The Effects Of Point Mutations Within The Interaction Sites Between GP41 Of Autographa Californica Multiple Nucleopolyhedrovirus And Two Host Proteins On Viral Replication

AcMNPV gp41 is one of the 37 core genes of baculovirus,homologues exists in all of the 73 baculovirus genomes that have been sequenced.AcMNPV GP41 is a glycosylated protein that is localized between the viral capsid and the capsule.The laboratory screened out Spodoptera exigua protein which interacted with GP41:CHCHCD2(the coiled-coil-helix-coiled-coil-helix domain-containing protein2)and GARS(glycyl-tRNA synthetase);it was found that the interaction site between GP41 and CHCHD2 was between 161-210AA and the interaction site with GARS was between 81-330AA.In this paper,the effects of these virus-host protein interactions on viral replication were investigated by point mutations within the interaction site between AcMNPV GP41 and the two host proteins and the phenotypic analysis of corresponding AcMNPV mutantsFirstly,the codon of 181 AA(leucine,Leu/L)、182AA(proline,Pro/P)、187 AA(lysine,Lys/K)、203 AA(glutamate,Glu/E)、206 AA(lysine,Lys/K)、208 AA(leucine,Leu/L)which located in interaction sites between the AcMNPV GP41 and CHCHD2、GARS were selected to mutate the codon of Amino acid(Ala/A).The mutated GP41 coding sequence was inserted into the yeast two-hybrid bait vector pGBKT7,the recombinant plasmids were transformed into yeast competent state and then mated to AH 109 yeast strain containing the cDNA clones of the CHCHD2 and GARS coding sequences.Four mutants,LPK/AAA、EKL/AAA、L181/A、L208/A,made a loss of interaction between GP41 and CHCHD2、GARS.gp41K187/A、gp41K206/A might make a loss of interaction between GP41 and CHCHD2,and decrease interaction between GP41 and GARS.gp41P182/A and gp41E203/A might make a loss of interaction between GP41 and CHCHD2,though has little effect on interaction between GP41 and GARS.In order to investigate whether coding sequence mutations in the interaction site between GP41 and host proteins CHCHD2、GARS affect the proliferation of the virus,eight gp41 mutants were inserted into the polh site of vAcgp41ko together with a copy of the green fluorescent protein gene(egfp)or a copy of the polyhedrin gene(polh)via the Bac-to-Bac system,to constructed AcMNPV mutants with vAcgp41LPK/AAA-egfp、vAcgp41EKL/AAA-egfp、vAcgp41L181/A-egfp、vAcgp41P182/A-egfp、vAcgp41K187/A-egfp、vAcgp41E203/A-egfp、vAcgp41K206/A-egfp、vAcgp41L208/A-egfp and AcMNPV mutants with polh vAcgp41LPK/AAA-PH、vAcgp41EKL/AAA-PH、vAcgp41L181/A-PH、vAcgp41P182/A-PH、vAcgp41K187/A-PH、vAcgp41E203/A-PH、vAcgp41K206/A-PH、VAcgp41L208/A-PH.These AcMNPV bacmid mutants transfected or infected Sf9 cells separately,and the results showed that the amount of infectious BV produced by mutants containing gp41P182/A、gp41EKL/AAA、gp41E203/A or gp41K206/A was close to or even higher than that of the wild type and vAcgp4lrep-PH in the transfected cell culture medium.However mutants containing gp41LPK/AAA、gp41L181/A or gp41K187/A produced only a very small amount of infectious BV in the transfected cell culture medium;the mutants containing gp41L208/A produced more infectious BV than those of gp41LPK/AAA,gp41L181/A and gp41K187/A in the transfected cell culture medium,but less than wild type and gp41 knockout repair bacmid.And the rest had no significant effect on virus proliferation.These mutants can produce occlusion bodies in transfected cells.The supernatants from Sf9 cell transfected with vAcgp41L181/A-egfp、vAcgp41L208/A-egfp were determined by qPCR,the results showed that two mutants released a small amount of BV after 72hpt.The Sf9 cells were transfected with gp41 knockout mutant vAcgp41ko-PH,gp41 knockout repair mutant vAcgp41rep-PH,gp41L181/A mutant vAcgp41L181/A-PH,and the cells were collected at 96hpt for observation by electron microscopy.The results showed that in the nucleus of Sf9 cell transfected with vAcgp41rcp-PH,it could clearly see a variety of typical features in the process of the virus morphology at different time points,including the virogenic stroma,the nucleocapsid,the envelope-coated virions and the occlusion bodies containing the virions and the large number of nucleocapsid which was transported from the nucleus to the plasma;The virogenic stroma and nucleocapsid can be observed in the nucleus of cell transfected with vAcgp41ko-PH or The formation of Virogenic stroma and occlusion body lagged in the nucleus of cells transfected vAcgp41L181/A-PH.Vesicles and a small number of suspected nucleocapsid transported from nucleus were found in the nucleus of cells transfected vAcgp41L181/A-PH but no envelope-coated virions;a small amount of inclusion body were observed,but the occlusion body containing no virosomes.In the nucleus of cell transfected vAcgp41ko-PH,some vesicles containing many nucleocapsid capsules were observed,but no vesicles;no nucleocapsules were released from the nucleus and occlusion bodies were not observed.The experimental results of this paper show that AcMNPV GP41 181AA mutation blocked the interaction of GP41 with host proteins CHCHD2 and GARS,and blocked the release of BV and viral capsid capsules,and also blocked the formation of ODV.208AA mutation also blocked the interaction of GP41 with host proteins CHCHD2 and GARS,and blocked the release of BV.187AA mutation weakened the interaction of GP41 with CHCHD2 and GARS significantly,as well as a significant decrease in infectious BV production.These experimental results indicate that the interaction of GP41 with the host protein CHCHD2 and GARS may be related to the morphogenesis of virus.