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

To investigate the effect of SARS-CoV N protein on the MBL-binding capability of MASP-2, purified MBL and MASP-2 (24) were incubated in mannan-coated microplate wells at 4C to avoid MASP-2 activation, and the dynamics of MASP-2: MBL binding were assessed 5 using an anti-MASP2 antibody. Compared with human serum albumin (HSA), a highconcentration component in binding buffer used as a negative control for N protein, the binding of MASP-2 to MBL was significantly enhanced in the presence of SARS-CoV N protein at a relatively low concentration (1%~0.1% of MASP-2) and Ca 2+ , and the potentiation was effectively abrogated by anti-N protein antibody (Fig. 2C) . Moreover, SARS-CoV N protein 10 bearing the 116-124 or 321-323 deletion or N protein from the less pathogenic human coronavirus 229E-CoV showed little or no effect on MASP-2:MBL binding compared with the full-length SARS-CoV N protein (Fig. 2C) . These results indicated that SARS-CoV N proteinpotentiated MASP-2 activation is dependent not only on MASP-2 association but also on N protein dimerization. 15 MASP-2 cleaves complement components C4 and C2 to generate C3 convertase in the lectin pathway of the complement system upon activation. Next, the effect of the SARS-CoV N protein on LP complement activation was assessed by C4 cleavage. Purified C4 was incubated with MASP-2, Mannan, and MBL in the presence/absence of equal molar N protein, and C4 cleavage was observed to be significantly potentiated by the SARS-CoV N protein in the 20 presence of mannan and MBL ( Fig. 2D and Fig. S2A ). Accordingly, mutants of SARS-CoV N protein (Δ116-124 and Δ321-323) as well as N protein from H229E-CoV failed to promote MASP-2-mediated C4 hydrolysis ( Fig. 2E and Fig. S2B) . Notably, an anti-MASP-2 monoclonal antibody or C1INH, an inhibitor of MASP-2 (25), blocked SARS-CoV-potentiated C4 hydrolysis, suggesting that N protein-potentiated C4 cleavage was dependent on MASP-2 activation (Fig. 2E 25 and Fig. S2B ). Moreover MERS-CoV N was also found to potentiate C4 cleavage (Fig. S2C ). These results indicated that N protein prompts C4 cleavage and therefore complement activation by MASP-2 association and activation.