Abstract
Purpose :
In addition to the respiratory tract, several studies have suggested the ocular surface as a possible infection route for SARS-CoV-2. While the expression level of the virus entry factors ACE2 and TMPRSS2 are relatively low in ocular cells, including the conjunctiva and cornea, these cells are still infected by the virus. To investigate the entry mechanism and possible alternative receptors for SARS-CoV-2 infection in ocular tissues, we studied the infection of the human retinal pigment epithelium ARPE-19 cell line with spike pseudotyped virus and assessed the infection in presence of antibodies and inhibitors targeting different endocytic mechanisms.
Methods :
ARPE-19 cells were cultured in DMEM + sodium pyruvate medium (+ 5% FBS). SARS-CoV-2 spike protein pseudovirions were produced in Expi293F™ suspension cells using a 2nd generation lentiviral packaging system. For pseudovirion infection assays, cells were seeded in 24-well plates and pre-treated with different concentrations of inhibitors for 4-6h followed by pseudovirion infection for 16h. For antibody treatments, cells were infected in the presence of different antibodies for 24h. Viral infectivity titers were measured 48-72h post-transduction based on the ratio of GFP positive-infected cells. We evaluated inhibitor cytotoxicity using the CyQUANT™ MTT cell proliferation assay kit.
Results :
Our results clearly ruled out the role of ACE2 and other proposed alternate receptors for viral entry in ARPE-19 cells using antibody blocking experiments. We found that cholesterol-depleting agents significantly block viral entry and spike-mediated membrane fusion in ARPE-19 cells. Both antibody blocking and chemical inhibitors experiments targeting the lipid raft-mediated endocytic pathway involving dynamin and caveolin-1 showed promising results in inhibiting pseudovirion infection in ARPE-19 cells. We further showed that clathrin- and flotillin-specific antibodies did not inhibit pseudovirion infection in ARPE-19 cells.
Conclusions :
SARS-CoV-2 employs a variety of strategies beyond ACE2 and TMPRSS2 receptors to enter human cells. We demonstrate that the internalization of spike pseudovirions in ARPE-19 cells is via a cholesterol-sensitive, dynamin-dependent raft pathway, possibly implicating caveolae. Based on our studies, we propose an entry mechanism and a potential candidate receptor for SARS-CoV-2 in ARPE-19 cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.