Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enzyme 2 (ACE2) is still viewed as the main cell entry receptor mediating SARS-CoV-2 internalization. It has been reported that wild-type mice, like other rodent species of the Muridae family, cannot be infected with SARS-CoV-2 due to differences in their ACE2 receptors. On the other hand, the consensus heparin-binding motif of SARS-CoV-2's spike protein, PRRAR, enables the attachment to rodent heparan sulfate proteoglycans (HSPGs), including syndecans, a transmembrane HSPG family with a well-established role in clathrin- and caveolin-independent endocytosis. As mammalian syndecans possess a relatively conserved structure, we analyzed the cellular uptake of inactivated SARS-CoV-2 particles in in vitro and in vivo mice models. Cellular studies revealed efficient uptake into murine cell lines with established syndecan-4 expression. After intravenous administration, inactivated SARS-CoV-2 was taken up by several organs in vivo and could also be detected in the brain. Internalized by various tissues, inactivated SARS-CoV-2 raised tissue TNF-α levels, especially in the heart, reflecting the onset of inflammation. Our studies on in vitro and in vivo mice models thus shed light on unknown details of SARS-CoV-2 internalization and help broaden the understanding of the molecular interactions of SARS-CoV-2.

Boeckeler Instruments Inc Tucson AZ 85714 USA

Delong Instruments a s 612 00 Brno Czech Republic

Department of Medicine Albert Szent Györgyi Clinical Center Faculty of Medicine University of Szeged H 6720 Szeged Hungary

Institute of Biochemistry Biological Research Centre H 6726 Szeged Hungary

Institute of Biophysics Biological Research Centre H 6726 Szeged Hungary

Laboratory of Proteomics Research Biological Research Centre H 6726 Szeged Hungary

Pharmacoidea Ltd H 6726 Szeged Hungary

Theoretical Medicine Doctoral School Faculty of Medicine University of Szeged H 6720 Szeged Hungary

Zobrazit více v PubMed

Lai C.C., Shih T.P., Ko W.C., Tang H.J., Hsueh P.R. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. Int. J. Antimicrob. Agents. 2020;55:105924. doi: 10.1016/j.ijantimicag.2020.105924. PubMed DOI PMC

Israfil S.M.H., Sarker M.M.R., Rashid P.T., Talukder A.A., Kawsar K.A., Khan F., Akhter S., Poh C.L., Mohamed I.N., Ming L.C. Clinical characteristics and diagnostic challenges of COVID-19: An update from the global perspective. Front. Public Health. 2020;8:567395. doi: 10.3389/fpubh.2020.567395. PubMed DOI PMC

Goyal M., Tewatia N., Vashisht H., Jain R., Kumar S. Novel corona virus (COVID-19); Global efforts and effective investigational medicines: A review. J. Infect. Public Health. 2021;14:910–921. doi: 10.1016/j.jiph.2021.04.011. PubMed DOI PMC

Wen W., Chen C., Tang J., Wang C., Zhou M., Cheng Y., Zhou X., Wu Q., Zhang X., Feng Z., et al. Efficacy and safety of three new oral antiviral treatment (molnupiravir, fluvoxamine and Paxlovid) for COVID-19: A meta-analysis. Ann. Med. 2022;54:516–523. doi: 10.1080/07853890.2022.2034936. PubMed DOI PMC

Hoffmann M., Kleine-Weber H., Schroeder S., Kruger N., Herrler T., Erichsen S., Schiergens T.S., Herrler G., Wu N.H., Nitsche A., et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181:271–280. doi: 10.1016/j.cell.2020.02.052. PubMed DOI PMC

Scialo F., Daniele A., Amato F., Pastore L., Matera M.G., Cazzola M., Castaldo G., Bianco A. ACE2: The major cell entry receptor for SARS-CoV-2. Lung. 2020;198:867–877. doi: 10.1007/s00408-020-00408-4. PubMed DOI PMC

Shen X.R., Geng R., Li Q., Chen Y., Li S.F., Wang Q., Min J., Yang Y., Li B., Jiang R.D., et al. ACE2-independent infection of T lymphocytes by SARS-CoV-2. Signal Transduct. Target. Ther. 2022;7:83. doi: 10.1038/s41392-022-00919-x. PubMed DOI PMC

Karthika T., Joseph J., Das V.R.A., Nair N., Charulekha P., Roji M.D., Raj V.S. SARS-CoV-2 cellular entry is independent of the ACE2 cytoplasmic domain signaling. Cells. 2021;10:1814. doi: 10.3390/cells10071814. PubMed DOI PMC

Liu J., Lu F., Chen Y., Plow E., Qin J. Integrin mediates cell entry of the SARS-CoV-2 virus independent of cellular receptor ACE2. J. Biol. Chem. 2022;298:101710. doi: 10.1016/j.jbc.2022.101710. PubMed DOI PMC

Wan Y., Shang J., Graham R., Baric R.S., Li F. Receptor recognition by the novel Coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS Coronavirus. J. Virol. 2020;94:e00127-20. doi: 10.1128/JVI.00127-20. PubMed DOI PMC

Shou S., Liu M., Yang Y., Kang N., Song Y., Tan D., Liu N., Wang F., Liu J., Xie Y. Animal models for COVID-19: Hamsters, mouse, ferret, mink, tree shrew, and non-human primates. Front. Microbiol. 2021;12:626553. doi: 10.3389/fmicb.2021.626553. PubMed DOI PMC

Schuurs Z.P., Hammond E., Elli S., Rudd T.R., Mycroft-West C.J., Lima M.A., Skidmore M.A., Karlsson R., Chen Y.H., Bagdonaite I., et al. Evidence of a putative glycosaminoglycan binding site on the glycosylated SARS-CoV-2 spike protein N-terminal domain. Comput. Struct. Biotechnol. J. 2021;19:2806–2818. doi: 10.1016/j.csbj.2021.05.002. PubMed DOI PMC

Kim S.Y., Jin W., Sood A., Montgomery D.W., Grant O.C., Fuster M.M., Fu L., Dordick J.S., Woods R.J., Zhang F., et al. Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions. Antiviral Res. 2020;181:104873. doi: 10.1016/j.antiviral.2020.104873. PubMed DOI PMC

Clausen T.M., Sandoval D.R., Spliid C.B., Pihl J., Perrett H.R., Painter C.D., Narayanan A., Majowicz S.A., Kwong E.M., McVicar R.N., et al. SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2. Cell. 2020;183:1043–1057. doi: 10.1016/j.cell.2020.09.033. PubMed DOI PMC

De Pasquale V., Quiccione M.S., Tafuri S., Avallone L., Pavone L.M. Heparan sulfate proteoglycans in viral infection and treatment: A special focus on SARS-CoV-2. Int. J. Mol. Sci. 2021;22:6574. doi: 10.3390/ijms22126574. PubMed DOI PMC

Paiardi G., Richter S., Oreste P., Urbinati C., Rusnati M., Wade R.C. The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms. J. Biol. Chem. 2022;298:101507. doi: 10.1016/j.jbc.2021.101507. PubMed DOI PMC

Tumova S., Woods A., Couchman J.R. Heparan sulfate chains from glypican and syndecans bind the Hep II domain of fibronectin similarly despite minor structural differences. J. Biol. Chem. 2000;275:9410–9417. doi: 10.1074/jbc.275.13.9410. PubMed DOI

Hudak A., Letoha A., Szilak L., Letoha T. Contribution of syndecans to the cellular entry of SARS-CoV-2. Int. J. Mol. Sci. 2021;22:5336. doi: 10.3390/ijms22105336. PubMed DOI PMC

Letoha T., Keller-Pinter A., Kusz E., Kolozsi C., Bozso Z., Toth G., Vizler C., Olah Z., Szilak L. Cell-penetrating peptide exploited syndecans. Biochim. Biophys. Acta. 2010;1798:2258–2265. doi: 10.1016/j.bbamem.2010.01.022. PubMed DOI

Hudak A., Kusz E., Domonkos I., Josvay K., Kodamullil A.T., Szilak L., Hofmann-Apitius M., Letoha T. Contribution of syndecans to cellular uptake and fibrillation of alpha-synuclein and tau. Sci. Rep. 2019;9:16543. doi: 10.1038/s41598-019-53038-z. PubMed DOI PMC

Fuki I.V., Meyer M.E., Williams K.J. Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts. Pt 3Biochem. J. 2000;351:607–612. doi: 10.1042/bj3510607. PubMed DOI PMC

Tkachenko E., Rhodes J.M., Simons M. Syndecans: New kids on the signaling block. Circ. Res. 2005;96:488–500. doi: 10.1161/01.RES.0000159708.71142.c8. PubMed DOI

Couchman J.R., Gopal S., Lim H.C., Norgaard S., Multhaupt H.A. Fell-Muir Lecture: Syndecans: From peripheral coreceptors to mainstream regulators of cell behaviour. Int. J. Exp. Pathol. 2015;96:1–10. doi: 10.1111/iep.12112. PubMed DOI PMC

Hudak A., Veres G., Letoha A., Szilak L., Letoha T. Syndecan-4 is a key facilitator of the SARS-CoV-2 delta variant’s superior transmission. Int. J. Mol. Sci. 2022;23:796. doi: 10.3390/ijms23020796. PubMed DOI PMC

Sarrazin S., Lamanna W.C., Esko J.D. Heparan sulfate proteoglycans. Cold Spring Harb. Perspect. Biol. 2011;3:a004952. doi: 10.1101/cshperspect.a004952. PubMed DOI PMC

Capila I., Linhardt R.J. Heparin-protein interactions. Angew. Chem. Int. Ed. Engl. 2002;41:391–412. doi: 10.1002/1521-3773(20020201)41:3<390::AID-ANIE390>3.0.CO;2-B. PubMed DOI

Hileman R.E., Fromm J.R., Weiler J.M., Linhardt R.J. Glycosaminoglycan-protein interactions: Definition of consensus sites in glycosaminoglycan binding proteins. Bioessays. 1998;20:156–167. doi: 10.1002/(SICI)1521-1878(199802)20:2<156::AID-BIES8>3.0.CO;2-R. PubMed DOI

Vallet S.D., Clerc O., Ricard-Blum S. Glycosaminoglycan-protein interactions: The first draft of the glycosaminoglycan interactome. J. Histochem. Cytochem. 2021;69:93–104. doi: 10.1369/0022155420946403. PubMed DOI PMC

Raman R., Sasisekharan V., Sasisekharan R. Structural insights into biological roles of protein-glycosaminoglycan interactions. Chem. Biol. 2005;12:267–277. doi: 10.1016/j.chembiol.2004.11.020. PubMed DOI

Simon Davis D.A., Parish C.R. Heparan sulfate: A ubiquitous glycosaminoglycan with multiple roles in immunity. Front. Immunol. 2013;4:470. doi: 10.3389/fimmu.2013.00470. PubMed DOI PMC

Cardin A.D., Weintraub H.J. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis. 1989;9:21–32. doi: 10.1161/01.ATV.9.1.21. PubMed DOI

Mali M., Jaakkola P., Arvilommi A.M., Jalkanen M. Sequence of human syndecan indicates a novel gene family of integral membrane proteoglycans. J. Biol. Chem. 1990;265:6884–6889. doi: 10.1016/S0021-9258(19)39232-4. PubMed DOI

Billings P.C., Pacifici M. Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: Mechanisms and mysteries. Connect. Tissue Res. 2015;56:272–280. doi: 10.3109/03008207.2015.1045066. PubMed DOI PMC

Christianson H.C., Belting M. Heparan sulfate proteoglycan as a cell-surface endocytosis receptor. Matrix Biol. 2014;35:51–55. doi: 10.1016/j.matbio.2013.10.004. PubMed DOI

Letoha T., Hudak A., Kusz E., Pettko-Szandtner A., Domonkos I., Josvay K., Hofmann-Apitius M., Szilak L. Contribution of syndecans to cellular internalization and fibrillation of amyloid-beta(1-42) Sci. Rep. 2019;9:1393. doi: 10.1038/s41598-018-37476-9. PubMed DOI PMC

Parolini I., Sargiacomo M., Galbiati F., Rizzo G., Grignani F., Engelman J.A., Okamoto T., Ikezu T., Scherer P.E., Mora R., et al. Expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane. Retention of caveolin-2 at the level of the golgi complex. J. Biol. Chem. 1999;274:25718–25725. doi: 10.1074/jbc.274.36.25718. PubMed DOI

Saphire A.C., Bobardt M.D., Zhang Z., David G., Gallay P.A. Syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages. J. Virol. 2001;75:9187–9200. doi: 10.1128/JVI.75.19.9187-9200.2001. PubMed DOI PMC

Steinfeld R., Van Den Berghe H., David G. Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican. J. Cell Biol. 1996;133:405–416. doi: 10.1083/jcb.133.2.405. PubMed DOI PMC

Uhlen M., Bjorling E., Agaton C., Szigyarto C.A., Amini B., Andersen E., Andersson A.C., Angelidou P., Asplund A., Asplund C., et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol. Cell Proteomics. 2005;4:1920–1932. doi: 10.1074/mcp.M500279-MCP200. PubMed DOI

Uhlen M., Fagerberg L., Hallstrom B.M., Lindskog C., Oksvold P., Mardinoglu A., Sivertsson A., Kampf C., Sjostedt E., Asplund A., et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347:1260419. doi: 10.1126/science.1260419. PubMed DOI

Sjostedt E., Zhong W., Fagerberg L., Karlsson M., Mitsios N., Adori C., Oksvold P., Edfors F., Limiszewska A., Hikmet F., et al. An atlas of the protein-coding genes in the human, pig, and mouse brain. Science. 2020;367:eaay5947. doi: 10.1126/science.aay5947. PubMed DOI

Maggi E., Canonica G.W., Moretta L. COVID-19: Unanswered questions on immune response and pathogenesis. J. Allergy Clin. Immunol. 2020;146:18–22. doi: 10.1016/j.jaci.2020.05.001. PubMed DOI PMC

Sharun K., Dhama K., Pawde A.M., Gortazar C., Tiwari R., Bonilla-Aldana D.K., Rodriguez-Morales A.J., de la Fuente J., Michalak I., Attia Y.A. SARS-CoV-2 in animals: Potential for unknown reservoir hosts and public health implications. Vet. Q. 2021;41:181–201. doi: 10.1080/01652176.2021.1921311. PubMed DOI PMC

Jackson C.B., Farzan M., Chen B., Choe H. Mechanisms of SARS-CoV-2 entry into cells. Nat. Rev. Mol. Cell Biol. 2022;23:3–20. doi: 10.1038/s41580-021-00418-x. PubMed DOI PMC

Colombo V.C., Sluydts V., Marien J., Vanden Broecke B., Van Houtte N., Leirs W., Jacobs L., Iserbyt A., Hubert M., Heyndrickx L., et al. SARS-CoV-2 surveillance in Norway rats (Rattus norvegicus) from Antwerp sewer system, Belgium. Transbound. Emerg. Dis. 2021 doi: 10.1111/tbed.14219. PubMed DOI PMC

Bartlett A.H., Park P.W. Heparan sulfate proteoglycans in infection. In: Pavão M.S.G., editor. Glycans in Diseases and Therapeutics. Springer; Berlin/Heidelberg, Germany: 2011. pp. 31–62.

Cagno V., Tseligka E.D., Jones S.T., Tapparel C. Heparan sulfate proteoglycans and viral attachment: True receptors or adaptation bias? Viruses. 2019;11:596. doi: 10.3390/v11070596. PubMed DOI PMC

Bobardt M.D., Salmon P., Wang L., Esko J.D., Gabuzda D., Fiala M., Trono D., Van der Schueren B., David G., Gallay P.A. Contribution of proteoglycans to human immunodeficiency virus type 1 brain invasion. J. Virol. 2004;78:6567–6584. doi: 10.1128/JVI.78.12.6567-6584.2004. PubMed DOI PMC

Pizzato M., Baraldi C., Boscato Sopetto G., Finozzi D., Gentile C., Gentile M.D., Marconi R., Paladino D., Raoss A., Riedmiller I., et al. SARS-CoV-2 and the host cell: A tale of interactions. Front. Virol. 2022;1:815388. doi: 10.3389/fviro.2021.815388. DOI

Gadanec L.K., McSweeney K.R., Qaradakhi T., Ali B., Zulli A., Apostolopoulos V. Can SARS-CoV-2 virus use multiple receptors to enter host cells? Int. J. Mol. Sci. 2021;22:992. doi: 10.3390/ijms22030992. PubMed DOI PMC

Bayati A., Kumar R., Francis V., McPherson P.S. SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis. J. Biol. Chem. 2021;296:100306. doi: 10.1016/j.jbc.2021.100306. PubMed DOI PMC

Ghosh S., Dellibovi-Ragheb T.A., Kerviel A., Pak E., Qiu Q., Fisher M., Takvorian P.M., Bleck C., Hsu V.W., Fehr A.R., et al. beta-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. Cell. 2020;183:1520–1535. doi: 10.1016/j.cell.2020.10.039. PubMed DOI PMC

Prydz K., Saraste J. The life cycle and enigmatic egress of coronaviruses. Mol. Microbiol. 2022;117:1308–1316. doi: 10.1111/mmi.14907. PubMed DOI PMC

Cesar-Silva D., Pereira-Dutra F.S., Moraes Giannini A.L., Jacques G.d.A.C. The endolysosomal system: The acid test for SARS-CoV-2. Int. J. Mol. Sci. 2022;23:4576. doi: 10.3390/ijms23094576. PubMed DOI PMC

Bareford L.M., Swaan P.W. Endocytic mechanisms for targeted drug delivery. Adv. Drug Deliv. Rev. 2007;59:748–758. doi: 10.1016/j.addr.2007.06.008. PubMed DOI PMC

Zimmermann P., Tomatis D., Rosas M., Grootjans J., Leenaerts I., Degeest G., Reekmans G., Coomans C., David G. Characterization of syntenin, a syndecan-binding PDZ protein, as a component of cell adhesion sites and microfilaments. Mol. Biol. Cell. 2001;12:339–350. doi: 10.1091/mbc.12.2.339. PubMed DOI PMC

Chen K., Williams K.J. Molecular mediators for raft-dependent endocytosis of syndecan-1, a highly conserved, multifunctional receptor. J. Biol. Chem. 2013;288:13988–13999. doi: 10.1074/jbc.M112.444737. PubMed DOI PMC

Karnovsky M.J., Karnovsky M.J., Karnovsky M.J., Karnovsky M.L., Karnovsky M.J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron-microscopy. J. Cell Biol. 1965;27:137.

Zsiros O., Nagy G., Patai R., Solymosi K., Gasser U., Polgar T.F., Garab G., Kovacs L., Horcsik Z.T. Similarities and Differences in the Effects of toxic concentrations of cadmium and chromium on the structure and functions of thylakoid membranes in Chlorella variabilis. Front. Plant Sci. 2020;11:1006. doi: 10.3389/fpls.2020.01006. PubMed DOI PMC

Millonig G. A modified procedure for lead staining of thin sections. J. Biophys. Biochem. Cytol. 1961;11:736–739. doi: 10.1083/jcb.11.3.736. PubMed DOI PMC

Nakase I., Niwa M., Takeuchi T., Sonomura K., Kawabata N., Koike Y., Takehashi M., Tanaka S., Ueda K., Simpson J.C., et al. Cellular uptake of arginine-rich peptides: Roles for macropinocytosis and actin rearrangement. Mol. Ther. 2004;10:1011–1022. doi: 10.1016/j.ymthe.2004.08.010. PubMed DOI

Nakase I., Tadokoro A., Kawabata N., Takeuchi T., Katoh H., Hiramoto K., Negishi M., Nomizu M., Sugiura Y., Futaki S. Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis. Biochemistry. 2007;46:492–501. doi: 10.1021/bi0612824. PubMed DOI