Ubiquitin-Mediated Proteasomal Degradation Of IRF3 Induced By LMP1 Enables EBV To Escape Immune Surveillance And Promote Tumor Progression

Natural immunity is the body’s first line of defence against pathogen infection,and the c GAS-STING pathway is an important component of this.This pathway recognises abnormal double-stranded DNA in the cytoplasm,activates the expression of type I interferon(IFN)and induces the body’s anti-pathogen and anti-tumour immune response.Over the course of long evolution,pathogens have developed a variety of strategies to evade host immune surveillance and maintain their own survival.EBV,a type IV human herpesvirus,is an important oncogenic virus that can drive the development of nasopharyngeal carcinoma.Latent membrane protein 1(LMP1),a key oncogenic protein encoded by EBV during latent infection,can remodel host cell signalling and ensure the survival of the virus itself while inducing malignant transformation of host cells.Our previous work found that free EBV DNA could be detected in EBV latently infected cell lines,but no immune responses such as elevated host cell interferon levels were observed.Therefore,we first investigated whether free EBV DNA could activate the host cell’s natural immune response against the virus in the latently infected EBV state.The m RNA and protein expression of type I IFN in EBV/LMP1 positive nasopharyngeal carcinoma cells were not significantly increased after transfection with interferon-stimulated DNA(ISD)compared with EBV/LMP1 negative nasopharyngeal carcinoma cells,suggesting that EBV(LMP1)may interfere with the host cell c GAS-STING interferon signaling pathway.To investigate the reason for the insensitivity of EBV/LMP1 positive nasopharyngeal carcinoma cells to DNA stimulation,we examined the protein expression and activation of the core molecules of the c GAS-STING pathway by WB assays and found that STING and TBK1 were in an activated state in EBV/ LMP1 positive nasopharyngeal carcinoma cells,while the expression,phosphorylation and dimerization levels of the downstream key transcription factor IRF3 were significantly reduced.EBV/LMP1-positive nasopharyngeal carcinoma cells that returned IRF3 expression showed a significant increase in type I IFN expression after ISD stimulation,demonstrating that EBV(LMP1)blocks the c GAS-STING signalling pathway to evade the host’s natural immune surveillance by reducing IRF3 expression.How does EBV(LMP1)lead to low expression of IRF3 protein? We treated cells with the protein synthesis inhibitor actinomycin(CHX)and showed that the rate of degradation of IRF3 protein was significantly accelerated in EBV-positive nasopharyngeal carcinoma cells.After treating cells with MG132,an inhibitor of the ubiquitin-proteasome pathway,Bafilomycin A1,an inhibitor of autophagy lysosomes,and ZVAD,an inhibitor of pan-Caspase activity,respectively,the results showed that MG132 treatment restored IRF3 expression,while Baf-A1 and Z-VAD treatment did not significantly alter IRF3 protein expression.Next,using proteasome inhibitors MG132 and Bortezomib to verify again,the protein level of IRF3 was significantly increased in EBV/LMP1-positive nasopharyngeal carcinoma cells,indicating that EBV(LMP1)induces IRF3 degradation through the proteasomal pathway.Co-IP experiments combined with ubiquitination level assays confirmed that EBV(LMP1)induces ubiquitination modification of IRF3 at position K48,which is then degraded by the proteasome system.Co-IP capture of IRF3-interacting proteins,detected by mass spectrometry,revealed the E3 ubiquitin ligase TRIM21.The results showed that TRIM21 expression was reduced in EBV/ LMP1-positive nasopharyngeal carcinoma cells,suggesting that the E3 ubiquitin ligase function of TRIM21 was not a major influence on the low expression of IRF3 in the context of EBV infection.In addition to its E3 ubiquitin ligase function,TRIM21 was reported in the literature to prevent PIN1-mediated degradation of IRF3 ubiquitination through intercalation with IRF3.This suggests that EBV(LMP1)reduces TRIM21 expression,depriving IRF3 of TRIM21 protection,and PIN1 binds to IRF3 to induce IRF3 degradation via the proteasome pathway.We further investigated the role of low IRF3 expression on tumour progression.Analysis of the GEO database showed that IRF3 m RNA expression was low in nasopharyngeal carcinoma samples.The expression of IRF3 was also found to be significantly lower in nasopharyngeal carcinoma biopsies than in nasopharyngitis samples,and the expression of IRF3 was negatively correlated with that of LMP1 in nasopharyngeal carcinoma and nasopharyngitis tissue sections.Nasopharyngeal carcinoma belongs to a specific subtype of head and neck squamous carcinoma,and our analysis using the Kaplan-Meier Plotter database revealed that patients with head and neck squamous carcinoma with high tissue IRF3 expression had a longer overall survival.Cellular level studies showed that reversion to IRF3 expression significantly reduced the proliferative and antiapoptotic capacity of EBV/ LMP1-positive nasopharyngeal carcinoma cells and significantly delayed cellular G1/S phase evolution,as well as significantly increased sensitivity to cisplatin treatment.Thus,in a model of nasopharyngeal carcinoma,EBV(LMP1)inhibited IRF3 expression and enhanced the malignant phenotype of the tumor.How does IRF3 affect the malignant phenotype of tumors? We used the h TFtarget website to identify target genes downstream of the transcription factor IRF3 that are associated with cell survival,proliferation,apoptosis and cycle,and validated the transcriptional target of IRF3,CDKN2C(p18).p18 is a key negative regulator of cell cycle progression that specifically blocks cells in the G1 phase.The present study demonstrated that IRF3 exerts its tumour suppressor function by regulating the transcription of CDKN2C(p18).In summary,this study found that EBV(LMP1)blocked c GASSTING pathway-mediated intracellular DNA sensing signals by inducing ubiquitinated degradation of IRF3.EBV(LMP1)mediated low expression of IRF3,enhanced tumour cell proliferation,resistance to apoptosis and tolerance to cisplatin,and accelerated the cell cycle transition from G1 to S phase.p18,a transcriptional target of IRF3,was found to play a significant role in the development of the tumour suppressor.p18,a transcriptional target of IRF3,was lowly expressed in EBV/LMP1-positive nasopharyngeal carcinoma cells,contributing to an increased rate of G1/Sphase transition and the construction of an S-phase-like microenvironment conducive to viral replication.Therefore,this study proposes a novel mechanism by which EBV-LMP1 uses the host cell ubiquitin-proteasome system to degrade IRF3 and block the c GAS-STING pathway during latent infection,thereby assisting EBV immune escape and tumour progression,providing a new theoretical basis for an in-depth understanding of the molecular mechanisms of EBV tumourigenesis.