For best viewing of the website please use Mozilla Firefox or Google Chrome.
Citation: Hang Yang, Huijun Yuan, Xiaohui Zhao, Meng Xun, Shangrui Guo, Nan Wang, Bing Liu, Hongliang Wang. Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells [J].VIROLOGICA SINICA, 2022, 37(3) : 380-389.  http://dx.doi.org/10.1016/j.virs.2022.03.003

Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells

  • Corresponding author: Hongliang Wang, hongliangwang@xjtu.edu.cn
  • Received Date: 10 November 2021
    Accepted Date: 04 March 2022
    Available online: 07 March 2022
  • The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.

  • 加载中
  • 10.1016j.virs.2022.03.003-ESM.docx
    1. Alotaibi, M.H., Bahammam, S.A., 2021. Determining the correlation between comorbidities and mers-cov mortality in Saudi Arabia. J. Taibah. Univ. Med.Sci. 16, 591-595.

    2. Barocchi, M.A., Masignani, V., Rappuoli, R., 2005. Opinion:cell entry machines:a common theme in nature? Nat. Rev. Microbiol. 3, 349-358.

    3. Bayati, A., Kumar, R., Francis, V., McPherson, P.S., 2021. Sars-cov-2 infects cells following viral entry via clathrin-mediated endocytosis. J. Biol. Chem. 100306, 296.

    4. Bolte, S., Cordelieres, F.P., 2006. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213-232.

    5. Chu, H., Chan, J.F., Yuen, T.T., Shuai, H., Yuan, S., Wang, Y., Hu, B., Yip, C.C., Tsang, J.O., Huang, X., Chai, Y., Yang, D., Hou, Y., Chik, K.K., Zhang, X., Fung, A.Y., Tsoi, H.W., Cai, J.P., Chan, W.M., Ip, J.D., Chu, A.W., Zhou, J., Lung, D.C., Kok, K.H., To, K.K., Tsang, O.T., Chan, K.H., Yuen, K.Y., 2020. Comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19:an observational study. Lancet. Microbe. 1, e14-e23.

    6. de Wit, E., van Doremalen, N., Falzarano, D., Munster, V.J., 2016. Sars and mers:recent insights into emerging coronaviruses. Nat. Rev. Microbiol. 14, 523-534.

    7. Guy, J.L., Jackson, R.M., Acharya, K.R., Sturrock, E.D., Hooper, N.M., Turner, A.J., 2003. Angiotensin-converting enzyme-2 (ace2):comparative modeling of the active site, specificity requirements, and chloride dependence. Biochemistry 42, 13185-13192.

    8. Guy, J.L., Jackson, R.M., Jensen, H.A., Hooper, N.M., Turner, A.J., 2005. Identification of critical active-site residues in angiotensin-converting enzyme-2 (ace2) by sitedirected mutagenesis. FEBS J. 272, 3512-3520.

    9. Haga, S., Yamamoto, N., Nakai-Murakami, C., Osawa, Y., Tokunaga, K., Sata, T., Yamamoto, N., Sasazuki, T., Ishizaka, Y., 2008. Modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace2 induces tnf-alpha production and facilitates viral entry. Proc. Natl. Acad. Sci. U. S. A. 105, 7809-7814.

    10. Hamming, I., Cooper, M.E., Haagmans, B.L., Hooper, N.M., Korstanje, R., Osterhaus, A.D., Timens, W., Turner, A.J., Navis, G., van Goor, H., 2007. The emerging role of ace2 in physiology and disease. J. Pathol. 212, 1-11.

    11. Haucke, V., 2005. Phosphoinositide regulation of clathrin-mediated endocytosis.Biochem. Soc. Trans. 33, 1285-1289.

    12. He, K., Marsland III, R., Upadhyayula, S., Song, E., Dang, S., Capraro, B.R., Wang, W., Skillern, W., Gaudin, R., Ma, M., Kirchhausen, T., 2017. Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic. Nature 552, 410-414.

    13. Hoffmann, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S., Schiergens, T.S., Herrler, G., Wu, N.H., Nitsche, A., Muller, M.A., Drosten, C., Pohlmann, S., 2020. Sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271-280 e278.

    14. Hu, B., Ge, X., Wang, L.F., Shi, Z., 2015. Bat origin of human coronaviruses. Virol. J. 12, 221.Inoue, Y., Tanaka, N., Tanaka, Y., Inoue, S., Morita, K., Zhuang, M., Hattori, T., Sugamura, K., 2007. Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted.J. Virol. 81, 8722-8729.

    15. Itoh, T., Takenawa, T., 2004. Regulation of endocytosis by phosphatidylinositol 4,5-bisphosphate and enth proteins. Curr. Top. Microbiol. Immunol. 282, 31-47.

    16. Kaksonen, M., Roux, A., 2018. Mechanisms of clathrin-mediated endocytosis. Nat. Rev.Mol. Cell Biol. 19, 313-326.

    17. Karthika, T., Joseph, J., Das, V.R.A., Nair, N., Charulekha, P., Roji, M.D., Raj, V.S., 2021.Sars-cov-2 cellular entry is independent of the ace2 cytoplasmic domain signaling.Cells 10, 1814.

    18. Koch, J., Uckeley, Z.M., Doldan, P., Stanifer, M., Boulant, S., Lozach, P.Y., 2021. Tmprss2 expression dictates the entry route used by sars-cov-2 to infect host cells. EMBO J. 40, e107821.

    19. Kumari, S., Mg, S., Mayor, S., 2010. Endocytosis unplugged:multiple ways to enter the cell. Cell Res. 20, 256-275.

    20. Lambert, D.W., 2009. The Cell Biology of the Sars Coronavirus Receptor, AngiotensinConverting Enzyme 2. Molecular Biology of the SARS-Coronavirus (chapter 2).

    21. Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., Zhang, Q., Shi, X., Wang, Q., Zhang, L., Wang, X., 2020. Structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor. Nature 581, 215-220.

    22. Li, W., Moore, M.J., Vasilieva, N., Sui, J., Wong, S.K., Berne, M.A., Somasundaran, M., Sullivan, J.L., Luzuriaga, K., Greenough, T.C., Choe, H., Farzan, M., 2003. Angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus. Nature 426, 450-454.

    23. Li, G., Su, B., Fu, P., Bai, Y., Ding, G., Li, D., Wang, J., Yang, G., Chu, B., 2021a. Npc1-regulated dynamic of clathrin-coated pits is essential for viral entry. Sci. China Life Sci. 65, 341-361.

    24. Li, X., Zhu, W., Fan, M., Zhang, J., Peng, Y., Huang, F., Wang, N., He, L., Zhang, L., Holmdahl, R., Meng, L., Lu, S., 2021b. Dependence of sars-cov-2 infection on cholesterol-rich lipid raft and endosomal acidification. Comput. Struct. Biotechnol. J. 19, 1933-1943.

    25. Lu, J.C., Chiang, Y.T., Lin, Y.C., Chang, Y.T., Lu, C.Y., Chen, T.Y., Yeh, C.S., 2016. Disruption of lipid raft function increases expression and secretion of monocyte chemoattractant protein-1 in 3t3-l1 adipocytes. PLoS One 11, e0169005.

    26. Marks, M.S., Ohno, H., Kirchnausen, T., Bonracino, J.S., 1997. Protein sorting by tyrosinebased signals:adapting to the ys and wherefores. Trends Cell Biol. 7, 124-128.

    27. McMahon, H.T., Boucrot, E., 2011. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 12, 517-533.

    28. McPherson, P.S., Kay, B.K., Hussain, N.K., 2001. Signaling on the endocytic pathway.Traffic 2, 375-384.

    29. Miettinen, H.M., Matter, K., Hunziker, W., Rose, J.K., Mellman, I., 1992. Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization. J. Cell Biol. 116, 875-888.

    30. Mirre, C., Monlauzeur, L., Garcia, M., Delgrossi, M.H., Le Bivic, A., 1996. Detergentresistant membrane microdomains from caco-2 cells do not contain caveolin. Am. J.Physiol. 271, C887-C894.

    31. Mlcochova, P., Kemp, S.A., Dhar, M.S., Papa, G., Meng, B., Ferreira, I., Datir, R., Collier, D.A., Albecka, A., Singh, S., Pandey, R., Brown, J., Zhou, J., Goonawardane, N., Mishra, S., Whittaker, C., Mellan, T., Marwal, R., Datta, M., Sengupta, S., Ponnusamy, K., Radhakrishnan, V.S., Abdullahi, A., Charles, O., Chattopadhyay, P., Devi, P., Caputo, D., Peacock, T., Wattal, C., Goel, N., Satwik, A., Vaishya, R., Agarwal, M., Indian, S.-C.-G.C.,, Genotype to Phenotype Japan C, Collaboration, C.-N.B.C.-, Mavousian, A., Lee, J.H., Bassi, J., Silacci-Fegni, C., Saliba, C., Pinto, D., Irie, T., Yoshida, I., Hamilton, W.L., Sato, K., Bhatt, S., Flaxman, S., James, L.C., Corti, D., Piccoli, L., Barclay, W.S., Rakshit, P., Agrawal, A., Gupta, R.K., 2021. Sars-cov-2 b.1.617.2 delta variant replication and immune evasion. Nature 599, 114-119.

    32. Montesano, R., Roth, J., Robert, A., Orci, L., 1982. Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature 296, 651-653.

    33. Morse, E.M., Brahme, N.N., Calderwood, D.A., 2014. Integrin cytoplasmic tail interactions. Biochemistry 53, 810-820.

    34. Ng Sht, M.L., See, E.E., Ooi, E.E., Ling, A.E., 2003. Early events of sars coronavirus infection in vero cells. J. Med. Virol. 71, 323-331.

    35. Orlandi, P.A., Fishman, P.H., 1998. Filipin-dependent inhibition of cholera toxin:evidence for toxin internalization and activation through caveolae-like domains.J. Cell Biol. 141, 905-915.

    36. Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L., Guo, R., Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X., Chen, J., Hu, K., Jin, Q., Wang, J., Qian, Z., 2020. Characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov. Nat. Commun. 11, 1620.

    37. Planas, D., Veyer, D., Baidaliuk, A., Staropoli, I., Guivel-Benhassine, F., Rajah, M.M., Planchais, C., Porrot, F., Robillard, N., Puech, J., Prot, M., Gallais, F., Gantner, P., Velay, A., Le Guen, J., Kassis-Chikhani, N., Edriss, D., Belec, L., Seve, A., Courtellemont, L., Pere, H., Hocqueloux, L., Fafi-Kremer, S., Prazuck, T., Mouquet, H., Bruel, T., Simon-Loriere, E., Rey, F.A., Schwartz, O., 2021. Reduced sensitivity of sars-cov-2 variant delta to antibody neutralization. Nature 596, 276-280.

    38. Posor, Y., Eichhorn-Grunig, M., Haucke, V., 2015. Phosphoinositides in endocytosis.Biochim. Biophys. Acta 1851, 794-804.

    39. Rahman, F.I., Ether, S.A., Islam, M.R., 2021. The "delta plus" covid-19 variant has evolved to become the next potential variant of concern:mutation history and measures of prevention. J. Basic Clin. Physiol. Pharmacol. 33, 109-112.

    40. Sanders, D.W., Jumper, C.C., Ackerman, P.J., Bracha, D., Donlic, A., Kim, H., Kenney, D., Castello-Serrano, I., Suzuki, S., Tamura, T., Tavares, A.H., Saeed, M., Holehouse, A.S., Ploss, A., Levental, I., Douam, F., Padera, R.F., Levy, B.D., Brangwynne, C.P., 2021.

    41. Sars-cov-2 requires cholesterol for viral entry and pathological syncytia formation.Elife 10, e65962.

    42. Sorkin, A., von Zastrow, M., 2009. Endocytosis and signalling:intertwining molecular networks. Nat. Rev. Mol. Cell Biol. 10, 609-622.

    43. Stan, R.V., 2005. Structure of caveolae. Biochim. Biophys. Acta 1746, 334-348.

    44. Tang, T., Bidon, M., Jaimes, J.A., Whittaker, G.R., Daniel, S., 2020. Coronavirus membrane fusion mechanism offers a potential target for antiviral development.Antivir. Res. 178, 104792.

    45. Tipnis, S.R., Hooper, N.M., Hyde, R., Karran, E., Christie, G., Turner, A.J., 2000. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J. Biol. Chem. 275, 33238-33243.

    46. Towler, P., Staker, B., Prasad, S.G., Menon, S., Tang, J., Parsons, T., Ryan, D., Fisher, M., Williams, D., Dales, N.A., Patane, M.A., Pantoliano, M.W., 2004. Ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis.J. Biol. Chem. 279, 17996-18007.

    47. Tran, D., Carpentier, J.L., Sawano, F., Gorden, P., Orci, L., 1987. Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway.Proc. Natl. Acad. Sci. U. S. A. 84, 7957-7961.

    48. Turner, A.J., Tipnis, S.R., Guy, J.L., Rice, G., Hooper, N.M., 2002. Aceh/ace2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors. Can. J. Physiol. Pharmacol. 80, 346-353.

    49. von Kleist, L., Stahlschmidt, W., Bulut, H., Gromova, K., Puchkov, D., Robertson, M.J., MacGregor, K.A., Tomilin, N., Pechstein, A., Chau, N., Chircop, M., Sakoff, J., von Kries, J.P., Saenger, W., Krausslich, H.G., Shupliakov, O., Robinson, P.J., McCluskey, A., Haucke, V., 2011. Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146, 471-484.

    50. Walls, A.C., Park, Y.J., Tortorici, M.A., Wall, A., McGuire, A.T., Veesler, D., 2020.Structure, function, and antigenicity of the sars-cov-2 spike glycoprotein. Cell 181, 281-292 e286.

    51. Wang, H., Yang, P., Liu, K., Guo, F., Zhang, Y., Zhang, G., Jiang, C., 2008. Sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway. Cell Res. 18, 290-301.

    52. White, J.M., Whittaker, G.R., 2016. Fusion of enveloped viruses in endosomes. Traffic 17, 593-614.

    53. Who, 2021. Tracking Sars-Cov-2 Variants. WHO. https://www.who.int/en/activities/tra cking-SARS-CoV-2-variants/. (Accessed 15 November 2021).

    54. Wrapp, D., Wang, N., Corbett, K.S., Goldsmith, J.A., Hsieh, C.L., Abiona, O., Graham, B.S., McLellan, J.S., 2020. Cryo-em structure of the 2019-ncov spike in the prefusion conformation. Science 367, 1260-1263.

    55. Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., Zhou, Q., 2020. Structural basis for the recognition of sars-cov-2 by full-length human ace2. Science 367, 1444-1448.

    56. Yang, N., Ma, P., Lang, J., Zhang, Y., Deng, J., Ju, X., Zhang, G., Jiang, C., 2012. Phosphatidylinositol 4-kinase iiibeta is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry. J. Biol. Chem. 287, 8457-8467.

    57. Yang, H., Zhao, X., Xun, M., Ma, C., Wang, H., 2021. Reverse genetic approaches for the generation of full length and subgenomic replicon of ev71 virus. Front. Microbiol. 12, 665879.

    58. Zoncu, R., Perera, R.M., Sebastian, R., Nakatsu, F., Chen, H., Balla, T., Ayala, G., Toomre, D., De Camilli, P.V., 2007. Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc. Natl. Acad. Sci. U. S. A. 104, 3793-3798.

  • 加载中

Article Metrics

Article views(3874) PDF downloads(15) Cited by()

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells

      Corresponding author: Hongliang Wang, hongliangwang@xjtu.edu.cn
    • a Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, 710061, China;
    • b School of Pharmacy, Xi'an Jiaotong University Health Science Center, Xi'an, 710061, China;
    • c BioBank, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, China;

    Abstract: The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.

    Reference (58) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return