The Mechanism Of ZZEF1 Promoting Type Ⅰ Interferon Signaling Pathways Through The Regulation Of HECTD3

When the host is invaded by pathogenic microorganisms,the innate immune system mainly induces the immune responses of the organism through the signaling pathways mediated by innate immune cells and innate immune molecules,and clears the pathogens to maintain the homeostasis.type Ⅰ interferons are the most important components of the innate immune signaling pathway.type Ⅰ interferons play essential roles in the host defense against infections by limiting the transmission of infectious pathogens especially viruses,by inducing an antimicrobial state in infected and adjacent cells.Therefore,it is crucial to study the regulatory mechanisms of type Ⅰ interferons.Previous study revealed that the E3 ubiquitin ligase HECTD3 plays an important role in the host innate immune response during bacterial infection.We demonstrated that HECTD3 promotes the TRIF and STING dependent type Ⅰ interferon production by mediating TRAF3 K63-linked polyubiquitination.However,the underlying mechanism of how upstream signals regulate the HECTD3-mediated type Ⅰ interferon signaling pathway is remain unclear.To explore how HECTD3 is regulated by upstream signals,we performed mass spectrometry analysis of the HECTD3 immunoprecipitation(IP)products under Acinetobacter baumannii and herpes simplex virus type 1 infectious condition,and identified a potential target protein ZZEF1.Co-IP experiments confirmed the interaction between ZZEF1 and HECTD3.To investigate the role of ZZEF1 in the type Ⅰ interferon signaling pathway,first of all,ZZEF1 was overexpressed in MSF cells by transfection pCDNA3.1-ZZEF1-V5,and the expression of Cxc19,Cxcl10 and 116 that induced by double-stranded RNA analogues poly(I:C)and A.baumannii was detected by RT-qPCR.Afterwards,ZZEF1 expression was knocked down by transfection of siRNA in L929 cells,and the expression of Cxcl9,Cxcl10 and Il6 that induced by DNA virus HSV-1,STING agonist 2’3’-cGAMP,IFNβ and poly(I:C)was detected by RT-qPCR.In the next part,the CRISPR/Cas9 technology was used to construct Zzef1-KO L929 cell lines,the expression of Cxcl9,Cxcl10 and Il6 induced by HSV-1,2’3’-cGAMP,IFNβand poly(I:C)was detected by RT-qPCR and the expression of pTBK1 and pIRF3 induced by HSV-1 was detected by Western Blot.These results showed that ZZEF1 promoted the type Ⅰinterferon signaling pathway mediated by TRIF,STING and MAVS.To explore the molecular regulation mechanism of ZZEF1 and HECTD3 in the type Ⅰinterferon signaling pathway,the expression of HECTD3 in ZZEF1-KD and KO cells was examined by Western Blot and RT-qPCR.We found that the HECTD3 protein expression level reduced while the transcriptional level of Hectd3 did not change significantly as the expression level of ZZEF1 decreased,suggesting that the stability of HECTD3 was mediated by ZZEF1.It has been reported that ZZEF1 plays a role similar to that of the E3 ubiquitin ligase RBR family.In order to further investigate whether ZZEF1 regulates the expression of HECTD3 through ubiquitination,the overexpression vectors of HECTD3,ZZEF1 and ubiquitin were transfected into HEK293T cells respectively,and the ubiquitination level of HECTD3 was detected by immunoprecipitation.It was found that ZZEF1 promoted the K3 3-linked polyubiquitination of HECTD3.The above results indicated that ZZEF1 stabilized the expression of HECTD3 by promoting the K3 3-linked ubiquitination of HECTD3.In order to confirm the antiviral capacity of ZZEF1,viral titers in control and Zzef1 KO L929 cells after HSV-1 infection were measured by virus plaque formation,and it was found that HSV-1 viral titers in ZZEF1 knockout L929 cells were increased,suggesting that the ability of L929 cells against HSV-1 reduced due to ZZEF1 knockout.In this study,we first found that ZZEF1 could stabilize HECTD3 by regulating the K33linked ubiquitination of HECTD3,thus promoted TRIF and STING-dependent type Ⅰ interferon signaling pathways,and played an anti-infection role during HSV-1 infection.This study enriched the knowledge of the innate immune regulatory network and provided potential targets for the development of anti-infective drugs.