The Molecular Mechanism Of YAP Protein Regulating HTLV-1 Viral Transcription

Adult T-cell leukemia(ATL)is a malignant tumor disease caused by Human T-cell leukemia virus type 1(HTLV-1)infection.HTLV-1 mainly affects the function of host cells by encoding regulatory proteins.HTLV-1 mainly encodes some structural genes such as gag,pol,env,and regulatory genes such as Tax,HBZ,Rex,p12,p13 and p30.Tax protein plays an important role in the transcription of HTLV-1virus and promotes the occurrence of ATL.Hippo/YAP signaling pathway was first discovered in Drosophila,and YAP as a key protein of Hippo pathway,also plays an important role in tumor regulation.However,the molecular mechanism of how YAP protein affects HTLV-1 viral transcription has not been clarified yet.Previous studies have shown that during the occurrence of ATL,the Hippo pathway is inhibited while the YAP protein is highly expressed,and YAP interacts with Tax protein.Therefore,we wonder whether YAP affects the transcription of HTLV-1 virus by regulating Tax,the key protein encoded by HTLV-1 virus,and then causes tumorigenesis? In order to confirm this conjecture,this study mainly explored the regulatory effect of YAP protein on Tax-mediated HTLV-1 viral transcription,and further analyzed the molecular mechanism and physiological significance of the regulatory effect.The main research contents are shown as follows:Part Ⅰ: Regulation of YAP protein on promoter of HTLV-1 virusIn order to explore the effect of YAP protein on the promoter activity of HTLV-1virus,we used Jurkat,293 T cells and ATL-T stable strains overexpressing/silenced YAP to detecte and analyze 5’LTR and 3’LTR promoter activity of HTLV-1mediated by YAP regulation Tax through reporter gene technology.Firstly,dual-luciferase reporter gene assay indicated that YAP inhibited the activity of the5’LTR promoter activated through Tax,but YAP had no effect on the activity of the3’LTR promoter activated by Tax.In addition,YAP alone had no effect on activity of5’LTR and 3’LTR promoter.Subsequently,dual-luciferase reporter gene assay was used to detect that YAP homologous TAZ also inhibited the activity of 5’LTR promoter activated by Tax,and TAZ alone had no effect on the activity of 5’LTR promoter.Then,we explored the regulatory mechanisms of YAP on different signaling pathways.The experimental results showed that YAP alone did not activate NF-κB and AP-1 signaling pathways,but YAP further enhanced the Tax-activated NF-κB signaling pathway,while YAP inhibited the Tax-activated AP-1 signaling pathway.It is suggested that YAP has different regulatory mechanisms in different signal pathways of Tax activation,which indicates that YAP has different regulatory effects on the activity of HTLV-1 promoter mediated by Tax is reasonable.Using ATL-T stable strain of overexpressing/silenced YAP,the results showed that overexpressing YAP in ATL-T cells further depressed Tax-mediated 5’LTR promoter activity,and obviously inhibited YAP protein expression in ATL-T further activated Tax-mediated 5’LTR promoter activity.Moreover,293 T cell were treated with YAP inhibitor VP,which significantly activated the activity of the 5’LTR promoter mediated by Tax.The above results indicated that YAP inhibited the activity of5’LTR promoter activated by Tax,but YAP had no effect on the activity of HTLV-1 3’LTR promoter.Part Ⅱ : Molecular mechanism of YAP regulating promoter activity of HTLV-1 virusTo explore the molecular mechanism of YAP regulating the activity of HTLV-1virus promoter,we found that Tax,CREB and p300 bound to YAP protein,and TAZ protein also bound to Tax by Co-IP.In order to further analyzed the binding sites of YAP and Tax,we used YAP mutants and Tax to conduct immunoprecipitation analysis,and the results showed that YAP protein was mainly involved in the interaction with Tax protein through YAP aa150-300 domain.And the Tax protein interacted with YAP mainly through the Tax aa1-319 domain.It was subsequently found that YAP did not affect the interaction between Tax and CREB,while YAP inhibited the combination of Tax and p300.The dual-luciferase reporter gene assay showed that YAP inhibited the activity of Tax-mediated 5’LTR promoter mainly by affecting p300.Further immunofluorescence experiments were carried out to analyze the intracellular localization of Tax and p300 by YAP,and it was found that YAP inhibited the intracellular co-localization of Tax and p300,and inhibited Tax recruitment p300 into nucleus.In addition,the dual-luciferase reporter gene assay showed that YAP inhibited Tax-mediated 5’LTR promoter activity without YAP protein phosphorylation pathways.Part Ⅲ: YAP regulates HTLV-1 virus gene transcription and its physiological effectsIn order to study the regulation of HTLV-1 gene transcription by YAP and its physiological phenomenon,we first treated ATL-T cell line with VP.It was found that after VP treatment inhibited YAP,the expression level of Tax gene increased,while the expression level of HBZ gene was not affected.Then,we used the ATL-T stable strain overexpressing/silenced YAP,Western blot analysis showed that the overexpressing YAP inhibited the expression of HTLV-1 virus encoding protein p19,p24,gp46,while silenced YAP enhanced the expression of HTLV-1 virus encoding protein.And q RT-PCR results also showed that overexpressing YAP inhibited the expression of HTLV-1 virus encoding p19,p24,gp46 gene.Finally,the virus infection experiment showed that HTLV-1 virus infection ability was inhibited in ATL cell lines with high expression of YAP,while HTLV-1 virus infection ability was significantly enhanced in ATL cell lines with silenced YAP,indicating that YAP had an inhibitory effect on HTLV-1 virus infection ability by inhibiting viral transcription.In summary,it was found that YAP inhibited the activity of HTLV-1 5’LTR promoter mediated by Tax,while YAP had no effect on the activity of HTLV-13’LTR promoter.YAP inhibited the activity of 5’LTR promoter activated by Tax through p300.YAP inhibited the interaction between Tax and p300,and YAP inhibited Tax recruitment p300 into nucleus.In addition,we found that the expression level of Tax gene increased after YAP function loss.However,HTLV-1 encoding genes p19,p24 and gp46 were significantly down-regulated at both gene and protein levels after overexpression of YAP.Finally,YAP inhibited viral infection by inhibiting viral transcription.This study found that YAP inhibited the activity of HTLV-1 5’LTR promoter,thus affecting viral transcription.This study provided a new idea for the pathogenesis of ATL,and it is helpful for the clinical diagnosis and treatment of ATL.