The Phosphorylation Of Several Amino Acids In VP2 Of Mink Enteritis Virus And Its Roles In Viral Amplification

The eclipse phase of the viral replicative cycle has been intensively studied. During eclipse phase, there are multiple interactions between viral proteins and host cell proteins. Therefore, to understand viral amplification process, it is vital to study the changes on structures and functions of viral proteins in eclipse phase. Post-translational modifications, especially phosphorylation, play an important role in regulating protein interactions. Recent reports have indicated that phosphorylation of capsid proteins has significant influence in interactions between viral proteins and host cell proteins.As an autonomous parvovirus within the genus Parvovirus of the family Parvoviridae, mink enteritis viruses (MEV) are among the smallest known viruses with an ssDNA genome enclosed within an icosahedral capsid. Mink viral enteritis, caused by MEV, is an acute infectious disease threatening mink production worldwide. At present, the researches of MEV are focused on prevention and cure of viral enteritis and preparation of MEV vaccine. There is no report on relationship between phosphorylation and MEV amplification.In this thesis, based on the reverse genetic system of MEV, a series of researches have been carried out and the main results are as follows:In order to understand more about MEV, making use of virology research methods and molecular biology methods, the morphology, growth curve and eclipse phase of MEV were defined. Then the structure and characteristics of MEV VP2 were analyzed by bioinformatics software. Meanwhile, to give service to the future experiments, hyperimmune serum of MEV was prepared by immunizing rabbits and the antibody titer was 1:512000.To identify the phosphorylation residues in MEV VP2, making use of web analytics (NetPhos 2.0 Server), we demonstrate that there were 24 potential phosphorylated amino acids in MEV VP2. Meanwhile, purified VP2 was obtained from eukaryotic expressing system and analyzed by LC-MS/MS. Four potential phosphorylated sites were identified, including Ser221 that was also detected by web analytics. Protein structure prediction and simulation of MEV VP2 and mutant MEV VP2 showed that the phosphorylation sites were distributed in loops. The infectious virus clones used in this study were derived from MEV infectious virus clone pB-MEV using site-directed mutation. We obtained 21 mutant MEV infectious clones with single point mutation or multisite mutation. After transfected into F81 cells,7 MEV mutants were rescued.To determine whether phosphorylation of Ser221 in VP2 affects MEV amplification, we compared growth curves of MEV-S221A and MEV-WT in F81 cells. Results showed that the growth of MEV-S221A was delayed compared with MEV-WT and produced lower levels of infectious virus. MEV-WT reached a peak titer of 10-5.5 TCID50/ml at 96 h, whereas MEV-S221A produced approximately two logs lower by 96 h. There was a sharp drop in the curve of MEV-S221A at 36 h. We next detected viral genome replication and VP2 expression of MEV-S221A by using molecular biology method. Results demonstrated that both the rising tendency of viral genomic copies and levels of VP2 expression were almost the same between MEV-WT and MEV-S221A, suggesting that mutation at Ser221 did not affect the replication of viral genome and expression of VP2.In summary, our study provides the first evidence that phosphorylation of MEV VP2 is important to a productive viral life cycle. We also hypothesize that Ser221 of MEV VP2 plays an important role in virion assembly or viral exocytosis, and this provides another avenue for future research.