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 binding between the N protein of SARS-CoV and MASP-2, and to delineate the interacting domains of the two proteins, lysates of human 293T cells expressing Flag-tagged full-length MASP-2, the N-terminal CUB1-EGF-CUB2 region, or the C-terminal 5 CCP1-CCP1-SP region (Fig. S1A ) were subjected to anti-Flag immunoprecipitation using anti-Flag antibody-conjugated agarose beads. The immunoprecipitates were next incubated with 293T cell lysates expressing GFP-tagged SARS-CoV N protein (GFP-SARS N) or truncated mutants in the presence of 2 mM CaCl 2 or 1 mM EDTA. The adsorbates were probed with anti-Flag or anti-GFP antibodies by immunoblotting. Associations between Flag-MASP-2 and GFP- 10 SARS N were observed only in the presence of CaCl 2 (Fig. 1A) , in agreement with the requirement of Ca 2+ for MASP-2-MBL binding and MASP-2 auto-activation. The CCP1-CCP2-SP region of MASP-2 ( Fig. 1A ) and the N-terminal domain (residues 1-175) of the N protein ( Fig. S1A and Fig. S1B ) were crucial for the association, whereas negative controls, or other truncated regions did not bind, and GFP-tagged full-length or truncated N protein did not co- 15 immunoprecipitate with mouse IgG conjugated beads ( Fig. 1A and Fig. S1B ).