Study On The Regulation Mechanism Of Silkworm ADA3 Protein In The Process Of Baculovirus Infection

Bombyx mori nucleopolyhedrovirus (BmNPV) is a family of baculovirus that specifically infects the silkworm (Bombyx mori),a lepidopteran model insect.The infection of the virus normally causes a serious economic loss to the silk industry,but the interaction mechanism between the BmNPV and silkworm has not yet been fully elucidated.In order to understand the interaction mechanism between them,we analyzed the differential proteomics and acetylomics of host cells infected with BmNPV.And the results showed that the expression level of Bombyx mori ADA3(alteration/deficiency in activation 3,BmADA3) protein significantly increased by 66% after viral infection and the acetylation level significantly reduced by 34%.Moreover,many studies have demonstrated that the protein has various biological functions,such as playing an important role in cell proliferation and apoptosis.But the biological functions of ADA3 in silkworm,especially its role in the interaction mechanism between virus and host,have not been reported.Therefore,Bombyx mori ADA3 was selected as the subject to study the regulation during the process of BmNPV infection based on the analysis of its own biological function.Firstly,bioinformatic analysis was applied to analyze Bombyx mori ada3 gene and the results showed that the full length of the ORF (open reading frame) was 1383 bp encoding 460 amino acids with a carboxyl-terminal conserved domain,ADA3.ada3 gene was then cloned and induced for expression in Escherichia coli and finally,purified to produce polyclonal antibody.BmN cells were transfected with the constructed transient expression vector pIEx-1-ada3 and the result of Western Blot showed that,the expression of the target protein started at 24 h post transfection and peaked at 48 h post transfection.MTT assay identified that overexpression of ADA3 protein significantly enhanced cell viability.Western Blot DNA fragmentation assays and flow cytometry analysis further demonstrated that overexpression of ADA3 protein could inhibit host cell apoptosis to some extent.Additionally,subcellular localization analysis showed that ADA3 protein was mainly located in the nucleus of Bombyx mori.The results of Western Blot also showed a downward trend in the expression of Cyclin D1 protein in ADA3 overexpressed cells,but the expression of Cyclin D1 increased gradually with the prolongation of viral infection time,indicating that overexpression of Bombyx mori ADA3 protein may promote transition from Gi to S phase of cell cycle.qPCR was used to analyze the replication of viral genome.The results revealed that overexpression of ADA3 protein could inhibit viral replication to some extent,especially in the early phases of viral replication.Finally,BmN cells were transfected with the constructed transient expression vectors that mimic acetylation and deacetylation,and the result of DNA fragmentation showed that the deacetylation modification at K124 site could significantly improve the apoptotic level of BmN cells.In summary,this study shows that Bombyx mori ADA3 protein has many biological functions such as enhancing cell viability,inhibiting apoptosis,promoting the transition from G1 to S phase,and inhibiting the viral replication,and deacetylation modification at K124 site has a significant regulatory effect on host cell apoptosis induced by baculovirus infection.This study will lay the groundwork for an in-depth study of the interaction mechanism between the baculovirus and its host,and will also provide new ideas for the prevention and control of silkworm virus diseases.