The Mechanism Of Aconitase Deacetylation In Regulating Bombyx Mori Nucleopolyhedrovirus Infection

As one of China’s most widely reared economic insects,the Bombyx mori is an essential part of agricultural production and a model insect in insect genetics and immunology research.Bombyx mori nucleopolyhedrovirus(Bm NPV)is the primary pathogen that infects silkworms and causes significant damage to the development of sericulture.Therefore,the interaction mechanism between the Bombyx mori and Bm NPV has been a hot research topic.Viral proliferation depends on various vital activities in the host cell,including energy metabolism.A previous study has shown that Bm N cells significantly downregulated the 56 th lysine acetylation modification of the tricarboxylic acid cycle Bombyx mori aconitase 2(Bm ACO2)by 28.3% after Bm NPV infection.At present,the interaction between host and Bm NPV in terms of energy metabolism needs to be better studied.Significantly,the mechanism of the acetylation modification of Bm ACO2 in the Bm NPV infection has not been reported.In order to reveal the effect of Bm ACO2 deacetylation modification on energy metabolism and the potential regulatory role in viral infection,we construct Bm ACO2 deacetylation mutants to investigate the effects of deacetylation modification on cellular metabolic functions and virus proliferation.In this study,we analyzed the effect of Bm NPV infection on host energy metabolism.The cellular energy metabolism was enhanced after 48 h p.i.by upregulating the gene expression of TCA cycle metabolic enzymes.Primarily,we found that the protein expression and enzyme activity of Bm ACO2 were promoted.Then,the lysine(K)at position 56 was site-directed to mutate to arginine(R)by overlapping PCR to mimic the deacetylation modification of Bm ACO2.Wild-type and deacetylation modified mutant were transfected in Bm N cells and expressing normally,indicating the deacetylation modification of K56 site did not affect the stability of the Bm ACO2.The immunoprecipitation experiment revealed that the acetylation levels of the 56 th site lysine of Bm ACO2 were significantly down-regulated after Bm NPV infection,consistent with the previous acetylome in Bm N cells,indicating that the deacetylation modification of Bm ACO2 played an essential role in the viral infection of the host.Subsequently,the enzyme activity assay revealed that the deacetylation modification of Bm ACO2 significantly down-regulated the enzyme activity,inhibiting the activities of two critical upstream and downstream rate-limiting enzymes of the TCA cycle.It also reduced intracellular ATP production and decreased the mitochondrial membrane potential.It was suggested that the deacetylation of Bm ACO2 resulted in a decrease in TCA cycle flux and reduced the levels of cellular energy metabolism.Finally,the effect of Bm ACO2 K56 deacetylation modification on the replication and proliferation of Bm NPV were analyzed.It revealed that decreased cellular energy metabolism inhibited the expression of key genes for Bm NPV DNA replication and assembly,thereby suppressing the proliferation of Bm NPV.In summary,Bm NPV invasion induces the expression of TCA cycle metabolic enzyme genes to provide energy for its replication and assembly.However,stimulation of Bm NPV causes the host to down-regulate the acetylation levels of Bm ACO2 K56,decreasing the metabolic enzyme activity to counteract the metabolic activation caused by Bm ACO2 expression.The present study reveals that the deacetylation of Bm ACO2,a product of the interaction between Bombyx mori and Bm NPV,plays a crucial role in host defence by affecting energy metabolism.Our research will provide a new theoretical basis for elucidating the mechanism of Bm NPV-host interaction.