Structural And Functional Analysis OF Baculovirus AcMNPV Pk-1Gene

During the interaction of viruses and cells, the protein phosphorylation of viruses and cells are close related to life cycle. Many viruses have activity of protein kinase in their purified virion. This kind of protein kinase is named virus associated protein kinase (VAPK). The VAPK in AcMNPV is PK-1, which is encoded by virus. It is a very conservative gene, which is homologous to protein kinase. The pk-1of AcMNPV is essential for virus replication and nucleocapsid assembly. In this study, we aimed to find the functional domain of pk-1, study the affect of PK-1kinase activity on virus replication, and explore the affect of the pk-1on different promoters.This thesis contained four chapters:In chapter one, we presented a brief introduction about baculovirus, including the classification, replication, genome of baculovirus. We also introduced the background of AcMNPV pk-1and the purposes and meanings of this study.In chapter two, the functional domain of AcMNPV pk-1gene was analysed and identified. Through bac-to-bac operating system, truncated pk-1genes were inserted into pk-1gene knockout bacmids. Transfection-infection assay indicated that those which contained the51-183amino acid fragment of PK-1could rescue the absence of pk-1. Electron microscopy revealed that nucleocapsid assembly of the rescued recombined viruses were normal, while the nucleocapsid assembly of the unrescued recombined viruses were anomalous. One-step growth curves demonstrated that all rescued recombined viruses could reach high titers at96h. In chapter three, the function of AcMNPV pk-1gene was studied. We obtained vAcA pk-1-D137A by inserting mutant pk-1gene into pk-1gene knockout bacmid. Transfection-infection assay indicated that the kinase activity of PK-1was crucial to AcMNPV replication. Different promoters were added to the upstream of egfp, and then each fragment containing promoter and egfp was inserted into pk-1gene knockout bacmids. Transfection assay demonstrated that the absence of pk-1did not affect the function of different promoters.In chapter four, conclusion and discussion of the results.