Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells

• Published in: International journal of molecular sciences 2023-07, Vol.24 (14), p.11860
• Main Authors: Uppal, Sheetal, Postnikova, Olga, Villasmil, Rafael, Rogozin, Igor B, Bocharov, Alexander V, Eggerman, Thomas L, Poliakov, Eugenia, Redmond, T Michael
• Format: Article
• Language: English
• Subjects: Angiotensin-Converting Enzyme 2 - pharmacology; Antibodies; Apoptosis; Biosynthesis; Cell culture; Cholesterol; Cholesterol - metabolism; Clathrin; Clathrin - metabolism; COVID-19; Cyclodextrins; Dextrins; Dynamin II; Enzymes; Health aspects; Homeostasis; Humans; Infection; Infections; LDL receptor; LDLR; Lipids; Lipoproteins, LDL - pharmacology; Low density lipoproteins; low-density lipoprotein receptor; Physiological aspects; Proteins; S-protein; SARS-CoV-2; SARS-CoV-2 - metabolism; Scientific equipment and supplies industry; Severe acute respiratory syndrome coronavirus 2; Spike Glycoprotein, Coronavirus - pharmacology; spike protein; Viral antibodies; Viral infections; Virus Internalization; Viruses
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms241411860

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.