Study On Function Of The Phosphorylation Sites In Baculovirus Protein VLF-1

vlf-1 is a core gene of baculovirus, which presents in all baculoviruses as a multi-functional gene. The VLF-1 encoded by vlf-1 does not only mediate expression of very late gene, but also involves in the nucleocapsid assembly, which is located in the end of nucleocapsid as a structural protein. Deletion experiments showed that the baculovirus knocked out vlf-1 would produce defective nucleocapsid which blocked the replication and infection of the virus.It is found that there are two sites of phosphorylation on VLF-1 in HearNPV virion. Phosphorylation is an important means to regulate the function of proteins. The protein phosphorylation of viruses and cells relates to life cycle of the virus closely during the interaction between viruses and cells. Therefore, we speculated that phosphorylation of VLF-1 might play an important role in its function.Sequence alignment found that AcMNPV VLF-1 also had two serine sites located in the similar region with the phosphorylated sites of HearNPV VLF-1. Because of the close relationship between two kinds of viruses, it is deduced that those sites of VLF-1 could be phosphorylated during AcMNPV infection and phosphorylation in those sites would play an important role in viral infection. In this study, those sites of VLF-1 in AcMNPV were mutated to analyze the function of VLF-1 losing phosphorylation, which was established for further study the role of phosphorylation in the replication of baculovirus AcMNPV, and would provide a reference for the study of phosphorylation of other viruses.In this study, two possible phosphorylation sites S218 and S233 on vlf-1 gene were mutated to alanine site using PCR. On the other hand, vlf-1 knockout AcBacmid containing the AcMNPV genome in Escherichia coli was generated by lambda homologous recombination. Furthermore, wt vlf-1 and mutated vlf-1 (S218A or S233A) containing native promoter region were transposed into polh locus on vlf-1 knockout AcBacmid by Bac-to-Bac expression system. Infectivity of recombinant viruses in Sf9 cells was observed by optical microscope, fluorescence microscope and electron microscope. One step growth curve of recombinant virus was drawn to qualitative analysis the effects of VLF-1 phosphorylation site mutations on viral replication. Finally, SDS-PAGE was used to analysis the effect of VLF-1 phosphorylation site mutation on the expression of very late gene polyhedrin.Those results observed by light and fluorescence microscopy showed that the recombinant viruses which phosphorylation sites were mutated could be replicated in Sf9 cells and the expression of reporter protein GFP as well as the fluorescence diffusion were observed, but polyhedra did not be observed. One step growth curve analysis determined that the infectivity of those mutative viruses was lower than wild-type or rescued baculovirus in culture cells. Subsequent aberrant nucleocapsids were observed in Sf9 cells infected with the mutative viruses using electron microscopy. However, normal nucleocapsids were also observed in the same cells, which ensure the infectivity of the mutative viruses. SDS-PAGE results showed that the mutation of phosphorylation site of VLF-1 greatly reduced the expression of very late gene polyhedrin in infection of those recombinant viruses compared with the expression level in wild type virus.Experimental results show that phosphorylation of VLF-1 plays an important role in the replication of baculovirus AcMNPV. The mutation of VLF-1 leads to the defect of nucleocapsid assembly, which cause the decrease in infectivity of those recombinant viruses. Contrasting to the results from vlf-1 deletion experiment, we speculate that mutation of phosphorylation sites of VLF-1 may affect the interaction between VLF-1 and other viral structural proteins. Further, the abnormal VLF-1 may influence nucleocapsid assembly. Meanwhile, the mutation may also affected the activity of VLF-1 as very late expression factor, which resulted in the decrease of the expression of very late protein POLH. Generally speaking, the phosphorylation sites of VLF-1 play an important role in both nucleocapsid assembly and the expression of very late gene.