Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells
Abstract
:1. Introduction
2. Results
2.1. Spike Pseudovirions Can Infect ARPE-19 Independently of ACE2
2.2. Perturbation of Cholesterol Homeostasis Inhibits Spike-Pseudovirion Infection of ARPE-19
2.2.1. Cyclodextrins
2.2.2. Oxysterol 25-HC
2.3. Clathrin-Dependent and Flotillin-Dependent Endocytic Pathways Are Not Major Pathways in Internalization of SARS-CoV-2 Spike Pseudotyped Lentiviral Particles
2.4. Caveolae-Mediated LDL Transcytosis Is Highjacked by Spike Pseudotyped Lentivral Particles to Infect ARPE19 Cells in Our SARS-CoV-2 Infection Model
2.5. LDL Receptor Overexpression in HeLa (hLDLR-HeLa) Greatly Increased Spike Pseudotyped Lentivral Particles Uptake in Our Model of SARS-CoV-2 Infection
2.6. Modes of Evolution of ACE2 and LDLR
3. Discussion
4. Materials and Methods
4.1. Antibodies, Inhibitors, and Reagents
4.2. Cells and Culture
4.3. Immunoblot Analysis
4.4. Production of SARS-CoV-2 Spike Protein Pseudovirions
4.4.1. Measurement of Physical and Infectious Viral Titer
4.4.2. Pseudovirus Infection Assay
4.5. Cell Viability Assay
4.6. siRNA Treatment and Pseudovirion Infection of ARPE-19 Cells
4.7. Multiple Alignments Analyses
4.8. Statistical Analysis of Data
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Antibody | Catalog No. | Company Name |
---|---|---|
Normal Mouse IgG (1 mg/mL) | 12-371 | Millipore Sigma (Burlington, MA, USA) |
Normal Rabbit IgG (1 mg/mL) | 12-370 | Millipore Sigma (Burlington, MA, USA) |
Mouse anti-EGFR monoclonal clone LA22 | 05-104 | Millipore Sigma (Burlington, MA, USA) |
ACE-2 mouse monoclonal IgG2A clone 171606 | MAB933 | R&D Systems (Minneapolis, MN, USA) |
ACE-2 mouse monoclonal IgG2A clone 535919 | MAB9332-100 | R&D Systems (Minneapolis, MN, USA) |
ACE-2 goat polyclonal IgG (0.2 mg/mL) | AF933 | R&D Systems (Minneapolis, MN, USA) |
Mouse anti-human monoclonal Neuropilin-1 (0.34 mg/mL) | MAB3870 | R&D Systems (Minneapolis, MN, USA) |
Mouse monoclonal Ultra-Leaf™ Purified anti-human CD147 (2 mg/mL) | 306221 | Biolegend® (San Diego, CA, USA) |
Axl rabbit polyclonal IgG (0.34 mg/mL) | PA5-34658 | Thermo Fisher Scientific (Waltham, MA, USA) |
Caveolin-1 rabbit polyclonal IgG (1 mg/mL) | PA1-064 | Thermo Fisher Scientific (Waltham, MA, USA) |
Vimentin (V9) mouse monoclonal (0.5 mg/mL) | sc-6260 | Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) |
Vimentin chicken polyclonal IgY | NB-300-223 | Novus Biologicals (Centennial, CO, USA) |
Dynamin I/II rabbit polyclonal IgG | 2342 | Cell Signaling Technology® (Danvers, MA, USA) |
Flotilin-1 rabbit polyclonal IgG (0.34 mg/mL) | 3253 | Cell Signaling Technology® (Danvers, MA, USA) |
Clathrin HC (C-20) goat polyclonal IgG (200 µg/mL) | sc-6579 | Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) |
LDL Receptor Mouse monoclonal [1B10H10] antibody (1 mg/mL) | ab204941 | Abcam (Waltham, MA, USA) |
LDL Receptor Mouse monoclonal clone C7 | MABS26 | Millipore Sigma (Burlington, MA, USA) |
SR-B1 rabbit polyclonal IgG | NB400-113 | Novus Biologicals (Centennial, CO, USA) |
Secondary IRDye 800CW Goat Anti-Rabbit | 925-32211 | LI-COR Biosciences, Lincoln, NE, USA |
Secondary IRDye 680RD Goat Anti-Mouse | 926-68070 | LI-COR Biosciences, Lincoln, NE, USA |
Secondary IRDye 680RD Donkey Anti-Goat | 925-68074 | LI-COR Biosciences, Lincoln, NE, USA |
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Uppal, S.; Postnikova, O.; Villasmil, R.; Rogozin, I.B.; Bocharov, A.V.; Eggerman, T.L.; Poliakov, E.; Redmond, T.M. Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells. Int. J. Mol. Sci. 2023, 24, 11860. https://doi.org/10.3390/ijms241411860
Uppal S, Postnikova O, Villasmil R, Rogozin IB, Bocharov AV, Eggerman TL, Poliakov E, Redmond TM. Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells. International Journal of Molecular Sciences. 2023; 24(14):11860. https://doi.org/10.3390/ijms241411860
Chicago/Turabian StyleUppal, Sheetal, Olga Postnikova, Rafael Villasmil, Igor B. Rogozin, Alexander V. Bocharov, Thomas L. Eggerman, Eugenia Poliakov, and T. Michael Redmond. 2023. "Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells" International Journal of Molecular Sciences 24, no. 14: 11860. https://doi.org/10.3390/ijms241411860