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

乙型冠状病毒是一个正链包膜RNA病毒家族，包括严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）。我们最近的研究主要集中在两个SARS-CoV-2编码蛋白，开放阅读框（ORF） 3a和8。先前对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的研究也得出了相同的结论。ORF3a是一种跨膜蛋白，包含几个保守基序，包括一个富含半胱氨酸的基序。我们已经证明SARS-CoV-2 ORF3a低聚物插入血浆和溶酶体膜以及一些未定义的细胞囊泡。其在细胞中的表达通过凋亡和坏死导致细胞死亡；溶酶体损伤;细胞内囊泡形成增多；破坏宿主细胞自噬。它还与SARS-CoV-2病毒粒子从受感染细胞的输出有关。我们研究了几种半胱氨酸到丙氨酸取代突变的影响，常见的Q57H突变的影响，以及各种c端截断的影响。我们已经确定半胱氨酸-133上的二硫键形成是ORF3a寡聚化的必要条件。Q57H突变改变了ORF3a转染细胞的形态，触发微刺形成。c端截断逐渐改变ORF3a的细胞内定位，导致大部分截断的蛋白被限制在内质网上。ORF3a与多种病毒和宿主蛋白相互作用。ORF3a与包膜蛋白(E)以及NSP3之间的相互作用已通过共免疫沉淀和荧光寿命成像（FILM）显微镜得到证实。我们已经记录了ORF3a与以下宿主蛋白之间的相互作用：溶酶体通道蛋白TCP2，溶酶体膜蛋白TMEM106，内质网（ER）和质膜氯离子通道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在ER中共定位。ORF3a的表达稳定了细胞内HMOX1蛋白水平。这些结果表明ORF3a可能会影响ERC8， ERC8是一种重要的E3连接酶，有助于指导蛋白质通过内质网的运输，并且已知会使HMOX1泛素化，从而影响其表达。TRC8与ORF3a一起定位于ER膜上。TRC8过表达通过er吞噬降低ORF3a蛋白水平，这一过程与自噬有关。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。相比之下，TRC8对内质网定位的另外两种SARS-CoV-2编码蛋白NSP4和NSP6的水平没有影响。除了增加细胞内囊泡外，ORF3a还增加了细胞外囊泡的产生。在细胞系中发现含有S、E、N、ORF3a、ORF7a和ORF8蛋白的胞外囊泡。ORF3a上调甾醇调节元件结合蛋白（SREBPs）的水平，SREBPs是调节参与脂质合成的基因的转录因子，这可以解释表达ORF3a的细胞内和细胞外囊泡的增加。在合作研究中，已经生产了重组E蛋白和ORF3a，并将其引入磷脂双层纳米盘中，以进行最终的结构研究。ORF3a和E蛋白在爪蟾卵母细胞中单独或共同表达，以评估单个通道的活性和潜在的相互作用。最后，ORF3a抗体已在家兔体内产生。

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.