The Mechanism Of Fructose-1,6-bisphosphate Aldolase Acetylation In Regulating Bombyx Mori Baculovirus Infection

As a model organism,the study of the interaction mechanism between silkworm and its important pathogenic microorganism,Bombyx mori nucleopolyhedrovirus(Bm NPV),has always been considered one of the research hotspots in the field of insect virology.At the same time,they are also an important model to study the mechanism of virus-host interaction.As obligate intracellular parasites,the proliferation process of virus strictly depends on a variety of life activities of host cells including energy metabolism.In the previous study,the acetylation level of fructose-1,6-bisphosphate aldolase(ALDO)at K42 residue was found up-regulated by 1.25-fold 36 h post Bm NPV invasion.At present,there are few reports on the relationship between Bm NPV and the energy metabolism of silkworm host cells,especially the regulatory role and mechanism of post-translational modification of Bm ALDO are unknown.Therefore,in order to further reveal the potential regulatory role of Bm ALDO acetylation during viral infection,this work used overlapping PCR technology and site-directed mutagenesis to explored the effect of Bm ALDO acetylation on its function and viral proliferation.First,the target gene Bmaldo was cloned form the Bm N genome through primer design,the overlapping PCR was performed for site-directed mutagenesis of 42-position lysine to mimic deacetylation(arginine,K/R),and then were inserted into the silkworm transient expression vector.The initial enzyme activity analysis showed that the acetylation of Bm ALDO at K42 significantly up-regulated its enzyme activity.The further glycolysis flux assay found that the acetylation of K42 significantly up-regulated the glycolysis flux,manifested as increased glucose uptake,lactate and ATP yield.Transcript level and protein level detection showed that the acetylation of K42 significantly promoted the expression of glucose transporter(Glut1),hexokinase 2(HK2)and lactate dehydrogenase(LDH),indicating that the acetylation of K42 increase glycolysis flux by up-regulating the expression of related genes.Then,the changes of host cellular glycolysis pre-and post-viral invasion were further examined,and the results showed that Bm NPV significantly up-regulated the host glycolysis flux 36 h post viral infection.The immunoprecipitation experiments further proved that acetylation level of Bm ALDO was significantly increased 36 h post viral invasion.In addition,fluorescence observation,q PCR and WB detection showed that the acetylation of Bm ALDO K42 significantly promoted viral proliferation at both DNA replication and protein levels.Further research found that under the premise that2-deoxyglucose(2-DG)significantly inhibited Bm NPV-induced up-regulation of glycolysis,2-DG significantly inhibited viral proliferation at both DNA replication and protein levels.In conclusion,Bm NPV can increase the host glycolysis flux by up-regulating the acetylation level of Bm ALDO,and the glycolysis is an indispensable condition for optimal proliferation of virus.This study revealed the potential function of protein post-translational modifications in the process of virus-induced host metabolic changes,which will provide a novel theoretical basis for elucidating the machanism of virus-host interaction.