Identification Of Key Sites Of Interaction Between AcMNPV GP41 And CHCHD2 Or GARS Proteins Of Sf9 Cells And Their Effects On Virus Replication

Autographa californica multiple nuclepolyhedrovirus(AcMNPV)GP41 is an O-glycosylated protein that is localized between the envelope and nucleocapsid.Studies have shown that gp41 is a necessary gene for nucleocapsid to obtain the envelope,but the specific mechanism is still unclear.Previously,two proteins of Sf9 cells,which are homologs of a coil-coil-helix-coil-coil-helix domain protein 2(CHCHD2)and a glycyl-tRNA synthetase(GARS)respectively,were identified to interact with AcMNPV GP41,by yeast two-hybrid assays.The region of GP41 CHCHD2 and GARS interacted with were located between GP41 AA161-210 and AA81-330,respectively.In this study,we try to identify the residues of GP41 required for interaction between GP41 and the host proteins,and to invesgate effects of interaction between GP41 and CHCHD2 and GARS.First,we verified the GP41-CHCHD2 and GP41-GARS interactions by immunoprecipitation and bimolecular fluorescence complementary assays.In the immunoprecipitation assays,GP41 and HA-CHCHD2 were detected in the precipitates pulled down by HA-specific antibodies from the cytolysate of the cells transfected with a chchd2 transient expression plasmid and infected with AcMNPV.In the bimolecular fluorescence complementary assays,yellow fluorescence was observed in Sf9 cells infected by the reporter virus expressing GP41-YFP C155 and YFP N173-CHCHD2 fusion proteins and the cells infected by the virus expressing GP41-YFP C155 and YFP N173-GARS fusion proteins,respectively.The yellow fluorescence mainly distributed around the nucleus of cells infected by the reporter virus expressing GP41-YFP C155 and YFP N173-CHCHD2,while the yellow fluorescence spreaded in the cytoplasm of the cells infected by the virus expressing GP41-YFP C155 and YFP N 173-GARS.We selected five conserved amino acid codon K137,M145,L152,L242 and L290 from GP41 AA81-330 for point mutation.The results of yeast two-hybrid experiments showed that K137A x CHCHD2 and M145A×CHCHD2 heterozygotes only grew a small number of colonies on the four-deficient selection medium plate;L290A×CHCHD2 and L290A x GARS plates did not grow colonies;K137A×GARS,M145A×GARS,L152A×GARS and L152A×CHCHD2 plates' colonies were significantly reduced,L242A×CHCHD2 and L242A×GARS plates were observed with a large number of colonies.In bimolecular fluorescence complementary assays,yellow fluorescence was observed in the Sf9 cells transfected by the reporter bacmids expressing GP41 K137A/M145A-C155 and N173-GARS fusion proteins,not observed in the cells transfected with the bacmids expressing GP41 K137A/M145A/L290A-C155 and N173-CHCHD2 fusion proteins,and so is the bacmid expressing GP41 L290A-YFP C155 and YFP N173-GARS fusion proteins.These phenomena indicated that K137,M145 and L290 is critical to the interaction between GP41 and CHCHD2,and L290 is also critical to the interaction between GP41 and GARS.Mutations at these sites made GP41 to lose its interaction with CHCHD2 or GARS.To determine effects of GP41 K137,M145,L152A,L242 and L290 on virus replication,AcMNPV recombinants with gp41 mutated at the indicated positions and with polh or egfp marker were constructed and used for infection-infection of Sf9 cells.In the cell cultures transfected with individual the polh-containing wild type,gp41-knockout recovered and gp41 L152A or L242A mutant,occlusion bodies were found in the majority of cells,while occlusion bodies were only found in a few cells in the cultures transfected with the ones containing gp41 K137A,M145A or L290A mutant.There was no any occlusion body-containing cell found in the cultures inoculated with supernatants from tansfection with individual AcMNPV recombinants containing gp41 K137A,M145A or L290A.Similarly,green fluorescence was not observed in the cell cultures inoculated with supernatants from transfection by the egfp-containing AcMNPV recombinants with gp41 K137A,M145A or L290A mutant.These phenomena suggested that the AcMNPV gp41 K137A,M145A and L290A mutants do not produce infectious budding virions,but do produce occlusion bodies.To investigate the effect of GP41 interacting protein GARS on virus replication,we interfered the expression of GARS gene in Sf9 cells infected with AcMNPV by RNAi,and detected the effect of interference on the proliferation of budding virions.Reverse transcription quantitative PCR analysis showed that the gars mRNA level in Sf9 cells infected by the recombinant virus containing reverse repeated gars sequence reduced 66%compared to the control.The peak level of the recombinant virus was 20%of the one of the wild virus.These results suggested that AcMNPV virus replication depended on the normal expression of the host gars gene.The experimental results above demonstrate:(1)AcMNPV GP41 interacts with Sf9 cell proteins CHCHD2 and GARS.(2)K137,M145 and L290 are the key sites of GP41 interacting with CHCHD2,and L290 is the key site of GP41 interacting with GARS.(3)Interaction between GP41 and CHCHD2 and GARS are required for virus replication.(4)AcMNPV replication depends on gars gene expression of host cells.