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

新型冠状病毒SARS-CoV-2于2019年底起源于中国武汉，导致大流行性冠状病毒疾病COVID-19，自那时以来对美国和世界其他地区产生了重大的公共卫生和经济影响。该疾病是相当异质性的，靶向内脏器官，具有许多可能的并发症、发病率和显著的全球性死亡率。此外，SARS-CoV-2病毒可以靶向眼睛，引起病毒性结膜炎，正如武汉早期病例所述。据估计，1/8的COVID-19病例有某种形式的眼部受累，使其成为视觉研究和NEI感兴趣的主题。SARS-CoV-2刺突糖蛋白（S蛋白）在其进化过程中，在681-684位残基处获得了一个新的4个氨基酸的插入片段-PRRA，该片段在其他谱系B -CoV如SARS-CoV中不存在，由一个新的12碱基RNA序列编码，该序列包含串联的稀有密码子。我们的基本假设是，这段RNA序列构成了一个核糖体暂停位点，具有类似于提前终止密码子的性质。或者，这些位点可能涉及大的多结构域蛋白（如S蛋白）翻译的暂停或解析，以允许连续结构域的正确折叠。该PRRA位点是弗林蛋白酶切割位点，其在SARS-CoV-2的毒力中也起主要作用。我们的诱变实验表明，插入可能会产生一个双刃武器（重叠弗林蛋白酶和翻译暂停位点的组合），使SARS-CoV-2更容易感染其新的宿主（人）。这强调了核糖体暂停的重要性，以允许蛋白质表达的有效调节，也是共翻译亚结构域折叠。这些结果已于上一报告期公布。使问题复杂化的是SARS-CoV-2变体的出现，包括弗林蛋白酶位点的突变（α变体中的-HRRA和δ变体中的-RRRA）。目前占主导地位的Omicron变体具有-HRRA-弗林蛋白酶位点。此外，Omicron变体在S蛋白中含有N679 K突变。我们还希望确定SARS-CoV-2使用哪些受体进入眼细胞，因为它们似乎与其他细胞（如肺细胞）上的受体不同。 在过去的一年中，我们一直在研究SARS-CoV-2刺突蛋白假型化的慢病毒颗粒进入眼细胞的机制，发现使用LDLR的小窝介导的内吞作用是SARS-CoV-2病毒在眼细胞系ARPE-19中内化的途径。我们取得了以下进展： 我们发现，虽然血管紧张素转换酶2（ACE 2）在ARPE-19细胞中表达，但通过抗体处理阻断ACE 2并不能阻止SARS-CoV-2刺突假病毒体的感染，也不能阻止细胞外波形蛋白和其他富含胆固醇的脂筏蛋白的抗体阻断。接下来，我们通过显示用不同的环糊精和氧固醇25-羟基胆固醇（25-HC）孵育细胞抑制ARPE-19的假病毒体感染来暗示胆固醇稳态在感染中的作用。然而，25-HC的作用可能不是通过胆固醇生物合成，因为与洛伐他汀孵育对感染没有明显影响。此外，我们确定25-HC不太可能对LXR受体产生激动作用，因为LXR激动剂GW 3965在高达5微摩尔GW 3965时对ARPE-19细胞的感染没有显着影响。我们探讨是否涉及内吞途径，但确定网格蛋白依赖性和浮体蛋白依赖性筏不参与。此外，20微摩尔氯丙嗪，网格蛋白介导的内吞作用（CME）的抑制剂，也几乎没有影响。相反，抗发动蛋白I/II抗体阻断了SARS-CoV-2刺突假病毒体的进入，动力蛋白GT3活性的非竞争性抑制剂dynasore也是如此。此外，抗小窝蛋白-1抗体显著阻断ARPE-19的刺突假型慢病毒感染。然而，制霉菌素，一个经典的抑制剂小窝依赖性内吞作用，不影响感染，而吲哚美辛抑制只有在10微摩尔在48小时的时间点。最后，我们发现抗LDLR抗体阻断假病毒体感染的程度与抗小窝蛋白-1和抗发动蛋白I/II抗体相似，而与乱序对照siRNA相比，用LDLR特异性siRNA转染导致刺突假型慢病毒感染减少。因此，我们得出结论，SARS-CoV-2刺突假病毒体感染ARPE-19细胞是一个动力蛋白依赖的过程，主要由LDLR介导。 此外，我们与A. V. Bocharov博士（NIH-CC）和托马斯Eggerman（NIH-CC）合作，在LDL稳定转染的HeLa细胞系中测试了我们的刺突假型慢病毒。这项工作已提交出版，并在本报告所述期间出版。 我们的下一步是在由H9胚胎干细胞（ESC）产生的全眼人类视网膜类器官上测试我们的假设。我们已经发现，类器官在分化2个月时对刺突假型慢病毒感染敏感，但在分化3个月后则不敏感。我们将通过免疫荧光显微镜观察类器官表面病毒和LDLR受体的共定位，并尝试用抗LDLR，抗发动蛋白和抗小窝蛋白1抗体阻断类器官的感染。目前占主导地位的Omicron变体具有-HRRA-弗林蛋白酶位点。此外，Omicron变体在S蛋白中含有N679 K突变。我们已经合成了原始S蛋白中弗林蛋白酶位点的Omicron版本，并将使用该构建体产生慢病毒假病毒粒子，用于感染ARPE-19细胞和SEAM类器官。我们希望了解弗林蛋白酶位点是否对LDLR受体识别和S蛋白内化很重要。这些研究是与纽约西奈山伊坎医学院的Tim Blenkinsop博士合作完成的。这项工作正在进行中。

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 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. In the past year, we have been investigating the mechanism of entry into ocular cells by lentiviral particles pseudotyped with SARS-CoV-2 spike protein, finding that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. We have made the following progress: 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. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, we determined that it was not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 micromolar GW3965. We probed whether endocytic pathways were implicated but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 micromolar chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 micromolar at the 48 h time point. Finally, 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. Additionally, in collaboration with Dr. A.V. Bocharov (NIH-CC) and Thomas Eggerman (NIH-CC), we tested our spike-pseudotyped lentiviruses in LDLR-stable transfected HeLa cell lines. This work was submitted for publication and was published in this reporting period. Our next step 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.