SARS-CoV-2-encoded ORF8 protein possesses complement inhibitory properties

Hyperactivation of the complement system, a major component of innate immunity, has been recognized as one of the core clinical features in severe covid-19 patients. However, how the virus escapes the targeted elimination by the network of activated complement pathways still remains an enigma. Here, we identified SARS-CoV-2-encoded ORF8 protein as one of the major binding partners of human complement C3/C3b components and their metabolites. Our results demonstrated that preincubation of ORF8 with C3/C3b in the fluid phase has two immediate functional consequences in the alternative pathway; this preincubation inhibits factor I-mediated proteolysis and blocks factor B zymogen activation into active Bb. ORF8 binding results in the occlusion of both factor H and factor B from C3b, rendering the complexes resistant to factor I-mediated proteolysis and inhibition of pro-C3-convertase (C3bB) formation, respectively. We also confirmed the complement inhibitory activity of ORF8 in our hemolysis-based assay, where ORF8 prevented human serum-induced lysis of rabbit erythrocytes with an IC₅₀ value of about 2.3 μM. This inhibitory characteristic of ORF8 was also supported by in-silico protein-protein docking analysis, as it appeared to establish primary interactions with the β-chain of C3b, orienting itself near the C3b CUB (C1r/C1s, Uegf, Bmp1) domain like a peptidomimetic compound, sterically hindering the binding of essential cofactors required for complement amplification. Thus, ORF8 has characteristics to act as an inhibitor of critical regulatory steps in the alternative pathway, converging to hasten the decay of C3-convertase and thereby, attenuating the complement amplification loop.