Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

Ting Gao; Mingdong Hu; Xiaopeng Zhang; Hongzhen Li; Lin Zhu; Hainan Liu; Qincai Dong; Zhang Zhang; Zhongyi Wang; Yong Hu; Yangbo Fu; Yanwen Jin; Kaitong Li; Songtao Zhao; Yongjiu Xiao; Shuping Luo; Lufeng Li; Lingfang Zhao; Junli Liu; Huailong Zhao; Yue Liu; Weihong Yang; Jing Peng; Xiaoyu Chen; Ping Li; Yaoning Liu; Yonghong Xie; Jibo Song; Lu Zhang; Qingjun Ma; Xiuwu Bian; Wei Chen; Xuan Liu; Qing Mao; Cheng Cao

The impact of SARS-CoV N protein on complement activation via the lectin pathway was further investigated by complement deposition assays. Purified C4 was incubated with 30 immobilized MBL-MASP-2 complex in the presence of indicated N protein. The N protein of SARS-CoV, MERS-CoV and SARS-CoV-2 but not the H229E-CoV N potentiated C4b deposition, which was dependent on the activity of MASP-2, in a dose-dependent manner ( Fig. 2F-2H ). Then, immobilized mannan was incubated with C1q-depleted serum (to eliminate the classical pathway) (26) in the presence/absence of SARS-CoV N protein, and the deposited C3 35 fragments (C3b, iC3b and C3dg) were detected by an anti-activated C3 antibody. In concert with C4b deposition, the deposition of activated C3 was evidently increased along with the increase of SARS-CoV N protein levels up to ~40 nM (Fig. S2D) , suggesting an enhanced activity of C3 convertase. In addition, SARS-CoV N protein had little or no effect on activated C3 deposition in calcium-free buffer containing EGTA, which suggests that SARS-CoV N protein-potentiated 40 C3 activation occurs through the LP but not the AP pathway, in which C3 activation is Ca 2+independent (Fig. S2E ). As shown in Figure S2D and S2E, C3b deposition was decreased in the presence of a high concentration of N protein, possibly due to further cleavage of C3b by soluble inhibitors in serum (such as factor H and factor I) when the surface is coated with high densities of C3b (27, 28) . We further tested the deposition of the C5b-9 complex. As a result of amplified 6 complement cascades, significantly increased deposition of the complex was induced by SARS-CoV N protein at a much lower concentration (Fig. S2F ), similar to that observed in patient sera (22) . Activated complement plays a crucial role in the efficient phagocytosis of pathogens and cellular debris by C3b or C5b-mediated opsonization (29) . To study complement-dependent 5 phagocytosis, E. coli and mouse peritoneal macrophages were incubated together in diluted serum with or without SARS-CoV N protein. Bound C3 or its large fragment C3b, the product after C3 cleavage, was stained with FITC-labeled anti-C3c antibody, and the FITC-positive macrophages containing C3-conjugated E. coli were counted under a microscope. In concert with complement activation, complement-dependent phagocytosis by mice peritoneal macrophage in 10 the presence of mouse serum containing complement component including MASP-2 was enhanced remarkably by SARS-CoV N protein compared with the HSA control (Fig. S2G ). These findings indicated that SARS-CoV N protein effectively promoted the activation and opsonic effect of the complement system. 15