Sars-cov N Protein With Masp2 Of Interactions And Biological Significance

Since the first case was reported in late 2002 in Guangdong Province, China, Severe Acute Respiratory Syndrome (SARS) infected more than 8000 people and caused more than 700 deaths in the world in the following months with a mortality rate of nearly 10%. Clinical presentation showed that the SARS coronavirus (SARS-CoV) induced severe activation of the host immune system and acute inflammation. Respiratory insufficiency leading to acute respiratory distress syndrome (ARDS) and respiratory failure was the main cause of death among fatal cases of SARS. The majority of recent studies have focused on SARS-CoV-associated cellular receptors, which mediate the infection of target cells via the surface protein of SARS-CoV. Research on virucidins and vaccines for SARS has also progressed rapidly. However, the main cause of severe acute inflammation, especially severe pulmonary inflammation, remains unclear.In our previous study, we showed with yeast two-hybrid experiments using SARS-CoV nucleocaspid (N) protein as bait protein that MBL-associated serine protease 2 (MASP2 )potentially interacts with SARS-CoV N protein. MASP2 is a main serine protease of the lectin pathway in the complement system. After mannan-binding lectin(MBL) identified surface carbohydrates of pathogens, MBL undergoes a conformation change that results in the auto-activation of MASP2. Then, activated MASP2 is able to hydrolyze complement C4 and C2, successively. C4 and C2 subsequently generate C3 convertase, thereby initiating the downstream reaction of complement activation.We endeavored to reveal the effects of SARS-CoV on the severe immune response and acute inflammation of the host by studying the interaction between SARS-CoV N protein and MASP2. With these studies, we would be able to make a contribution to antiviral drug development.We found that SARS-CoV N protein interacts with MASP2 in vitro by immunoprecipitation, and this interaction depends on the presence of Ca2+. We hypothesize that SARS-CoV N protein binds to MASP2 in the serum of SARS patients because SARS-CoV N has been found high levels in serum, and could be used as an early diagnostic marker for SARS. We confirmed this result by immunoprecipitation in both human serum and mouse serum; therefore, we could establish the mouse as an animal model for subsequent functional studies. Next, we constructed shortened mutants, and we found that the RNA-binding domain of SARS-CoV N protein and the CUB1-EGF-CUB2 domains of MASP2 interacted with one another, and the latter was important for MASP2 autoactivation. As a result, SARS-CoV N might play a role in MASP2 autoactivation.To directly study the effect of SARS-CoV N protein on MASP2 function in vitro, we purified the SARS-CoV N protein by strong acidic cation exchange and molecular sieve chromatography. We obtained pure MASP2 protein by prokaryotic expression and inclusion-body protein renaturation, and we obtained MBL protein from human plasma by affinity chromatography. We found that the SARS-CoV N protein helps MASP2 engage in homodimerization by its interaction with MASP2, especially in the presece of MBL and mannan. We detected MASP2 binding to MBL-coated plates by Enzyme-linked immunosorbent assay(ELISA), and we determined that the SARS-CoV N protein induced binding between MASP2 and MBL. We added purified MASP2, SARS-CoV N protein, and mannan-activated MBL to an in vitro biochemical reaction system with the necessary ions, detected the hydrolyzed MASP2 by western blotting, and found that the SARS-CoV N protein helps MBL activate MASP2. Next, we added purified C4 to the reaction system, and in the same way, we found that C4 hydrolysis was enhanced by the SARS-CoV N protein, especially in the presence of MBL and mannan. Then, we detected serum MBL-MASP2 function by C4b-deposition assay, and we confirmed that the SARS-CoV N protein had an enhancing effect on MBL-MASP2 activity.To extend the findings regarding the SARS-CoV N protein to the human serum system, we used C1q-depleted human serum as a complement pool to eliminate the classic pathway, and we used different reaction buffers to separate the MBL pathway and the alternative pathway. Then, we detected activated C3 and C5b-9 in the solid phase by a complement deposition assay and found that activation of the MBL pathway was almost twice that of its normal activity in the presence of SARS-CoV N protein.To confirm the effects of the SARS-CoV N protein on the host immune system, we used an inflammatory mouse model. We used an adenoviral vector to express SARS-CoV N protein in mice and found that the level of foreign SARS-CoV N protein in mouse serum was nearly the same as in SARS patient serum by western blotting and ELISA. Furthermore, we demonstrated that LPS could activate complement C4 and C3, while both C1INH and MASP2 antibodies had inhibitory effects. After activating the mouse complement system by tail vein injection of LPS, we obtained blood samples by cutting the tail each hour. We quantified the levels of the inflammatory factor LTB4, which is produced by the downstream reaction of complement activation. We found that LTB4 levels peaked in the first 1~2 hours and began to diminish after 3 hours. After 5 hours, LTB4 was at a level lower than normal concentrations. C1INH and silybin inhibited the generation of LTB4, and silybin was more effective when used more than once. After expressing the SARS-CoV N protein and injecting LPS through the tail vein, we found that the levels of LTB4 in mouse serum doubled when we expressed SARS-CoV N protein, while C1INH and silybin still inhibited the production of LTB4. Then, we grouped the animals, injected adenovirus-SARS-CoV N or adenovirus-null (as a control), used LPS as a activator of the complement system, recorded the number of live mice, and found that LPS-treated mice that expressed SARS-CoV N protein died within 14 hours, while 70% of the LPS-treated mice previously infected by adenovirus-null were still alive after 24 hours; in addition, silybin could protect animals from death. Pulmonary histopathological examinations showed that mice expressing SARS-CoV N protein suffered severe pulmonary injury, with thickened alveolar walls and increased white blood cell count. These symptoms were much more serious than in mice treated only with LPS or with adenovirus-SARS-CoV N, while silybin could relieve the symptoms.We found for the first time that the SARS-CoV nucleocaspid protein interacts with serum proteins associated with the immune system and affects the activation of complement system. Our study revealed one mechanism by which SARS causes severe inflammation and pulmonary injury at the molecular level. Furthermore, we obtained intriguing results with our serum reaction system and animal models. This study may offer clues to the pathogenicity of SARS and the interaction between the host innate immune system and the virus while also contributing to knowledge regarding major infectious disease control and prevention. .