The Study Of Interaction Between Autographa Californica Multiple Nucleopolyhedrovirus AC132 And Several Proteins

Autographa californica multiple nucleopolyhedrovirus(AcMNPV)orf132(ac132)is located within a cluster of five ORFs(orfl29 to 133),which are oriented in the same direction on the genome of AcMNPV.Homologs of ac132 are present in all sequenced Group I genomes,and ac132 is expressed as a late gene.AC 132 was detected in the fractions of nucleocapsids of both BV and ODV in our previous studies.Deletion of ac132 resulted in almost no infectious BV production,decreasing the occlusion bodies containing multiple nucleocapsids.AC 132 co-immunoprecipitated with envelope protein ODV-E18 and the viral DNA-binding protein P6.9.In this study,we focused on AC 132 and attempted to elucidate the function of AC 132 by analysing the interactions between AC 132 and several viral and host proteins.1.The screening of the interacting proteins with AC132 by yeast two-hybrid assays.The interacting proteins with AC 132 were screened from the cDNA library of Sf9 cells infected at 18h.p.i with AcMNPV using yeast two-hybrid assays.Two positive clones were obtained,which respectively contain a part of the coding sequences of the homologues of aspartate aminotransferase and ADP/ATP transferase.2.Verification of the nucleotide sequence of leukotriene A-4 hydrolase from Spodoptera frugiperda.Several positive clones have been screened from the cDNA library of Sf9 cells which likely interact with AC132 using yeast two-hybrid assays in our laboratory previously.The analyses of the positive clones revealed that the nucleotide sequence of leukotriene A-4 hydrolase from Spodoptera frugiperda had high similarity to its known homologues,but its coding sequence in aa118 was stoped by TGA,while the corresponding coden in its homologes was TGG encoding tryptophan.To test the cause of the variation,the total RNA from Sf9 cells was purified,and the 5'-terminal sequence of transcriptional product of the gene was obtained by using 5'RACE.Sequencing results show that the 118th coden of No.7 is TGG.3.The detections of interacting regions between AC 132 and its interacting proteins.Various host proteins interacting with AC 132 have been screened using yeast two-hybrid assays in our laboratory,such as leukotriene A-4 hydrolase(No.7),the protein 2 containing cysteine-rich hydrophobic domain(No.20),40S ribosomal protein(No.69),and the receptor for activated protein kinase C(No.96).To further determine the interacting regions in AC132,AC 132 was gradually truncated from the N-and C-terminal of coding sequence,respectively.Yeasts carrying full length or truncations of AC 132 were respectively mated to yeasts carrying the coding sequence of interacting proteins with AC132.As a result,the interacting regions between AC132 and No.7,69,and 96 were respectively located in aa 111?219,aa 91?219,aa 71?219 of the C-terminal of AC 132,and the interacting regions between AC 132 and No.20 was located in aa 51?90.4.The analyses of colocations between AC 132 and several AcMNPV proteins.Recombinant bacmids vAcac132:egfp-e18:rfp expressing AC132-EGFP and E18-RFP fusion proteins,vAcac132:egfp-e25:rfp expressing AC132-EGFP and E25-RFP fusion proteins,vAcac132:egfp-vp39:rfp expressing AC132-EGFP and VP39-RFP fusion proteins,vAcac132 egfp-39k:rfp expressing AC132-EGFP and 39K-RFP fusion proteins,and vAcac132:egfp-iel;rfp expressing AC 132-EGFP and IE1-RFP fusion proteins were respectively constructed through the recombinant fragments containing the ac132 fused with egfp,and e18,e25,vp39,39k or iel fused with rfp,inserting in polh locus of pMON 14272 by using Bac-to-Bac system.Sf9 cells transfected with recombinant bacmids were observed under laser confocal microscope at 48 hpt,showing that AC 132 colocated with three structural proteins E18,E25 and VP39,and the colocation between AC132 and E18,VP39 were mainly in perinuclear and cytoplasm,a little in nucleus.The colocation between AC132 and E25 were mainly in nucleus.But no colocation between AC132 and two non-structural proteins,39K or IE 1 was observed.To analyse the effect of deletion of AC 132 on the subcellular locations of E18,VP39 and 39K,recombinant bacmids vAce18:rfp,vAcvp39:rfp,vAc39k:rfp,vAcac132ko-e18:rfp,vAcac132ko-vp39:rfp and vAcac132ko-39k rfp were constructed through the fusion genes e18-rfp,vp39-rfp and 39k-rfp inserting in polh locus of pMON14272 and vAcac132ko deleting ac132.Sf9 cells transfected with recombinant bacmids were observed for red fluorescence under laser confocal microscope,showing the locations of E18,VP39,and 39K in Sf9 cells infected recombinant bacmids of ac132 deletion are consistent with the locations in Sf9 cells infected with wild bacmids.These results suggested that deletion of AC132 likely has no effect on the subcellular locations of E18,VP39,or 39K.5.ODV produced from ac132 deletional mutant lost oral infection ability.A previous study in our laboratory found that deletion of ac132 resulted in almost no infectious BV production in Sf9 cells,low BV production in High5 cells,but OB containing viral pariticles were produced in the two kinds of cells.To analyse the effect of deletion of ac132 on ODV virulence,High5 cells infected with four bacmids,vAcac132ko-PH,vAcac132ko-gfp-PH,vAcac132ko-rep-PH,and vAcac132ko-rep-gfp-PH,were collected at 96 hpt.Third-instar Spodoptera exigua larva were fed respectively with four infected High5 cells,and observed.As a result,no symptom was observed in larva fed with High5 cells infected with vAcac132ko-PH or vAcac132ko-gfp-PH,while all larva fed with High5 cells infected with VAcac132ko-rep-PH,or vAcac132ko-rep-gfp-PH were liquefied and dead after six days.The hemolymphs from larva infected with four viruses after six days were sampled and observed under microscope,respectively,showing that no typical inclusion body was observed in hemolymphs from larva infected with vAcac132ko-PH,and no green fluorescence was observed in hemolymphs from larvae infected with vAcac132ko-grp-PH.Inclusion bodies and green fluorescences were easily observed in hemolymphs from larva infected with vAcac132ko-rep-PH and vAcac132ko-rep-gfp-PH,respectively.Fifth-instar Spodoptera exigua larva were infected respectively with four virus suspensions,and observed,showing no infectal symptom was observed in larva infected with vAcac132ko-PH or vAcac132ko-gfp-PH suspensions,most of which grown to pupation,except a few accidently dying.All laeva infected with vAcac132ko-rep-PH and vAcac132ko-rep-gfp-PH suspensions died and liquefied after seven days of infection.These results implied that the ODV produced from ac132 deletional mutant lost oral infection ability.