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

National Institute Of Allergy And Infectious Diseases

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Abstract

Our studies have focused on two SARS-CoV-2 encoded proteins, open reading frame (ORF) 3a and 8. 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. 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 mouse CD4 and CD8 T cells do not. We also assessed the binding of ORF8 to human PBMC lymphocytes using a recombinant SARS-CoV-2 ORF8. The results showed that SARS-CoV-2 ORF8 predominantly targets human B cell subsets in PBMC, triggering calcium mobilization, de-phosphorylation of pERM and cell polarization. Additionally, it is internalized in discrete vesicles preserved with wheat germ agglutin (WGA). ORF8 also modulated BCR signaling molecules independently of BCR engagement, disrupting the normal regulation of transcription factors IRF4 and Pax5 expression in B cell differentiation. This disruption ultimately led to reduced plasmablast differentiation and the suppression of humoral immune responses. These findings suggest an inhibitory role of SARS-CoV-2 ORF8 in human B cell differentiation and humoral immune responses during COVID-19 pathogenesis.