Role of SARS-CoV-2 Spike Protein and Accessory ORFs in the immune pathogenesis of COVID-19

β-冠状病毒是一个正链包膜RNA病毒家族，包括严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）。 我们最近的研究集中在两个SARS-CoV-2编码蛋白，开放阅读框（ORF）3a和8。以前的研究SARS-CoV-1确定ORF 3a作为疾病发病机制的一个重要因素，最近的研究SARS-CoV-2也得出了同样的结论。ORF 3a是一种跨膜蛋白，包含几个保守的基序，包括富含半胱氨酸的基序。 我们已经证明SARS-CoV-2 ORF 3a寡聚体插入到血浆和溶酶体膜以及一些不确定的细胞囊泡中。其在细胞中的表达通过细胞凋亡和坏死引起细胞死亡;溶酶体损伤;细胞内囊泡形成增加;和宿主细胞自噬的破坏。它还涉及SARS-CoV-2病毒粒子从感染细胞中的排出。我们已经研究了几个半胱氨酸到丙氨酸取代突变的影响，常见的Q57 H突变的影响，和各种C-末端截短的影响。我们已经确定，在半胱氨酸-133形成二硫键是ORF 3a寡聚化的必要条件。Q57 H突变改变了ORF 3a转染细胞的形态，引发微峰形成。C-末端截短逐渐改变ORF 3a的细胞内定位，导致大多数截短的蛋白质局限于ER。ORF 3a与多种病毒和宿主蛋白相互作用。ORF 3a与包膜蛋白（E）以及NSP 3之间的相互作用已通过免疫共沉淀和荧光寿命成像（FILM）显微镜验证。我们已经记录了ORF 3a和以下宿主蛋白之间的相互作用：溶酶体通道蛋白TCP 2，溶酶体膜蛋白TMEM 106，内质网（ER）和质膜氯离子通道CLCC 1，和ER蛋白HMOX 1。HMOX 1是一种调节宿主免疫细胞活性的酶，可以抑制病毒复制和炎症途径。SARS-CoV-2 ORF 3a和HMOX 1共定位于ER。ORF 3a表达稳定细胞内的HMOX 1蛋白水平。这些结果表明ORF 3a可能影响ERC 8，这是一种重要的E3连接酶，有助于引导蛋白质通过ER，并且已知泛素化HMOX 1，影响其表达。TRC 8与ORF 3a一起定位于ER膜上。TRC 8过表达通过ER-吞噬降低ORF 3a蛋白水平，ER-吞噬是一种与自噬相关的过程。TRC 8还减少了其他SARS-CoV 2蛋白，包括刺突蛋白（S），E蛋白，基质蛋白（M），ORF 8和NSP 3。相比之下，TRC 8对ER定位的NSP 4和NSP 6（SARS-CoV-2编码的另外两种蛋白质）的水平没有影响。除了增加胞内小泡外，ORF 3a还增加胞外小泡的产生。含有S、E、N、ORF 3a、ORF 7a和ORF 8蛋白的细胞外囊泡在细胞系中表达后被发现。ORF 3a上调固醇调节元件结合蛋白（SREBP）的水平，所述固醇调节元件结合蛋白是调节参与脂质合成的基因的转录因子，这为ORF 3a表达细胞中细胞内和细胞外囊泡的增加提供了解释。在合作研究中，重组E蛋白和ORF 3a已经被生产并引入磷脂双层纳米盘中用于最终的结构研究。ORF 3a和E蛋白在非洲爪蟾卵母细胞中单独或一起表达，以评估单个通道活性和潜在的相互作用。 最后，针对ORF 3a的抗体已经在兔子中产生。 SARS-CoV-2基因组编码免疫原性分泌蛋白ORF 8。在细胞培养上清液和COVID-19患者血清中检测到细胞外ORF 8。此外，COVID-19患者会产生ORF 8反应性抗体。ORF 8在哺乳动物细胞系中的表达揭示了一个主要的胞浆蛋白与一些ER定位。我们用亲和层析和凝胶过滤纯化了哺乳动物表达的蛋白，并将其荧光标记，当注射到小鼠体内时，ORF 8与淋巴管内皮细胞结合并引发局部炎症。由于ORF 8缺乏内在的化学引诱物活性，其注射可能触发局部化学引诱物的产生。单核细胞和B细胞是人PBMC中结合ORF 8的主要细胞群体。ORF 8诱导人骨髓源性巨噬细胞分泌TNF、IL 6、IL-10、IL-12、IL-1、IL-23、CXCL 10、CCL 17和CXCL 10。 小鼠边缘区B细胞与ORF 8蛋白结合。 其他B细胞亚群也结合，而CD 4和CD 8 T细胞不结合。ORF 8蛋白抑制人外周血B细胞分化向B细胞培养物中添加ORF 8不影响细胞死亡或早期BCR信号传导。它略微降低了B细胞增殖，但显著降低了浆母细胞的生成（45%），并降低了IgG 1、IgG 2、IgG 3、伊加和IgM分泌水平。描述这项工作的手稿正在编写中。与对B细胞抗体产生的影响一致，ORF 8蛋白改变了重要的B转录因子水平，下调IRF 4/Pax-5比率，如通过细胞内流式细胞术评估的。

Beta-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our recent studies have focused on two SARS-CoV-2 encoded proteins, open reading frame (ORF) 3a and 8. Previous studies of SARS-CoV-1 identified ORF3a as an essential factor for disease pathogenesis and more recent studies with SARS-CoV-2 have reached the same conclusion. ORF3a is a transmembrane protein that contains several conserved motifs including a cysteine-rich motif. We have shown that SARS-CoV-2 ORF3a oligomers insert into the plasma and lysosomal membranes as well as some undefined cellular vesicles. Its expression in cells causes cell death via apoptosis and necrosis; lysosomal damage; increased intracellular vesicle formation; and disruption of host cell autophagy. It is also implicated in the egress of SARS-CoV-2 virions from infected cells. We have investigated the effect of several cysteine-to-alanine substitution mutations, the impact of the common Q57H mutation, and the effect of various C-terminal truncations. We have determined that disulfide bond formation at cysteine-133 is integral for ORF3a to oligomerize. The Q57H mutation alters the morphology of ORF3a transfected cells triggering micro-spike formation. The C-terminal truncations progressively alter ORF3a intracellular localization resulting in the confinement of the most truncated proteins to the ER. ORF3a interacts with multiple viral and host proteins. Interactions between with ORF3a and the envelope protein (E) as well as NSP3 have been verified by co-immunoprecipitations and fluorescent lifetime imaging (FILM) microscopy. We have documented interactions between ORF3a and the following host proteins: lysosomal channel protein TCP2, the lysosomal membrane protein TMEM106, the endoplasmic reticulum (ER) and plasma membrane chloride channel CLCC1, and the ER protein HMOX1. HMOX1 is an enzyme that modulates host immune cell activity and, which can suppress viral replication and inflammatory pathways. SARS-CoV-2 ORF3a and HMOX1 co-localize in the ER. ORF3a expression stabilizes HMOX1 protein levels within cells. These results suggest that ORF3a may affect ERC8, an important E3 ligase that help direct protein traffic through the ER, and is known to ubiquitinate HMOX1, affecting its expression. TRC8 localizes on ER membrane with ORF3a. TRC8 overexpression reduces ORF3a protein levels via ER-phagy, a process related to autophagy. TRC8 also reduced other SARS-CoV2 proteins including the spike protein (S), the E protein, the matric protein (M), ORF8, and NSP3. In contrast, TRC8 had no effect on the levels of the ER localized NSP4 and NSP6, two other SARS-CoV-2 encoded proteins. Besides increasing intracellular vesicles ORF3a also enhanced extracellular vesicle production. Extracellular vesicles containing S, E, N, ORF3a, ORF7a, and ORF8 protein have been found following their expression in cell lines. ORF3a upregulated the levels of Sterol regulatory-element binding proteins (SREBPs), transcription factors that regulate genes involved in lipid synthesis providing an explanation for the increase in intracellular and extra cellular vesicles in ORF3a expressing cells. In collaborative studies recombinant E protein and ORF3a have been produced and introduced into phosopholipid bilayer nanodiscs for eventual structural studies. ORF3a and E protein have been expressed alone or together in Xenopus oocytes to assess individual channel activity and potential interactions. Finally, antibodies to ORF3a have been produced in rabbits. The SARS-CoV-2 genome encodes an immunogenic secreted protein ORF8. Extracellular ORF8 has been detected in cell culture supernatants and in the sera of COVID-19 patients. In addition, COVID-19 patients develop ORF8 reactive antibodies. The expression of ORF8 in mammalian cell lines revealed a largely cytosolic protein with some ER localization. We purified the mammalian expressed protein by affinity chromatography and gel filtration, and fluorescently labeled it. When injected in mice ORF8 binds to lymphatic endothelial cells and triggers local inflammation. Since ORF8 lacks intrinsic chemoattractant activity, its injection likely triggers local chemoattractant production. Monocytes and B cells are the predominant cell populations in human PBMC that bind ORF8. ORF8 induced human bone marrow derived macrophages to secrete TNF, IL6, Il-10, IL-12, IL-1, IL-23, CXCL10, CCL17, and CXCL10. Mouse marginal zone B cells avidly bind ORF8 protein. Other B cell subsets also bind, while CD4 and CD8 T cells do not. ORF8 protein inhibits peripheral blood human B cells differentiation. The addition of ORF8 to B cell cultures did not affect cell death or early BCR signaling. It slightly reduced B cell proliferation, but it significantly reduced the generation of plasmablasts (45%), and it reduced the levels of IgG1, IgG2, IgG3, IgA, and IgM secretion. A manuscript describing this work is in progress. Consistent with an effect on B cell antibody production ORF8 protein altered important B transcription factor levels downregulating the IRF4/Pax-5 ratio as assessed by intracellular flow cytometry.