The Mechanism Of TMED2 In CGAS-MITA Pathway

After DNA virus infection,cytoplasmic pattern recognition receptors cGAS(cyclic GMP-AMP synthase)recognizes and binds to viral DNA,which catalyzes the synthesis of cGAMP(cyclic dinucleotide cGMP-AMP)from ATP and GTP.The cGAMP,functions as a second messenger that activates the endoplasmic-reticulum(ER)– membrane adaptor MITA(Mediator of IRF3 activation).The activated MITA transports from the ER to perinuclear vesicles,where it recruits TBK1 and IKK,which phosphorylate and activate the transcription factor IRF3 and NF-?B,leading to the production of type I interferons(IFNs,interferons)and inflammatory cytokines,and subsequent initiation of the antiviral immune responses.MITA functions as a adaptor protein in this process,so the regulation mechanism of MITA activation is also extremely important.To identify potential molecules involved in cGAS-MITA pathway,we screened a cDNA library containing ~10000 clones by dual-luciferase reporter assays in this paper.We found that overexpression of TMED2(transmembrane emp24 protein transport domain containing 2)potentiated the cGAS-MITA-mediated activation of ISRE(IFN-stimulated response elements).To further explore the role of TMED2 in antiviral innate immune responses,we utilized the RNA interference and CRISP-Cas9 technology to knockdown or knockout of endogenous TMED2.Our results indicated that TMED2 knockdown or knockout inhibited HSV-1-induced the activation of IRF3 and NF-?B and subsequent IFN I and inflammatory cytokines transcription.TMED2-knockout cells harbor more HSV-1 titer than wild-type cells.However,TMED2 knockdown had no significant effect on SeV-induced innate immune signaling pathway.These results suggest that TMED2 functions specifically in anti-DNA virus innate immune signaling pathway.To further explore the role of TMED2 in anti-DNA virus signaling pathway.We found that TMED2 knockdown inhibited cGAS-MITA-induced ISRE activation,but hardly affected TBK1-induced ISRE activation.TMED2 knockdown had no marked effect on the expression of cGAS and ISD45-induced production of cGAMP.TMED2 knockdown impaired HSV-1-induced augment of MITA dimerization as well as the subsequent recruitment of TBK1 and IKK?.Overexpressed TMED2 interacted specifically with MITA,but not other key components in the DNA-sensing pathway.HSV-1 infection induced the interaction between endogenous TMED2 and MITA.These results suggested that HSV-1 infection induced TMED2 interacted specifically with MITA and subsequently activated MITA and MITA-mediated downstream signaling pathway,but had no significant effect on signaling events upstream of MITA.To further explore the molecular mechanism of TMED2.Co-immunoprecipitation indicated that the luminal domain of TMED2 interacted with the first two transmembrane domain of MITA.HSV-1 infection induced the oligomerization of TMED2,which mediated by its luminal domain.TMED2 knockdown inhibited HSV-1-induced MITA dimerization.Immunofluorescent assays showed that TMED2 overexpression potentiated MITA congregation in the perinuclear compartments.TMED2 knockdown markedly inhibited HSV-1-induced MITA congregation.These results indicate that HSV-1 infection induced the oligomerization of TMED2,which reinforced the dimerization of MITA and promoted the trafficking of MITA from the ER to perinuclear compartments.Therefore,we continued to explore the mechanism of TMED2 in MITA trafficking.Our results suggested that TMED2 promoted the translocation of MITA from the ribosome into the ER following translation by enhancing the interaction between MITA and TRAP?.In addition,TMED2 promoted the assembly of MITA into the COPII complex by enhancing the association of MITA with Sec24 C,leading to the trafficking of MITA from the ER to the Golgi.In summary,we identified TMED2 as a new regulator which potentiated MITA activation and trafficking.We also analyzed the molecular mechanism of TMED2 in MITA-mediated anti-DNA virus signaling pathway.These founds provide important support for constructing antiviral immune signal transduction regulatory network and clues for screening molecular targets for antiviral drugs.