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Principal Investigator: T. Michael Redmond
Organization: NATIONAL EYE INSTITUTE
Fiscal Year: 2024
Award: $386,457
Funding agency: National Eye Institute
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. Our fundamental hypothesis was that this RNA sequence constitutes a ribosomal pausing site, with properties similar to premature stop codons. Alternatively, such sites may be involved in pausing, or parsing, of translation of large multi-domain proteins (such as S protein) to allow for proper folding of successive domains. This -PRRA- site is a furin protease cleavage site that also plays a major role in the virulence of SARS-CoV-2. Our mutagenesis experiments suggest that the insert may create a double-edged weapon (a combination of overlapping furin and translation pausing sites) that has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and, also, of co-translational subdomain folding. These results were published in the prior reporting period. Complicating the issue has been the appearance of SARS-CoV-2 variants, including mutations at the furin site (-HRRA- in alpha variant and -RRRA- in delta variant). The dominant current Omicron variants have a -HRRA- furin site. In addition, the Omicron lineage variants contain the N679K mutation in S protein. We also wish to determine which receptors SARS-CoV-2 uses to enter ocular cells, as they appear to be different than those on other cells, such as lung cells. We constructed the N679K/R681H double mutant in pSelect-Soriginal-GFP construct described in the first publication.
In the past year, we have investigated the mechanism of entry into ocular cells by lentiviral particles pseudotyped with SARS-CoV-2 spike protein. We found that dynamin-dependent caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. We found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in ARPE-19 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. But, surprisingly, 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 concluded that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.
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 compared effect of original spike and N679K/P681R double mutant(omicron) pseudovirions on ARPE 19 cells viability and determined that the Omicron variant is less toxic to ARPE-19 cells. We investigated the internalization path for the Omicron variant in ARPE-19 cells and determined that it uses the same LDLR receptor and not ACE2 receptor as in lung cells. Monoclonal anti-SEB antibody, serendipitously recognizing the furin region of the spike protein, neutralizes original and Omicron spike cell internalization in a similar fashion. Currently, we are preparing this data for publication.
Our next step is to apply our knowledge to SEAM organoids and reproduce the infection with original and Omicron spike pseudovirions on SEAM organoids to discover which are the sensitive cells and the receptors involved.
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