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 beta-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. 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). We also wish to determine which receptors SARS-CoV-2 uses to enter ocular surface cells, as they appear to be different than those on other cells, such as lung cells. In the past year, we have been investigating aspects of the translational regulation of S protein. We have made the following progress: a) As indicated, S protein contains a unique 4 amino acid -PRRA- insertion sequence at amino acid residues that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence wildtype spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection compared to the optimized sequence. Further 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. A manuscript describing these results was published in this reporting period. Ongoing/future work will expand these findings as well as use ribosome profiling sequence analysis to characterize the mechanism. Analysis of the role of mutations at the furin site on S protein pseudovirion infectivity is also ongoing. b) Additionally, we are beginning studies aimed at identifying the ocular surface receptor for S protein. These studies are being done in collaboration with Dr. Tim Blenkinsop, Icahn School of Medicine at Mount Sinai, NYC. This work is ongoing.