.
Nio, Y., Hasegawa, H., Okamura, H., Miyayama, Y., Akahori, Y., and Hijikata, M. (2016). Liver-specific mono-unsaturated fatty acid synthase-1 inhibitor for anti-hepatitis C treatment. Antiviral Res 132, 262–267.
Nixdorf, R., Schmidt, J., Karger, A., and Mettenleiter, T.C. (1999). Infection of Chinese hamster ovary cells by pseudorabies virus. J Virol 73, 8019–8026.
Poh, M.K., Shui, G., Xie, X., Shi, P.Y., Wenk, M.R., and Gu, F. (2012). U18666A, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication. Antiviral Res 93, 191–198.
Pontes, B., Monzo, P., and Gauthier, N.C. (2017). Membrane tension: A challenging but universal physical parameter in cell biology. Semin Cell Dev Biol 71, 30–41.
Shin, S.Y., Lee, K.S., Choi, Y.K., Lim, H.J., Lee, H.G., Lim, Y., and Lee, Y. H. (2013). The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells. Carcinogenesis 34, 2080–2089.
Smith, D., Magri, A., Bonsall, D., Ip, C.L.C., Trebes, A., Brown, A., Piazza, P., Bowden, R., Nguyen, D., Ansari, M.A., et al. (2019). Resistance analysis of genotype 3 hepatitis C virus indicates subtypes inherently resistant to nonstructural protein 5A inhibitors. Hepatology 69, 1861–1872.
Snyers, L., Zwickl, H., and Blaas, D. (2003). Human rhinovirus type 2 is internalized by clathrin-mediated endocytosis. J Virol 77, 5360–5369.
Stapleford, K.A., and Miller, D.J. (2010). Role of cellular lipids in positivesense RNA virus replication complex assembly and function. Viruses 2, 1055–1068.
Subbarao, K., and Mahanty, S. (2020). Respiratory virus infections: understanding COVID-19. Immunity 52, 905–909.
Subtil, A., Gaidarov, I., Kobylarz, K., Lampson, M.A., Keen, J.H., and McGraw, T.E. (1999). Acute cholesterol depletion inhibits clathrincoated pit budding. Proc Natl Acad Sci USA 96, 6775–6780.
Targett-Adams, P., Boulant, S., Douglas, M.W., and McLauchlan, J. (2010). Lipid metabolism and HCV infection. Viruses 2, 1195–1217.
Trinh, M.N., Lu, F., Li, X., Das, A., Liang, Q., De Brabander, J.K., Brown, M.S., and Goldstein, J.L. (2017). Triazoles inhibit cholesterol export from lysosomes by binding to NPC1. Proc Natl Acad Sci USA 114, 89–94.
Tsai, B., and Qian, M. (2010). Cellular entry of polyomaviruses. In: Johnson, J., ed. Cell Entry by Non-Enveloped Viruses. Current Topics in Microbiology and Immunology. Berlin: Springer. 177–194.
Tscherne, D.M., Jones, C.T., Evans, M.J., Lindenbach, B.D., McKeating, J. A., and Rice, C.M. (2006). Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol 80, 1734–1741.
Wang, H., Yang, P., Liu, K., Guo, F., Zhang, Y., Zhang, G., and Jiang, C. (2008). SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res 18, 290–301.
Wang, J., Chu, B., Du, L., Han, Y., Zhang, X., Fan, S., Wang, Y., and Yang, G. (2015). Molecular cloning and functional characterization of porcine cyclic GMP-AMP synthase. Mol Immunol 65, 436–445.
Wang, J., Li, G.L., Ming, S.L., Wang, C.F., Shi, L.J., Su, B.Q., Wu, H.T., Zeng, L., Han, Y.Q., Liu, Z.H., et al. (2020a). BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses. PLoS Pathog 16, e1008429.
Wang, Y., Zhang, D., Du, G., Du, R., Zhao, J., Jin, Y., Fu, S., Gao, L., Cheng, Z., Lu, Q., et al. (2020b). Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. Lancet 395, 1569–1578.
Wisskirchen, K., Lucifora, J., Michler, T., and Protzer, U. (2014). New pharmacological strategies to fight enveloped viruses. Trends Pharmacol Sci 35, 470–478.
Xiang, S., Zhou, Z., Hu, X., Li, Y., Zhang, C., Wang, J., Li, X., Tan, F., and Tian, K. (2016). Complete genome sequence of a variant pseudorabies virus strain isolated in central China. Genome Announc 4.
Yin, X., Ambardekar, C., Lu, Y., and Feng, Z. (2016). Distinct entry mechanisms for nonenveloped and quasi-enveloped hepatitis E viruses. J Virol 90, 4232–4242.
Zeng, L., Wang, M.D., Ming, S.L., Li, G.L., Yu, P.W., Qi, Y.L., Jiang, D. W., Yang, G.Y., Wang, J., and Chu, B.B. (2020). An effective inactivant based on singlet oxygen-mediated lipid oxidation implicates a new paradigm for broad-spectrum antivirals. Redox Biol 36, 101601.
Zhang, N., Zhao, H., and Zhang, L. (2019). Fatty acid synthase promotes the palmitoylation of chikungunya virus nsP1. J Virol 93.
Zhou, P., Yang, X.L., Wang, X.G., Hu, B., Zhang, L., Zhang, W., Si, H.R., Zhu, Y., Li, B., Huang, C.L., et al. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273.
Acknowledgements
We thank Bao-Liang Song (Wuhan University, China) for providing CHO-K1 and CT43 cells; Ke-Gong Tian (Henan Agricultural University, China) for providing PRV HN1201; and Shao-Bo Xiao (Huazhong Agricultural University, China) for providing MHV A59.
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Compliance and ethics The author(s) declare that they have no conflict of interest. The animal experimental procedure was approved by the Ethics Committtee of Henan Agricultural University.
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Li, G., Su, B., Fu, P. et al. NPC1-regulated dynamic of clathrin-coated pits is essential for viral entry. Sci. China Life Sci. 65, 341–361 (2022). https://doi.org/10.1007/s11427-021-1929-y
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DOI: https://doi.org/10.1007/s11427-021-1929-y