Study Of The Effect Of Cytosolic DNA On Cellular Tetrazolium Reduction And Glucose Metabolism

The interaction of virus and host has attracting continuous attention for a long time.During virus infection,viral proteins and genomes would enter the host cells.These viral components would be recognized as foreign molecules and induce a series of cellular responses.Recently,accumulating evidences showed that DNA viruses injected their genomic DNA into the cytosol of myeloid cells,becoming cytosolic DNA,which could be recognized by cGAS and induce the expression of type I interferon,such as IFNbeta and IL-6,through activating STING-TBK1-IRF3 pathway.It has been reported that cGAS was barely expressed in non-myeloid cells,such as HEK293 T cells,where cytosolic DNA could induce apoptosis but not expression of IFNbeta or IL-6.The underlying mechanism is still unknown.Furthermore,whether cytosolic DNA has any effects other than innate immune response is still to be unveiled.On the other sides,cellular glucose metabolism is essential for cellular growth and proliferation.Glucose metabolism provides synthetic blocks for viral proteins and nucleic acids,and also energy.Meanwhile,host cell would undergo metabolic reprogramming to accommodate or combat with virus infection.The metabolic interaction of virus and host cells is still veiled.This study focused on cellular metabolic response upon cytosolic DNA stimulation by transfection of “interferon stimulating DNA,ISD”,which was a model used in the study of cytosolic DNA induced innate immune response.First,we confirmed that cytosolic DNA could inhibit cell growth in 24 hours.Interestingly,both ssISD and dsISD,but not poly(dA)had this effect.Annexin V/pI staining showed that most of the cells with cytosolic DNA stimulation for 24 hours were still alive.Next,we monitor the effect of cytosolic DNA on cellular metabolism using tetrazolium-based assay.Cytosolic DNA dramatically inhibited tetrazolium reduction within 5 hours.Consistently,live cell assay showed that transfected DNA entered cells within 2 hours.DNA or transfection reagent alone could not inhibit tetrazolium reduction,indicating that DNA entering is essential for this phenomenon.We also showed that various DNA sequences could inhibit tetrazolium reduction in many types of cell lines.Infection of herpes simplex virus 1(HSV-1),a DNA virus,also inhibited tetrazolium reduction.These data indicated that cytosolic DNA either from DNA transfection or viral infection could lead to deregulation of cellular metabolism.Furthermore,we showed that cytosolic DNA inhibited tetrazolium reduction,but not innate immune response in cGAS or STING knockout L929 cells,demonstrating that cGAS and STING were dispensable for cytosolic DNA induced metabolic response,and that cytosolic DNA induced metabolic response and innate immune response were independent.To further elucidate the biological meaning of tetrazolium reduction inhibiton,we performed following studies.Based on the fact that NADH or NAD(P)H that could come from cellular glucose metabolism might reduce tetrazolium,we proposed a hypothesis that the ability to reduce tetrazolium might reflect the cellular viability of glucose metabolism.Therefore,we turned to study the relationship between tetrazolium reduction and cellular glucose metabolism.Our results showed that the glucose concentration,but not serum or glutamine in the medium is the determinant for tetrazolium reduction.Short term of glucose starvation would not lead to cell death.Moreover,glucose metabolism inhibitor,2-DG,could block tetrazolium reduction,which resembled the effect of cytosolic DNA induced metabolic response.These data indicated that cytosolic DNA induced metabolic response might be a consequence of glucose metabolism inhibition.Indeed,we demonstrated that cytosolic DNA could inhibit glucose uptake and lactate production,leading to ATP depletion and activation of AMPK mediated energy stress.To unveil the molecular mechanism underlying cytosolic DNA induced cellular metabolic response,we purified and identified a dozen of putative cytosolic DNA binding proteins by biotin-streptavidin system followed by mass spectrum.The data inhand demonstrated that AUF1 could directly binds to ssDNA and dsDNA,but not poly(dA).AUF1 knockout could attenuate the inhibitory effect of cytosolic DNA on tetrazolium reduction.AUF1 has been reported to regulate the mRNA stabilities of glycolytic enzymes,such as GLUT1 and PFK2.Our data indicated that cytosolic DNA induced metabolic response might be mediated by AUF1.Taken together,our research demonstrated that cytosolic DNA could inhibit tetrazolium reduction and cellular glucose metabolism,leading to ATP depletion and activation of AMPK mediated cellular energy stress.In this process,AUF1 is a potential cytosolic DNA binding protein.Importantly,our study suggested that host cells might recognize invaded viral DNA and inhibit virus replication through inhibiting glucose metabolism and activating cellular energy stress.This might serve as metabolic barrier to viral infection,in collaboration with cytosolic DNA induced innate immune response.