Biochemistry of SARS-CoV-2 Spike Protein and its Ocular Surface Membrane Receptor

National Eye Institute

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Abstract

Novel coronavirus SARS-CoV-2, originating at the end of 2019 in Wuhan, China, causes the pandemic coronavirus disease COVID-19 that has had a major public health and economic impact in the USA and the rest of the world since then. The disease is quite heterogeneous and targets internal organs with many possible complications, morbidity, and significant world-wide mortality. Furthermore, the SARS-CoV-2 virus can target the eye causing viral conjunctivitis as was described in early cases in Wuhan. It has been estimated that 1/8 of COVID-19 cases have some form of ocular involvement, making it a subject of interest to vision research and to the NEI. In the course of its evolution, SARS-CoV-2 Spike glycoprotein (S protein) acquired a novel 4 amino acid insert -PRRA- at residues 681-684, absent in other lineage B -CoVs such as SARS-CoV, that is encoded by a novel 12-base RNA sequence which contains tandem rare codons. The current dominant Omicron variants have a -HRRA- furin site, rather than the -PRRA- site of the original. In addition, the Omicron variants contain the N679K mutation in their Spike (S) proteins. An important goal of this study is to determine which receptors both original and Omicron variant SARS-CoV-2 uses to enter ocular cells, as they appear to be different than those on other cells, such as lung cells. In the past year, we have continued investigating the mechanism of entry of the virus into ocular cells using lentiviral particles pseudotyped with SARS-CoV-2 spike protein. In a previous reporting period we found and published that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. We confirmed 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 concluded that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR. The current dominant Omicron variants have a -HRRA- furin site and the N679K mutation in their S protein. We have synthesized the Omicron version of the furin site in the original S protein and used this construct to generate lentiviral pseudovirions for infection of ARPE-19 cells. We wish to learn if the furin site is important for LDLR receptor recognition and the internalization of S protein. To determine if LDLR is the primary route of Omicron variant internalization in ARPE19 cells we utilized the N679K/P681H spike variant in the pSelect vector. We found that the N679K/P681H S protein variant pseudotyped lentiviruses are internalized through LDLR in ARPE-19 cells in similar degree to the original SARS-COV-2 variant and that the ACE2 receptor does not play any role in infection of ARPE-19 ocular cells by either the original or Omicron lentiviral pseudovirions. We demonstrated that the active form of vitamin D (calcitriol) blocks original spike pseudotyped lentiviral infection of ARPE-19 cells and not toxic to the cell at 25 µM-100 µM concentrations based on the MTT apoptosis assay. However, inactive forms of vitamin D (vitamin D3 and 25-hydroxyvitamin D3) were toxic at these concentrations and caused cell death. This effect is modulated in the presence of Amphoterecin B. We will continue to study the effect of vitamin D on infection and investigate the mechanism of the vitamin D action.