The Function And Mechanism Study Of Mitochondrial Solute Carrier Protein SLC25A23 In The Antiviral Innate Immunity

The innate immune system produces a series of cytokines to resist the virus invasion through the recognition of virus by pattern recognition receptors.RIG-I-like receptors(RLRs),including RIG-I and MDA5,are cytoplasmic innate sensors for viral RNA.RIG-I undergoes a conformational change after binding of pathogentic RNA to the helicase domain and is recruited to the mitochondrial antiviral signaling(MAVS)adaptor.The downstream signal transduction of MAVS eventually activates the transcriptional factor IRF3 and NF-?B.The activated IRF3 and NF-?B enter the nucleus and bind to the promoter region of IFN-?.Stimulating the expression of IFN-?,which ultimately exerts an antiviral effect by inducing the expression of IFN-sti mulated genes(ISGs).In this study,we used a genome-wide CRISPR/Cas9 library to screen the host factors which support VSV replication.From the result of the next-generation sequencing,we found that SLC25A23 was the most promising candidate to promote VSV replication.SLC25A23 is Calcium-dependent mitochondrial solute carrier.SLC25A23 act as a ATP-Mg/Pi exchanger that mediates the transport of Mg-ATP in exchange for phosphate,catalyzing the net uptake or efflux of adenine nucleotides into or from the mitochondria.We down-regulated SLC25A23 by si RNA in Hela-cas9 cells and the VSV replication was significantly reduced.Similar results were achieved in CRISPR/Cas9 mediated SLC25A23 knockdown.As we know,type? IFNs pathway is known to be the main target of viruses to antagonize the innate immune system.Then,we tested the type I IFNs pathway,we find that down-regulation of SLC25A23 enhanced Sendai virus(Se V)-induced IFN-?production at m RNA level.Next,we down-regulated SLC25A23 by si RNA in the U3 A cells,which are deficient with STAT1.The results suggest that down-regulation of SLC25A23 in the U3 A cells did not affect VSV replication.We hypothesize thatSLC25A23 may play an important role in IFN signaling to antagonize VSV infection and replication.The overexpression plasmids of SLC25A23 and IFN-?-luc,RL-TK dual fluorescent reporter plasmids and RIG-IN,MDA5,MAVS,TBK1,IKK?,IRF3/5D overexpression plasmids were co-transformed into HEK293 T cells,and then using the luciferase reporter system to detect IFN-? changes.We found that overexpression of SLC25A23 significantly inhibited IFN-? mediated by RIG-IN,MDA5,MAVS,TBK1 and IKK? linker molecules,but not IRF3/5D,suggesting that SLC25A23 may act on the upstream molecule of IRF3.Through Co-immunoprecipitation experiments,we found that SLC25A23 interacted with MAVS and TBK1.MAVS plays an important role in the RLR signaling pathway.Previous studies have shown that RNA virus infection can induce a conformational transition in MAVS,leading to the formation of prion protein-like functional aggregates,which plays a key role in the activation of RLR signaling pathway.SLC25A23 is located on mitochondria,so we hypothesized that SLC25A23 may regulate the natural immune antiviral response by modulating the production of type I interferon mediated by MAVS.Taken together,we used genome-wide CRISPR/Cas9 library screens to identify human genes with crucial roles in the replication of VSV.Despite previous screens of human host factors for the virus through genome-wide small interfering RNA.We discovered several genes that had not been linked to VSV replication before.And,we discovered that SLC25A23 plays an important role in the RNA virus mediated innate immune signaling pathway and anti-viral response of the body for the first time.In addition,we also found that SLC25A23 can target MAVS and TBK1,negatively regulate the production of IFN-? and exert antiviral effects.This discovery has provided new ideas and treatment points for the clinical treatment of viral infectious diseases.