Abstract
Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.
Highlights
Coronaviruses (CoVs) are a group of related enveloped RNA viruses of which some are known to cause respiratory tract infections in humans
Our study proposes that drugs altering both endocytic entry as well as endosomal acidification can assist in the clinical management of viral infections
We study the endocytosis of receptor binding domain (RBD) of SARS-CoV-2 Spike protein in gastric epithelial cells (AGS) in the presence and absence of angiotensin converting enzyme 2 (ACE2)
Summary
Coronaviruses (CoVs) are a group of related enveloped RNA viruses of which some are known to cause respiratory tract infections in humans. While fusion directly at the plasma membrane is well established for HIV and Influenza virus infections [5,6], both alternatives of entry are feasible for CoV infections depending on the availability of receptors and proteases at the host cell surface. CoVs require proteolytic processing of the viral envelope Spike protein by host cell proteases to gain entry [18,19] These viruses can directly fuse at the cell surface if the Spike protein is cleaved by a cell surface serine protease like TMPRSS2 [12,20], or utilize an endolysosomal route for fusion, where the Spike protein is primed by cysteine protease cathepsins [12,21,22,23]. An understanding of the entry pathways across various host cell types is important as it allows better interpretation of cell-based drug screens and translatability of cellular models of infection
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