Study On The Assembly Of Virus-like Particles And Subcellular Localization Of Pns12 Protein Of Rice Gall Dwarf Virus

Rice gall dwarf virus(RGDV),which was first described in Thailand in early 1980s, is a member of the genus Phytoreovirus in the family Reoviridae.RGDV causes a severe disease of rice and is responsible for significant yield losses in rice crops in Malaysia, China,Japan and South Korea and,which is emerging as great threat for rice production. Although the nucleotide sequence of each of the 12 genomie RNAs has been determined, almost the functions of viral genes have not been identified.In this dissertation,to study the characterization of the assembly of virus-like particles(VLPs)a Bac-to-Bac baculovirus expression system was adopted to express the RGDV P3 and P8 proteins in insect cells Spodoptera frugiperda(Sf9).The subcellular localization of Pns12 protein was also determined.The inner core gene S3 was amplified successfully by RT-PCR and cloned into pMD18-T vector.S3 gene was subcloned into pET-32a prokaryotic expression vector after the recombinant plasmid pMD-S3 was double digested with EcoRâ… and Hindâ…¢.The expression vectors were transformed into(Escherichia coli)E.coli Rosetta(DE3)â…¡.The S3 gene was expressed with the induction of IPTG.The molecular weight of express products accorded with the anticipated molecular weight of recombinant P3 protein by SDS-PAGE.By indirect ELISA the polyelonal antibody against RGDV P3 was produced in a rabbit at high titer of 1:20480,and its specificity was demonstrated by Western blot. This result shows that polyelonal antibody can be useful for studying on the assembly of core-like particles(CLPs)and virus-like particles(VLPs).The full-length S3 ORF(Open reading frames,ORF)and S8 ORF were inserted into the pFastBac vector,creating the donor plasmids pFB-S3 and pFB-S8,respectively.The full-length S3 ORF and S8 ORF were cloned into pFastBacDual simultaneously,creating the donor plasmid pFD-S3/S8 containing both S3 and S8 genes for the first time.After transformation,pFB-S3,pFB-S8 and pFD-S3/S8 was introduced into the competent cells (E.coli DH10Bac)to generate recombinant bacmids rbpFB-S3,rbpFB-S8,rbpFD-S3/S8, and then Sf9 cells were transfected to produce recombinant baculoviruses,yielding rvpFB-S3,rvpFB-S8,rvpFD-S3/S8,respectively.SDS-PAGE and western blot analysis of lysates from infected Sf-9 cells with either recombinant baculoviruses demonstrated that rvpFB-S3 showed one novel band of the size of expected P3 protein which is 116 kDa, rvpFB-S8 demonstrated 1 novel band of the size of expected P8 which is 47 kDa, rvpFD-S3/S8 demonstrated 2 novel bands of the size of expected P3 and P8.These indicate that RGDV structural proteins expressed in insect cells successfully.Recombinant baculovirus-infected Sf9 cells were mixed with cytoBuster protein extraction reagent;the supernatant was collected after centrifugation for 10 min at 10000 r/m.After staining with phosphotungstic acid,the supernatant of cells infected with rvpFB-S3 revealed abundant single-shelled core-like particles about 40 nm in diameter by electron microscopy.Co-expression of P3 and P8 in Sf9 cells produced double-shelled virus-like particles that resemble in both size and appearance RGDV particles purified from RGDV-infected rice plants;however there were no such structures when P8 alone was expressed.These results suggest that recombinant P3 and P8 have the ability to generate double-shelled particles in vitro.To determine the subcellular localization of Pns12 protein,the recombinant baculovirus vectors expressing RGDV S12 gene and its deletion mutants fused with GFP for localization in insect cells were constructed.Fusion protein of Pns12 with GFP expressed from recombinant baculovirus in Sf9 insect cells,directed the accumulation of fluorescence to the nucleus as observed by confocal microcopy.In contrast,similar construct with the mutated S12 gene or only GFP control accumulated within the nucleus and cytoplasm.RGDV Pns12 protein contains a putative nuclear localization sequence (NLS,²⁰²KRRPR₂₀₆).The deletion mutants of Pns12 having no putative NLS(Nuclear localization signal,NLS)or mutated NLS(²⁰²KRRPR₂₀₆→AAAAA)accumulated equably within in the cytoplasm and nucleus,indicate that Pns12 protein maybe have a functional NLS.To my knowledge,no nucleoplasmin protein has been reported forphytoreovirus.