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Amiodarone Alters Late Endosomes and Inhibits SARS Coronavirus Infection at a Post-Endosomal Level

Konrad Stadler^1,*, Huy Riem Ha², Vincenzo Ciminale³, Carlo Spirli^4,, Giulietta Saletti¹, Marco Schiavon⁵, Daniela Bruttomesso⁶, Laurent Bigler⁷, Ferenc Follath², Andrea Pettenazzo⁸ and Aldo Baritussio⁹

¹ Novartis Vaccines, Siena, Italy; ² Cardiovascular Therapy Research Laboratory, Department of Internal Medicine, University Hospital of Zurich, Zurich, Switzerland; ⁷ Institute of Organic Chemistry, University of Zurich, Zurich, Switzerland; ⁴ Venetian Institute of Molecular Medicine, University of Padova, Padova, Italy; ⁵ Divisione di Chirurgia Toracica, and Departments of ³ Oncological and Surgical Sciences, ⁶ Clinical Medicine, ⁸ Pediatrics, and ⁹ Medical and Surgical Sciences, University of Padova, Padova, Italy

Correspondence and requests for reprints should be addressed to Aldo Baritussio, M.D., Clinica Medica I, Department of Medical and Surgical Sciences, University of Padova, 35128 Padova, Italy. E-mail aldo.baritussio{at}unipd.it

Amiodarone interferes with the endocytic pathway, inhibits proteolysis, and causes the formation of vacuoles, but uptake and intracellular distribution of the drug, origin of vacuoles, and functional consequences of amiodarone accumulation remain unclear. Our objective was to study amiodarone uptake, clarify the origin of vacuoles, and investigate the effect of amiodarone on the life cycle of the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS), which, to enter cells, relies on the proteolytic cleavage of a viral spike protein by the endosomal proteinase cathepsin L. Using alveolar macrophages, we studied uptake of ¹²⁵I-amiodarone and ¹²⁵I-B2, an analog lacking the lateral group diethylamino-β-ethoxy, and analyzed the effects of amiodarone on the distribution of endosomal markers and on the uptake of an acidotropic dye. Furthermore, using Vero cells, we tested the impact of amiodarone on the in vitro spreading of the SARS coronavirus. We found that (1) amiodarone associates with different cell membranes and accumulates in acidic organelles; (2) the diethylamino-β-ethoxy group is an important determinant of uptake; (3) vacuoles forming upon exposure to amiodarone are enlarged late endosomes; (4) amiodarone inhibits the spreading in vitro of SARS coronavirus; and (5) trypsin cleavage of the viral spike protein before infection, which permits virus entry through the plasma membrane, does not impair amiodarone antiviral activity. We conclude that amiodarone alters late compartments of the endocytic pathway and inhibits SARS coronavirus infection by acting after the transit of the virus through endosomes.

Key Words: amiodarone • endocytic pathway • SARS coronavirus

CLINICAL RELEVANCE This article shows that vacuoles are enlarged late endosomes and that amiodarone inhibits in vitro severe acute respiratory syndrome coronavirus infection acting after the delivery of the viral genome into the cytoplasm.