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

乙型冠状病毒是包括严重急性呼吸综合征冠状病毒2(SARS-CoV-2)在内的一类正链包膜RNA病毒。我们最近的研究集中在SARS-CoV-2编码的两个蛋白，开放阅读框架(ORF)3a和8。以前对SARS-CoV-1的研究发现ORF3a是疾病发病的重要因素，最近对SARS-CoV-2的研究也得出了同样的结论。ORF3a是一种跨膜蛋白，含有几个保守的基序，其中包括一个富含半胱氨酸的基序。我们已经证明SARS-CoV-2 ORF3a寡聚体插入到血浆和溶酶体膜以及一些未知的细胞囊泡中。它在细胞中的表达通过细胞凋亡和坏死、溶酶体损伤、细胞内囊泡形成增加和宿主细胞自噬的破坏而导致细胞死亡。它还与SARS-CoV-2病毒粒子从感染细胞中排出有关。我们研究了几个半胱氨酸到丙氨酸替代突变的影响，常见的Q57H突变的影响，以及不同C末端截断的影响。我们已经确定半胱氨酸-133上二硫键的形成是ORF3a齐聚所必需的。Q57H突变改变了ORF3a转基因细胞的形态，触发了微棘波的形成。C末端的截断逐渐改变了ORF3a在细胞内的定位，导致大多数截断的蛋白质被限制在内质网。ORF3a与多种病毒和宿主蛋白相互作用。免疫共沉淀和荧光寿命成像(胶片)显微镜证实了ORF3a与包膜蛋白(E)和NSP3之间的相互作用。我们已经记录了ORF3a与以下宿主蛋白的相互作用：溶酶体通道蛋白TCP2，溶酶体膜蛋白TMEM106，内质网(ER)和质膜氯通道CLCC1，以及ER蛋白Hmox1。Hmox1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 ORF3a和Hmox1共定位于内质网。ORF3a的表达稳定了细胞内Hmox1的蛋白水平。这些结果表明，ORF3a可能影响ERC8，ERC8是一种重要的E3连接酶，有助于引导蛋白质通过内质网进行运输，并已知泛素化Hmox1，影响其表达。TRC8与ORF3a结合定位于ER膜上。TRC8过表达通过ER吞噬降低ORF3a蛋白水平，这是一个与自噬相关的过程。TRC8还降低了其他SARS-CoV2蛋白，包括刺突蛋白(S)、E蛋白、基质蛋白(M)、ORF8和NSP3。相反，TRC8对另两个SARS-CoV-2编码蛋白ER定位的NSP4和NSP6的水平没有影响。除了增加细胞内小泡外，ORF3a还促进了细胞外小泡的产生。随着S、E、N、ORF3a、ORF7a和ORF8蛋白在细胞中的表达，已发现含有这些蛋白的胞外小泡。ORF3a上调了Sterol调节元件结合蛋白(SREBPs)的水平，SREBPs是调节与脂质合成相关的基因的转录因子，这为表达ORF3a的细胞内和细胞外小泡的增加提供了解释。在合作研究中，重组E蛋白和ORF3a已经被生产出来，并被引入磷脂双层纳米盘中，用于最终的结构研究。ORF3a和E蛋白已经在非洲爪哇卵母细胞中单独或联合表达，以评估单个通道的活性和潜在的相互作用。最后，已经在兔子身上产生了针对ORF3a的抗体。 SARS-CoV-2基因组编码一种免疫原性分泌蛋白ORF8。已在细胞培养上清液和新冠肺炎患者的血清中检测到细胞外ORF8。此外，新冠肺炎患者还会产生ORF8反应性抗体。ORF8在哺乳动物细胞系中的表达表明，ORF8主要是胞浆蛋白，并有一定的ER定位。我们通过亲和层析和凝胶过滤纯化了哺乳动物表达的蛋白，并对其进行了荧光标记。当注射到小鼠体内时，ORF8与淋巴管内皮细胞结合，并引发局部炎症。由于ORF8缺乏内在的趋化活性，它的注射可能会触发局部趋化物质的产生。单核细胞和B细胞是人PBMC中结合ORF8的主要细胞群。ORF8诱导人骨髓巨噬细胞分泌肿瘤坏死因子、白介素6、白介素10、白介素12、白介素1、白介素23、CXCL10、CCL17和CXCL10。小鼠边缘区B细胞可与ORF8蛋白强烈结合。其他B细胞亚群也结合，而CD4和CD8T细胞不结合。ORF8蛋白抑制人外周血B细胞分化在B细胞培养中加入ORF8不影响细胞死亡或早期bcr信号转导。它能轻微抑制B细胞的增殖，但显著减少浆母细胞的生成(45%)，并降低IgG1、IgG2、IgG3、IgA和IgM的分泌水平。描述这项工作的手稿正在进行中。通过细胞内流式细胞术检测，ORF8蛋白改变了重要的B转录因子水平，下调了IRF4/Pax-5的比率，这与B细胞抗体的产生是一致的。

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.