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

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

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.