Abstract:
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.
摘要:
补体系统过度激活,先天免疫的主要组成部分,已被认为是严重covid-19患者的核心临床特征之一。然而,病毒如何通过激活的补体途径网络逃避靶向清除仍然是一个谜。这里,我们确定SARS-CoV-2编码的开放阅读框8(ORF8)蛋白是人补体C3/C3b成分及其代谢产物的主要结合伴侣之一。我们的结果表明,在液相中将ORF8与C3/C3b预孵育在替代途径(AP)中具有两个直接的功能后果;这种预孵育抑制因子I(FI)介导的蛋白水解并阻断因子B(FB)酶原激活为活性Bb。ORF8结合导致因子H(FH)和FB与C3b的闭塞,使复合物抵抗FI介导的蛋白水解和抑制pro-C3-转化酶(C3bB)的形成,分别。我们还证实了ORF8的补体抑制活性在我们的溶血为基础的测定,其中ORF8阻止人血清诱导的兔红细胞裂解,IC50值为约2.3μM。ORF8的这种抑制特性也得到了计算机蛋白质-蛋白质对接分析的支持,因为它似乎与C3b的β链建立了主要的相互作用,将自身定位在C3bCUB附近(C1r/C1s,Uegf,Bmp1)结构域,如拟肽化合物,空间阻碍补体扩增所需的必需辅因子的结合。因此,ORF8具有作为AP中关键调控步骤的抑制剂的特征,汇聚加速C3-转化酶的衰变,衰减补体扩增环。