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

​该病是一种异质性很强的疾病，以内脏为靶点，有许多可能的并发症、发病率和显著的全球死亡率。此外，正如武汉早期病例所描述的那样，SARS-CoV-2病毒可以靶向引起病毒性结膜炎的眼睛。据估计，1/8的COVID-19病例有某种形式的眼部受累，这使其成为视觉研究和NEI感兴趣的主题。在进化过程中，SARS-CoV-2刺突糖蛋白（S蛋白）在681-684残基处获得了一个新的4个氨基酸插入物- prra -，这是SARS-CoV等其他B - cov谱系中所没有的，该插入物由一个包含串联罕见密码子的12碱基RNA序列编码。我们的基本假设是，这个RNA序列构成了一个核糖体暂停位点，具有类似于过早停止密码子的特性。或者，这些位点可能参与大的多结构域蛋白（如S蛋白）翻译的暂停或解析，以允许连续结构域的适当折叠。这个- prra -位点是一个蛋白蛋白酶裂解位点，在SARS-CoV-2的毒力中也起着重要作用。使问题复杂化的是SARS-CoV-2变体的出现，包括furin位点的突变（α变体中的- hrra -和δ变体中的- rrra -）。目前主要的Omicron变体具有- hrra - furin位点。此外，Omicron变体在S蛋白中含有N679K突变。我们还希望确定SARS-CoV-2使用哪些受体进入眼表细胞，因为它们似乎与其他细胞（如肺细胞）上的受体不同。

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. 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 variants contain the N679K mutation in S protein. 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 adjacent O-glycosylation sites. It was published that the P681H and the P681R mutations resulted in decreased glycosylation of spike protein and increased furin cleavage. 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. Analysis of the role of mutations at the furin site on S protein pseudovirion infectivity is also ongoing. b) Additionally, we have infected ARPE-19 ocular cells with original spike-pseudotyped lentivirus and determined that its infection is independent of the ACE2 receptor. We performed studies aimed at identifying the ocular surface receptor for S protein and found that infection could be blocked by anti-LDLR antibodies, anti-Caveolin1, anti-Dynamin antibodies and cholesterol-depletion agents (25-hydroxycholesterol and beta-cyclodextrins). We did not see any inhibitory effect with anti-clathrin, anti-flotillin, anti-SRB1, anti-vimentin, anti-AXL, anti-neurophilin, anti-EGFR and anti-CD147 antibodies. We confirmed that Bafilomycin A1, a well-known endosomal/lysosomal acidification agent, effectively blocked pseudovirion infection of ARPE-19 cells. Based on our results, we propose that caveolae and LDLR receptor are the component of receptor-dependent endocytosis machinery that SARS-CoV-2 virus uses to infect certain tissues such as ocular cells. We suspect that virus binding to LDLR receptors highjacks LDL-based cholesterol transport in cells. This work is being prepared for submission for publication. To further confirm LDLR function as one of the receptors for SARS-CoV-2 we plan to study the effect of knockdown of LDLR on infection of ARPE-19 cells by original spike-pseudotyped lentiviruses. The next stage is to test our hypothesis on whole eye human retinal organoids generated from H9 embryonal stem cells (ESCs). We have found that organoids are susceptible to spike-pseudotyped lentivirus infection at 2 months but not after 3 months of differentiation. We will look at colocalization of virus and LDLR receptor on the surface of organoids by immunofluorescence microscopy and try to block infection of organoids with anti-LDLR, anti-dynamin, and anti-caveolin 1 antibodies. The dominant current Omicron variants have a -HRRA- furin site. In addition, the Omicron variants contain the N679K mutation in S protein. We have synthesized the Omicron version of the furin site in original S protein and will use this construct to generate lentiviral pseudovirions for infection of ARPE-19 cells and SEAM organoids. We wish to learn if the furin site is important for LDLR receptor recognition and internalization of 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. Additionally, in collaboration with Dr. A.V. Bocharov (NIH-CC), we will test our spike-pseudotyped lentiviruses in LDLR-, SR-BI-, SR-BII- and CD36-stable transfected HeLa cell lines.