Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2Research in context
Annarita Miluzio,
Alessandro Cuomo,
Chiara Cordiglieri,
Lorena Donnici,
Elisa Pesce,
Mauro Bombaci,
Matteo Conti,
Alessandra Fasciani,
Luigi Terracciano,
Lara Manganaro,
Mirco Toccafondi,
Alessandra Scagliola,
Stefania Oliveto,
Sara Ricciardi,
Renata Grifantini,
Raffaele De Francesco,
Sergio Abrignani,
Nicola Manfrini,
Stefano Biffo
Affiliations
Annarita Miluzio
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Alessandro Cuomo
Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, 20141, Milan, Italy
Chiara Cordiglieri
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Lorena Donnici
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Elisa Pesce
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Mauro Bombaci
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Matteo Conti
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Alessandra Fasciani
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Luigi Terracciano
Institute of Pathology, University Hospital Basel, 4031, Basel, Switzerland
Lara Manganaro
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Mirco Toccafondi
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Alessandra Scagliola
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Stefania Oliveto
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Sara Ricciardi
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy; Department of Biosciences, University of Milan, 20133, Milan, Italy
Renata Grifantini
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy
Raffaele De Francesco
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy; Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133, Milan, Italy
Sergio Abrignani
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy; Department of Clinical Sciences and Community Health, University of Milan, 20122, Milan, Italy
Nicola Manfrini
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy; Department of Biosciences, University of Milan, 20133, Milan, Italy; Corresponding author. National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy.
Stefano Biffo
National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy; Department of Biosciences, University of Milan, 20133, Milan, Italy; Corresponding author. National Institute of Molecular Genetics, ''Fondazione Romeo ed Enrica Invernizzi'', INGM, 20122, Milan, Italy.
Summary: Background: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. Methods: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. Findings: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. Interpretation: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. Funding: This work was supported by an unrestricted grant from “Fondazione Romeo ed Enrica Invernizzi” to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project – P.I. Manfrini Nicola.
