The Role Of E3 Ubiquitin-protein Ligase NEDD4L In Antiviral Innate Immune Response And The Underlying Mechanisms

Innate immune responses provide the first line of host defense against invading pathogens.Innate immune cells including dendritic cells(DCs)and macrophages recognize conserved molecular components of foreign microorganisms known as pathogen-associated molecular patterns(PAMPs)via pattern-recognition receptors(PRRs).PRRs can also detect damaged tissue or necrotic cells-derived immunostimulatory products,termed damage-associated molecular patterns(DAMPs),and this recognition is crucial for host defense.Virus infection can induce the production of type?interferon(IFN).This process is dependent on the recognization of virus by PRRs.Activation of Toll-like receptors(TLRs)triggers a range of intracellular signaling molecules,resulting in the production of nuclear factor-?B(NF-?B)-dependent inflammatory cytokines or interferon regulatory factor(IRF)-dependent type I IFNs.Retinoic acid-inducible gene I-like receptors(RLRs)are cytoplasmic RNA helicases,mainly responsible for the identification of ds RNA viruses.RLRs include retinoic acid-inducible gene I(RIG-I)and melanoma differentiation-associated protein 5(MDA5),that are indispensable for recognition of viruses and inhibition of viral replication and dissemination through the production of type I IFNs.Vesicular stomatitis virus(VSV)can be recognized by TLRs or RLRs.Type I IFNs can induce more than 200 kinds of antiviral gene expression that intervening in multiple stages of viral infection cycle and increasing sensitivity for the cytotoxic NK cells and CD8 + T cells.Ubiquitylation is a versatile post-translational modification involved many physiological processes.Ubiquitylation-induced degradation or activation of signaling molecules is a crucial mechanism for immune responses.Ubiquitylation as a key regulatory mechanism involves in many aspects of the immune system,including antigen presentation and the activation of the adaptive immune system,the recognition and clearance of pathogens by the innate immune system.The ubiquitination pathway comprises E1 ubiquitin-activating enzyme,E2 ubiquitin-conjugating enzyme,and E3 ubiquitin-protein ligases enzymes(E3s).Substrate specificity of the ubiquitination system is mainly mediated by E3 s which own a large family,and human genome encodes over 700 E3 s or E3 complexes.E3 s can be grouped into two main families,HECT(homologous to the E6-AP C terminus)type E3 s and RING(really interesting new gene)type E3 s.E3s are involved in the regulation of many signaling pathways during immune responses.For example,E3 s RNF125 conjugates K48-linked ubiquitin to RIG-I leading to its proteasomal degradation,thereby negatively regulating RIG-I signaling.A20 functions both as a RING E3 s and as a de-ubiquitinating enzyme,also acts as a key negative regulator of TLRs and RIG-I-mediated antiviral signaling.NEDD4L(neural precursor cell expressed developmentally down-regulated 4-like)belongs to the NEDD4 family of HECT ubiquitin ligases.The NEDD4 family is characterized by a unique modular structure: the amino terminal Ca2+ phospholipid binding(C2)domain,2-4 WW domains(protein-protein interaction domains)and the carboxyl terminal HECT domain.The number of WW domains is the main sign to distinguish the family members that with substrate specificity and involvement in distinct biological processes.NEDD4 L was originally identified as a developmentally down-regulated gene in the early embryonic central nervous system(CNS).NEDD4 L expressed in many tissues,especially has a dramatically high expression in liver,kidney,heart and lungs.Early studies have demonstrated that NEDD4 L played an important role in hypertension disease through ubiquitylating the sodium channel of cell membrane.Low level of NEDD4 L has been seen in many tumors.In colorectal cancer,NEDD4 L inhibits the classical Wnt signaling as a tumor suppressor.Several studies propose that NEDD4 L can potentially regulate TGF? signaling by targetingThe functions of NEDD4 L in the immune response especially in innate immunity have never been investigated,to our knowledge.It has reported that NEDD4 family members such as NEDD4 and ITCH are implicated in regulating the immune cells and antiviral immune responses.Noteworthily,NEDD4 L is the closest homolog of NEDD4 which is the prototypic member of the family,and is widely expressed in many tissues.However,whether NEDD4 L can regulate antiviral innate immune response remains unknown.In this study,we aimed to investigate the role of E3 ubiquitin-protein ligase NEDD4 L in antiviral innate immune response and the underlying mechanismsPart ?.NEDD4 L selectively promotes the production of type ? interferon induced by VSV in macrophagesWe found that VSV infection upregulated the expression of NEDD4 L in peritoneal macrophages derived from C57BL/6 mice in a time-dependent manner.To examine the effects of NEDD4 L on the production of VSV-induced cytokines,we utilized small interfering RNA(siRNA)to silence NEDD4 L expression in macrophages.We found that knockdown of NEDD4 L significantly suppressed the VSV-induced IFN-? production,while had no effect on TNF-? or IL-6.Next,we generated the NEDD4 L conditional KO mice to get the wild type(NEDD4Lfl/flLyz2cre-)and the NEDD4L-dificient(NEDD4Lfl/flLyz2cre+)mice.NEDD4 L expression in peritoneal macrophages was downregulated over 70% compared to that in WT mice,and was downregulated over 80% in bone marrow-derived macrophages.Then we tested whether NEDD4 L involved in development of immune system,we analyzed the number of immune cells derived from spleen,bone marrow and peripheral blood by FACS.The results showed that NEDD4 L had no effect in differentiation and development of immune cells.To further confirm the role of NEDD4 L in the production of VSV-induced cytokines,we observed the cytokines production after VSV infection in peritoneal macrophages from NEDD4Lfl/flLyz2cre-and NEDD4Lfl/flLyz2cre+ mice.It showed that NEDD4 L deficiency significantly suppressed the VSV-induced IFN-? production,but had no effect on TNF-? or IL-6 production.Taken together,these results suggest that NEDD4 L promotes the production of type?interferon by VSV infection,however,it does not affect the production of inflammatory cytokines.Part ?.The potential molecular mechanisms for the positive regulation of type?interferon production by NEDD4LAccording to the Part?result,we examined the activated signaling leading to type?interferon production by NEDD4 L after VSV infection.We detected the activation of the key signaling molecules of MAPKs,NF-?B and IRFs in macrophages isolated from NEDD4Lfl/flLyz2cre-and NEDD4Lfl/flLyz2cre+ mice after infected with VSV for different time interval by Western Blot.It showed that NEDD4 L deficiency significantly suppressed the phosphorylation of TBK1 and IRF3,while had no effect on the activation of MAPKs(JNK,ERK,and P38)and NF-?B(P65).Next,we investigated the molecular mechanism of NEDD4 L.After recognition of viral nucleic acids,RLRs and TLRs were triggered.The ubiquitin-protein ligase enzyme TRAF3,located in the upstream of TBK1 and a key regulator for the production of type I interferon,regulates RLRs or TLRs signal pathway via ubiquitylation.We analyzed the amino acid sequence of NEDD4 L and found that there were several TRAF binding sites in NEDD4 L.So we supposed that NEDD4 L upregulated phosphorylated TBK1 and IRF3 by interacting with TRAF3.Immunoprecipitation experiment showed that NEDD4 L could combine with TRAF3.To investigate which domain of NEDD4 L could interact with TRAF3,different plasmids encoding NEDD4 L and its mutants were constructed according to its known structural and functional domains: C2 domain deletion(NEDD4L-?C2),HECT domain deletion(NEDD4L-?HECT),C2 and WW domain deletion(NEDD4L-?CW),and cysteine residue 943 in HECT domain was mutated to alanine(NEDD4L-C943A).To detect the interaction,the plasmids encoding wild type NEDD4 L and its mutants and TRAF3 were co-transfected into HEK293 T cells.We found that the WW domain of NEDD4 L was essential for interacting with TRAF3.Then we tested the modulation of TRAF3 by NEDD4 L.We supposed that NEDD4 L increased VSV-induced type?interferon production through ubiquitylating TRAF3.The plasmids encoding TRAF3 or NEDD4 L or ubiquitin(Ub)were co-transfected into HEK293 T cells,the result indicated that NEDD4 L significantly enhanced the ubiquitylation of TRAF3.To further investigate the function of each domain of NEDD4 L in the enhancement of TRAF3 ubiquitylation,various combinations of plasmids encoding NEDD4 L or its mutants or Ub or TRAF3 were co-transfected into HEK293 T cells.We found that carboxyl terminal HECT deficiency(NEDD4L-?HECT)suppressed TRAF3 ubiquitylation,while the amino terminal C2 deficiency(NEDD4L-?C2)significantly increased NEDD4L-mediated TRAF3 ubiquitylation.These results suggest that the HECT domain ubiquitylates TRAF3 and C2 domain is a self-repression sequence.Next,we co-transfected vectors encoding TRAF3,NEDD4 L together with Ub or its mutants which were mutated at different lysine residues namely Ub-K11 R,Ub-K29 R,Ub-K48 R,and Ub-K63 R.The Ub-K29 R markedly suppressed the NEDD4L-mediated TRAF3 ubiquitylation,demonstrating that NEDD4 L could enhance the K29-linked ubiquitination of TRAF3.Taken together,these results demonstrate NEDD4 L directly interacts with TRAF3 through its WW domain,and polyubiquitinates TRAF3 by HECT domain in manner of K29-linked ubiquitination which activating the downstream signaling,leading to the enhanced production of type?interferon triggered by activated IRF3.Meanwhile,C2 domain functions as a self-repression sequence.Part ?.NEDD4 L enhances host defense against viral infection by promoting type?interferon productionWe further discussed the role of NEDD4 L in virus infectious diseases.We established the infective animal model by intraperitoneal injection of VSV.The level of IFN-? in serum of NEDD4Lfl/flLyz2cre+ mice were markedly lower than that in NEDD4Lfl/flLyz2 cremice.Next,we tested the VSV replication in spleen and liver of VSV infectious mice.It showed that VSV RNA replication and the VSV titers(TCID50)in spleen and liver of NEDD4Lfl/flLyz2cre+ mice was much higher than that in NEDD4Lfl/flLyz2cre-mice.These results reveal that in vivo,NEDD4 L can inhibit VSV replication by promoting the production of type?interferon and protect the host from virus infection.To sum up,we have demonstrated that NEDD4 L can selectively promote the production of type?interferon induced by VSV infection in vitro and in vivo.NEDD4 L interacts directly with TRAF3 through its WW domain,polyubiquitinates TRAF3 by HECT domain in manner of K29-linked ubiquitination and activates the downstreamsignaling,leading to the enhanced production of type?interferontriggered byactivatedIRF3,and enhances the resistance to VSV infection in mice.Meanwhile,C2 domain functions as a self-repression sequenceOur study reaveals that HECT E3 ubiquitin-protein ligaseNEDD4 L is responsible for regulating the antiviral innate immune response,enriching the understanding of regulatory mechanism for type ? interferon prodution,and provides a new target for immunotherapy for virus infectious diseases.