Study On Protein Kinase Regulating Polyhedra Assembly And Virus Infection

Virus needs to replicate and proliferate in its host. Many life cycle steps of virus may rely on the phosphorylation of viral proteins. A number of viruses especially the enveloped viruses (such as Retrovirus, Rerpes simplex virus, Orthomyxoviridae, Hepatotropic virus and Baculovirus), not only can use the protein kinase (protein kinase, PK) to function in the host cell, but also their purified virions contain protein kinase activity. Based on the specificity of amino acid substrate, PK can be divided into two categories:tyrosine kinases and serine/threonine kinases. Protein kinases of PK-1 is found from AcMNPV, its gene encoded by the virus. Sequence alignment result suggests that pk-1 is a kind of gene conserved. Our previous study found that pk-1 is an essential gene for viral infection, which is not only involved in the regulation of very late genes, but also the kinase activity plays a key role in the formation of the viral nucleocapsid. Viruses without of PK-1 could not form a normal infection, and typical hollow tubular sheath capsid appeares in the cell nucleus, which is transfected by the virus. In addition, the transfected cells are not also observed polyhedra formed. Therefore, the function of PK-1 has an important relevance to polyhedra formation.Polyhedrin gene (polk) is the highest conservative gene among all the structural proteins encoded by baculovirus, which is the earliest very late gene as well as most well studied. Its expression product is polyhedrin. Under natural conditions, the polyhedrin protein crystals can help to protect its embedded ODV virion, thus to maintain the stable infection capability of the virus. The expression of polh is related to the gene expression and proteins regulation of various baculovirus. The main target of this thesis is the polh gene of AcMNPV which belongs to the baculovirus species. By profiling the protein phosphorylation sites of homologous HearNPV polh, the polh phosphorylation sites of PK-1 was determined. Through the point mutation of polh target sites, making the action site of the kinase delete, thus the protein kinase can not properly fulfill its kinase activity. Then we can analyse the impact of protein kinase phosphorylation to the formation of AcMNPV polyhedra.The first chapter is about the literature review section, including two aspects:firstly, the introduction of baculovirus, respectively in terms of baculovirus classification, the virion morphology, life history, gene expression, genome and applications; secondly, introduction of the background of protein kinase and pk-1 gene. The purpose and significance are also expounded in this paper.The second chapter is about the polh gene of AcMNPV. Site S5, T9, T70 of polh gene were mutated to alanine by site-directed mutagenesis, polh gene of phosphorylation site mutants was obtained via site-directed mutagenesis by PCR amplification in order to construct the recombinant carrier of mutants. By inserting polh gene mutated into AcMNPV genome through Bac-to-Bac system, mutation-containing recombinant virus vAcPH-S5A, vAcPH-T9A, vAcPH-T70A were obtained. Transfection infection experiments suggest that AcMNPV infected cells can form a normal virus particles after the mutagenesis of phosphorylation sites of polh of S5, T9, T70; the amount of mutant viruses polyhedra is significantly less than the wild type. SDS-PAGE result shows that there’s no significant difference of the expression of polyhedrin protein between the mutant and wild-type virus. In summary, the assembly of polyhedra can be affected by phosphorylation, but it has no effect on the expression of polyhedrin.The third chapter is about the construction of recombinant AcMNPV Bacmid containing HearNPV pk-1 gene by Bac-to-Bac system. In order to lay the foundation for further study of the impacts of pk-1 from various baculovirus on the viral replication, as well as the formation of polyhedra with the pk-1-deficient AcMNPV. The HearNPV pk-1 was inserted into the AcMNPV genome which the pk-1 is knockout, the recombinant Bacmid vAcpFBI-P-Hpk-1 was obtained.The fourth chapter is a summary and discussion of the study.