Sars-cov N Protein And Cytochrome P450 (cyp4f3) Interaction

Nucleocapsid protein is one of structural proteins of SARS-CoV , Which is known to bind viral RNA to form the helical nucleocapsid. Also, Nucleocapsid protein of SARS-CoV is the major immunogenic antigen which was capable of generating strong humoral and cellular immune responses, suggesting an important application of SARS N protein in SARS diagnosis and prevention. The SARS N protein was shown to play important roles such as regulation of SARS-CoV RNA synthesis and nucleocapsid formation, disassembly of the viral core by binding to human cyclophilin A, and activation of AP-1 signal transduction pathway, which interferences host cell division and induces apoptosis in COS-1 cells in the absence growth factors. In our previous work, we discovered that the rabbits coimmuned with SARS-CoV N protein and S protein displayed more serious inflammation than those with single S protein. This indicated that the N protein be involved in pathogensis of SARS-CoV.To delinate the molecular approach by which N protein was involved in the pathogensis, full length SARS- CoV N protein was employed as bait protein to screen a cDNA library from human fetal liver in the yeast two hybridization system. The results indicated that SARS-CoV N protein may interact with human Cytochrome P450 family 4 peptide 3 (CYP4F3). In this study, the direct interaction between SARS-CoV N protein and CYP4F3 was demonstrated by immuno-coprecipitation and invtro binding assay. BIACORE assay indicated that the K_D value(affinity constant) between CYP4F3 and SARS-CoV N protein was 4.3×10^-11 (stronger than the affinity between antigen and antibody, which is approximately 10^-10). CYP4F3 can interact with both the 5' and 3' end of the SARS-CoV N protein. Furthermore, we discovered that theω-hydroxylase activity of CYP4F3 can be inhibited by binding with SARS-CoV N protein and then decrease the degradation of the substrate(LTB₄, an important inflammatory factor). Moreover, we assayed the serum LTB₄ level of various SARS patients, the results showed that the LTB₄ level of the patients' early serums was markedly higher than those ten days later and the LTB4 level of SARS patients was much higher than that of the common pneumonia patients. These all support our experimental result.The results found basis for mechanism of SARS-CoV .