Preliminary Study On The Nuclear Mechanism Of Baculovirus DNA Polymemse

The family Baculoviridae comprise a diverse group of insect-specific viruses,it is characterized by a large circular double-stranded,80 to 180 kb DNA genome packaged within a rod-shaped nucleocapsid and enclosed by a lipid envelope.Baculovirus replication is crucial for viral life cycle and results in production of progeny virus.Once baculovirus infects insect cells,the viral genome was released into nucleus of infected cells,baculovirus replication initiates in viroplasm of infected cells,and DNA polymerase?DNApol?plays a key role during baculovirus replication.However,it is unclear how the DNApol is transported into the nucleus of infected cells.In this study we analyzed the nuclear localization signal?NLS?role of betabaculovirus Pieris rapae granulovirus?PiraGV?DNApol and the interaction between AcMNPV DNApol and host protein,our data will contribute to understanding of the molecular mechanism of baculovirus proteins into the nucleus.The results are summiarized as follows:Bioinformatic analysis showed that PiraGV DNApol contains a potential NLS?4LFKRKLDEPPTPTHHTLVKAIKLS25?at the N-terminus,which does not match either the classical monopartite or the bipartite NLS consensus sequence.The NLS of PiraGV DNApol was fused to the green fluorescent protein?GFP?,and the resulting fusion protein could localize to the nucleus,confirming that the peptide sequence could functions as NLS.Multiple point mutation analysis revealed that the basic amino acid clusters 6KRK8 and 20KAIK23 in the NLS motif are essential for nuclear localization of PiraGV DNApol.To identify whether the NLS of PiraGV DNApol is functional in Spodoptera litura nucleopolyhedrovirus?SpltNPV?DNApol,the NLS of SpltNPV DNApol was replaced by that of PiraGV DNApol,generating plasmid pBlue-HA:Slpol^?nls:Prnls,and pBlue-HA:Slpol^?nls:Prnls was transfected into Sf-9 cells,immunofluorescence analysis showed that HA:Slpol^?nls:Prnls could not localize into the nucleus but stay in the cytoplasm.The NLS substituted DNApol HA:Slpol^?nls:Prnls was reinserted into dnapol-nullAcMNPVbacmid,resultinginBac-Ac?pol:Slpol^?nls:Prnls.Immunofluorescence analysis demonstrated that HA:Slpol^?nls:Prnls was located in cytoplasm of transfected cells.Viral growth curve showed that the virus titer of Bac-Ac?pol:Slpol^?nls:Prnls decreased by 2600-fold compared to that of Bac-Ac?pol:Slpol at 120 hpt.Real-time quantitative PCR analysis revealed that the copy number of the Bac-Ac?pol:Slpol^?nls:Prnls genome reduced by 19-fold compared to that of Bac-Ac?pol:Slpol at 24 h after transfection.No polyhedra was observed for Bac-Ac?pol:Slpol^?nls:Prnls in transfected cells,while the polyhedron were found in 3.1%of Bac-Ac?pol:Slpol-transfected cells.Together these results indicateded that replacement of NLS in SpltNPV DNApol results in reduction in infectious progeny virus and viral DNA replication and yield of occlusion bodies.Transcriptome analysis revealed that Sf9 cells infected by AcMNPV contain four importin?homologs,SfIMA1/SfIMA2,SfIMA4 and SfIMA7,which showed 46%/26%,69%and 64%amino acid sequence homology with human importin?1/?2,importin?4and importin?7,respectively.SfIMA1,SfIMA4 and SfIMA7 showed typical characteristics of importin?family proteins,including an importin?binding domain?IBB?at the N terminus,eight armadillo domain?ARM?repeats,and a C terminal Arm repeat sequence.Subcellular localization indicated that SfIMA1,SfIMA4 and SfIMA7could localize to the nuclear membrane of Sf9 cells.Yeast two-hybrid experiments confirmed that SfIMA1,SfIMA4 and SfIMA7 interacted with the C terminus of AcMNPV DNApol.Bimolecular fluorescence complementation?BIFC?experiments further confirmed that the C terminal of AcMNPV DNApol containing nuclear localization signals was co-expressed with each of SfIMA1,SfIMA4 and SfIMA7 in co-transfected cells.Together,our results suggested that three importin-?proteins,SfIMA1,SfIMA4 and SfIMA7,are important for nuclear import of AcMNPV DNApol in Sf9 cells.