HBZ Protein Suppresses The Innate Immune Response In ATL Cells By Regulating TRIM25

Human T-cell leukemia virus type 1(HTLV-1)is the pathogenic retrovirus that associated with adult T-cell leukemia(ATL).HTLV-1 virus can encode a series of viral proteins,such as p12,p13,Rex,Tax and HBZ,and have regulatory functions.Tax and HBZ proteins are considered to be two key proteins for HTLV-1 infection and maintenance.Both of them are involved in regulating many signaling pathways in vivo,such as TGF-β,NF-κB and AP-1.Pathogens can activate the host’s innate immune response when they invade their host,and RIG-I in retinoic acid inducible gene I(RIG-I)-like receptors(RLRs)can recognize RNA virus genomes and promote the synthesis and secretion of type I interferons(IFN-α/β)and other inflammatory factors,which are key for cells to initiate innate immunity against RNA virus infection.Studies have shown that tax inhibits the IFN-β signaling pathway to help HTLV-1-infected cells evade surveillance.However,because Tax has a high immunogenicity and is regulated by epigenetics or non-epigenetics in the late stage of viral infection,its expression is reduced or even not expressed.However,IFN-βsignaling pathway is still inhibited,suggesting that there are other molecular mechanisms in ATL that inhibit IFN-β signaling pathway.Subsequently,it was discovered that the viral protein HBZ inhibits the IFN-β signaling pathway by affecting the function of IRF3,but the specific mechanism remains undefined.Tripartite motif protein 25(TRIM25)-mediated RIG-I ubiquitin activation is considered to be a key step to initiate intracellular antiviral.However,many RNA viruses have evolved different countermeasures against TRIM25 to avoid natural Immune monitoring.Based on this,we speculate: In ATL cells,can HBZ regulate the IFN-β signaling pathway mediated by TRIM25 by affecting the function of TRIM25,thereby promoting viral infection and the occurrence of ATL? In order to prove the inference and clarify its molecular mechanism,this research carried out research from the following aspects:Part 1: TRIM25 is highly expressed in ATLFirst,we assessed TRIM25 protein expression by Western blot in HTLV-1negative control cell lines CEMT-4,Jurkat,MOLT-4,and HTLV-1 positive cell lines MT-1,MT-2,MT-4,ATL-2,ATL-T,ED.The results showed that TRIM25 protein was highly expressed in ATL positive cell lines.Subsequently,using Jurkat cell lines stably overexpressing HBZ,we showed that HBZ can significantly upregulate TRIM25 protein expression.Finally,using transient transfection assays,we found that HBZ was able to stabilize TRIM25 protein levels in a dose-dependent manner.The above results indicate that HBZ is a key factor that contributes to the high expression of TRIM25 in ATL cell lines.Part Ⅱ: HBZ inhibits LUBAC-mediated ubiquitination of TRIM25 and stabilizes TRIM25 protein levelsTo investigate the molecular mechanism underlying the high expression of TRIM25 protein in ATL cells,we first used western bolt to detect the effect of HBZ on the half-life of TRIM25 and found that HBZ can extend the half-life of TRIM25 to some extent.Through immunoprecipitation experiments,we found that HBZ inhibited the ubiquitination of TRIM25 in a dose-dependent manner.Subsequent immunoprecipitation experiments found that both HBZ and TRIM25 can bind to HOIP and HOIL-1L in the linear ubiquitin chain assembly complex(LUBAC),and HBZ significantly inhibits the binding of TRIM25 to HOIP and HOIL-1L.Finally,the immunoprecipitation results showed that HBZ can significantly inhibit the ubiquitination of TRIM25 induced by HOIL-1L.The above results indicate that HBZ inhibits the ubiquitination degradation pathway of TRIM25 by inhibiting the combination of TRIM25 and LUBAC,thereby stabilizing the protein level of TRIM25.Part Ⅲ : HBZ inhibits TRIM25-mediated IFN-β signaling pathway and its molecular mechanismTo investigate the regulatory effect of HBZ on TRIM25 mediated innate immune signaling,we first examined the function of TRIM25 in ATL cells by dual luciferase reporter gene assay.It revealed that TRIM25 can further activate innate immune responses in ATL cells in response to different stimuli.To further elucidate the molecular mechanism,we detected the interaction and colocalization between HBZ and TRIM25 by CO immunoprecipitation(coimmunoprecipitation)as well as immunofluorescence(IF)experiments,which showed that HBZ could bind to TRIM25 and that both mainly colocalized in the cytoplasm around the nucleus.Subsequently,we performed coimmunoprecipitation experiments using HBZ and TRIM25 mutants.It showed that the AD domain of HBZ binds TRIM25,whereas the CC and PS domains of TRIM25 interact with HBZ.We found that the CD domain of HBZ inhibits TRIM25 mediated IFN-β signaling in reporter gene assays.Finally,we found that HBZ does not affect the formation of TRIM25 dimerization,but significantly inhibits the combination of TRIM25 and RIG-I.In summary,HBZ can inhibit the activation of TRIM25 on RIG-I by affecting the combination of TRIM25 and RIG-I,and ultimately inhibit the IFN-β signaling pathway.Part Ⅳ: HBZ regulates TRIM25 to repress target gene expression and promote ATL genesisIn order to study whether HBZ affects the expression of downstream target genes in IFN-β signaling pathway by inhibiting TRIM25-mediated IFN-β signaling pathway,we did q RT-PCR experiment and found that when HBZ is expressed,IFN-βdownstream target gene IFN-β,IFIT1,IFIT2 was significantly down-regulated.Meanwhile,to illustrate the function of highly expressed TRIM25 in ATL cells,we constructed ATL-T stable lines with silenced TRIM25 by crispr-cas9 technology,and found that silencing TRIM25 significantly inhibited the growth of ATL-T cells by MTT assay,which suggested that highly expressed TRIM25 promoted the growth of ATL cells.In conclusion,we found that HBZ,a viral protein encoded by HTLV-1,stabilizes TRIM25 ubiquitination upregulation by LUBAC,inhibits the ubiquitination degradation pathway of TRIM25 to stabilize its protein level,and utilizes the pro proliferative growth function of TRIM25 to promote ATL development.In summary,in this study,we found that HBZ,a HTLV-1 encoded viral protein,on the one hand,stabilizes the protein level of TRIM25 by inhibiting its ubiquitination upregulation by Lubac,and uses the pro-proliferative growth function of TRIM25 to promote ATL development;On the other hand,HBZ suppresses TRIM25 mediated IFN-β signaling by inhibiting the RIG-I ubiquitination activation pathway to downregulate interferon stimulated gene expression,ultimately suppressing the cell’s innate immune response.This study not only elucidates the molecular mechanism underlying the regulation of TRIM25,a key protein in innate immunity,by HBZ,but also illustrates the physiological function of high TRIM25 expression in ATL.The results of this study will provide clues to elucidate the molecular mechanism of HTLV-1 viral carcinogenesis and provide new strategies and targets for the clinical treatment of ATL.