Regulation Study Of Citrate Synthase And Its Acetylation In The Process Of Bombyx Mori Baculovirus Infection

Baculovirus infection has an important impact on the physiological process of silk host cells.As the parasite of the host,the virus also depends to a large extent on the host cell to provide the resources needed for self-reproduction.In order to understand the interaction mechanism between the host cells of the silkworm and the Bombyx mori nucleopolyhedrovirus,our laboratory conducted a differential group analysis of the acetylation modification of the BmN cells before and after BmNPV infection,and found that BmNPV infection can cause changes in the acetylation modification levels of a variety of rate-limiting enzymes involved in catalyzing the main energy metabolism,among them,citrate synthase(CS),the key rate-limiting enzyme in the tricarboxylic acid cycle,is one of the enzyme proteins with the most significant acetylation modification level,and the acetylation level of lysine 336 had been increased 1.833 times.At present,there are few studies on citrate synthase in insect cells,and the possible regulatory mechanism of its acetylation modification in the process of virus infection has not yet been reported.In order to deeply analyze the mechanism of Bombyx mori citrate synthase(BmCS)acetylation modification in the process of BmNPV infection,this study takes BmCS the as the research object to explore the effects of its acetylation modification on its biological function and further explore the mutual regulation mechanism of citrate synthase and its post-translational modification between virus and host.In this study,the BmCS gene was first cloned from the whole genome of BmN cells.And fusion PCR technology was used to conduct site-directed mutagenesis on specific amino acid sites,to mutate lysine(K)to glutamine(Q)to simulate acetylation modification to construct a transient expression plasmid.Then through immunoprecipitation analysis,it is found that the acetylation modification of BmCS can be regulated by the silkworm deacetylase Sirt2.In order to further analyze the effect of BmCS acetylation modification on protein function,the successful transient expression vectors were used to transfect cells and the enzyme activity analysis found that the acetylation modification of K336 can significantly reduce the enzyme activity of BmCS.Next,using the ATP content detection kit and JC-1 staining to further study the effect of acetylation modification on cell energy metabolism,it was found that the acetylation of K336 can significantly reduce the ATP content and mitochondrial membrane potential in the cell.Similarly,the use of RNA interference to reduce the expression of BmCS also reduces the ATP content and mitochondrial membrane potential in the cell.And after virus infection,cellular ATP production is significantly up-regulated,while mitochondrial membrane potential decreases significantly after virus infection.And then,the effect on BmCS expression was detected at different time points of virus infection exhibited that BmCS expression was up-regulated transiently at 2 h and BmCS was also acetylated after virus infection.Then,this study used fluorescence microscopy,q PCR technology and virus titer analysis to detect the effects of BmCS expression reduction and acetylation at position K336 on the virus.The results showed that the decrease in BmCS expression and acetylation at position 336 both significant inhibit the BmNPV genome replication and the generation of progeny viruses,while BmCS overexpression can promote the proliferation of the virus.Finally,in order to analyze whether changes in the TCA cycle process will affect the process of virus infection of host cells,DCA inhibitors were used to treat BmN cells,and it was found that the acceleration of TCA cycle metabolism caused by DCA treatment significantly increased the ATP content and mitochondrial membrane potential in the cells;and BmNPV infection of DCA-treated BmN cells significantly promoted BmNPV genome replication,progeny virus production and proliferation.The main conclusion of this study: the acetylation modification of BmCS K336 and the decrease of its expression significantly affect cell energy production and mitochondrial membrane potential changes,and regulate the BmNPV genome replication and progeny virus production.It’s showed that changes in the TCA cycle process also affect cell energy metabolism and mitochondrial function,and are closely related to the proliferation of viruses.