Study On The Interaction And Pathogenesis Of MERS - CoV N Protein With Host Protein

MERS is an abbreviation of Middle East respiratory syndrome. According to the WHO, up to now, since the first MERS patient in Saudi Arabia reported in September, 2012, the global accumulated patient infected were 1690 cases which including deaths 603 and the fatality rate 35.6%. MERS pathogen, similar to SARS coronavirus, was called MERS-Coronavirus(MERS-Co V). The clinical data showed that number of lymphocytes was significantly reduced and acute inflammatory response was observed in patients with MERS-Co V. The primary cause of death generally regarded as respiratory failure caused by acute respiratory distress. At present, research work of MERS-Co V mainly focused on antibodies, vaccines, cell receptors and animal models, etc. However the reason of lymphocytes reduction and acute inflammation in patients were not entensively investigated.In our previous studies, SARS-Co V N protein was found interacted with Mannose Associated Serine Protease 2(MASP2) protein and translational enlongation factor 1?(EF-1A). By sequence alignment, We found the sequences of SARS N proein involove in EF1 A and MASP2 interaction were highly homology to MERS-Co V N. So MERS-Co V N protein was proposed to interact with MASP2 protein and EF-1A protein in host.MASP2 is a important serine protease in lectins pathway of the complement activation system. Once the(Mannose binding lection) MBL recognize mannan in the surface virus infected cell it recruits MASP2 and then activates enzyme activity of MASP2. Activated MASP2 could acivate the complement system by cleave complement C4 to C4 a and C4 b. Complement activation will lead to the, release inflammatory factor, and cause local or systemic inflammatory response. EF-1A, as a translation extension factor, catalyze aminoacyl-t RNA to the A site in ribosome. There are many functions of EF-1A except as translation extension factor, such as adjust factor about the assembly of cytoskeleton, microfilament and cytokinesis ring.This research was expected to reveal acute inflammation and lung injury durding MERS coronavirus infection from the interaction between MERS-Co V N protein and MASP2. And this paper was also expected to explain the lymphopenia in MERS patient from molecular level by the interaction between MERS-Co V N protein and EF-1A protein. From two aspects of mediating inflammation and decrease lymphocytes reveal the pathogenesis of MERS, this provides some theoretical guidance for virus prevention, treatment, diagnosis and drug research.MASP2 mainly exist in the serum, and concentration of nucleocapsid was also detected in serum of patients with MERS. The interaction in vitro between MERS-Co V N proteins and MASP2 was proved by immune coprecipitation and Alpha techniques. A mutant deleted 9 key amino acid that proved non-interaction with MASP2 by immune coprecipitation, was constructed to verify the 9 amino acids very important for interaction. The purpose that we purify MERS-Co V N protein from prokaryotic expression system in way of the AKTA system and strong cation exchange chromatography is research the effect of MERS-Co V N protein on MASP2 function in vitro. Purified MERS-Co V N protein can activate MASP2, which was certified by biochemical hydrolyzing reaction in vitro and Western blotting technology. Serum remove C1 q could rule out a classic way of activation complement system, and complement C4 can eliminate the bypass way of activation complement system. We conduct a complement deposition experiment using both serum remove C1 q and complement C4. The ELISA result confirmed that MERS-Co V N protein could enhance MASP2 activity in serum.To study MERS-Co V N protein’s effect on inflammation after activation of the complement system by activated MASP2, mice were administraded with LPS to stimulate acute inflammation simulate virus invasion and then challenged with adenovirus expressing MERS-Co V N protein, adenovirus expressing MERS-Co V N protein with 104-112 aa deletion, or adenovirus. Immediately after the viral infection, mice were gave C1 INH inhibitor, MASP2 antibody, or PBS as control. Administration of MERS-Co V N protein resulted more serious lung injury than the mice inject empty virus. However, the lung inflammatory or injury of these mice injected C1 INH inhibitor or MASP2 antibody was significantly improved. C1 INH inhibitor or MASP2 antibody could protect mice from inflammatory or injury. The survival curves showsed that there was 90% death in inflammatory mice expressed MERS-Co V N protein, however, and no mortality in mice expressing deleted 9 amino acids or injected empty virus was observed. Interestingly, fatality rate decreased to 30% or 50% when C1 INH inhibitor or MASP2 antibody were given to the mice challangeing with MERS-N protein expressing adenovirus. These results collectively suggested that MERS-Co V N protein could aggravate inflammation and reduce survival rate of inflammatory mice by interaction with MASP2.By immune-coprecipitation, MERS-Co V N protein was proved to combine with EF-1A. A series of the truncated and deleted mutants oft MERS N were constructed, It was demonstrated that N protein of MERS-Co V interacted with EF-1A by immuno coprecipitation experiment. It was also found that multiple nuclearated cells were induced by MERS-Co V N protein expression. It was demonstrated to be dependent on the interaction between EF-1A and MERS-Co V N protein, N mutants not binding with EF1 A could not induce such multiple nucleated cells The conclusion is that MERS-Co V N proteins induced multinucleated cells by interaction with EF-1A.To delinated the mechanisms, EF-1A was shown to interact with MERS-Co V N protein due to its C domain. However, we also verified EF-1A interaction with actin was dependent on the presence of the C terminal of the N protein. So, EF-1A protein was competitively combinated and then cytokinesis ring consisting of EF-1A and other proteins would be disturbed. Interaction between MERS-Co V N and EF-1A formed inactive actin polymers and prevent form developing correct cytokinesis ring, then interfered with the cell division and forming multinucleated cells. The reason of cytokinesis blockacd would affect cell proliferation, cell proliferation curve of three kinds of lymphoma cells verifyed that MERS-Co V N proteins inhibited the cell proliferation, which might accord with lymphocytes reduced in MERS patients.Finally, EF-1A, as a translation elongation factor, plays a role in the elongation of protein translation and interacts with MERS-Co V N protein. MERS-Co V N protein was confirmed to promote protein translation by protein translation system in vitro. But the promotion had nothing to do with the interaction of EF-1A.This article discovered that the MERS-Co V N protein could interact with MASP2 proteins in the serum complement system, and the interaction wolud affect its function. We reveal the partial mechanism about lung tissue damage in acute inflammatory reaction during MERS-Co V infection on the molecular level. The interaction between MERS-Co V N protein and EF-1A protein disrupts cytokinesis ring formation and inhibited lymphoid cell proliferation. The mechanism of reduction numbers of lymphocytes in MERS patients could be explained on the molecular level. The found reasons of acute inflammation and low immunity during virus infection could provide important reference value in prevention of MERS and drug research.