The Molecular Mechanism Of ENPP1 Upregulating LMNB1 Acetylation Modification In Inhibiting The Anterior Genome Promoter Transcriptional Activity Of HBV

Background and PurposeHepatitis B virus(HBV)infection is a major health problem affecting approximately 300 million people worldwide.Although prophylactic vaccine reduces new infections,there is still no cure for chronic hepatitis B(CHB).Current anti-HBV therapy can slow disease progression but not cure because they cannot eliminate or permanently silence HBV covalently closed circular DNA(cccDNA).The cccDNA minor chromosomes persist in the nuclei of infected hepatocytes,serving as a template for viral transcription.The interaction between host factors and cccDNA is essential for their formation,stability,and transcriptional activity.ENPP1 is a type of transmembrane glycoprotein with pyrophosphatase and phosphodiesterase activities,and ENPP1 promotes tumor growth and dissemination and is a potential immunotherapeutic target.Cyclosine monophosphate(cGAMP)acts as a second messenger in the natural immune response triggered by HBV,ultimately activating the interferon(IFN)signaling pathway.ENPP1 is able to hydrolyze cGAMP,and perhaps a link between ENPP1 and the native immunity triggered by HBV infection may also play a role in the transcriptional regulation of HBV.LMNB1 is a potential transcription factor as well as a drug target for its involvement in cell proliferation and senescence by affecting chromosomal distribution,gene expression,and DNA damage repair.This paper focuses on the regulatory role and molecular mechanism of ENPP1 and LMNB1 in HBV replication and transcription,and its results further enrich the regulatory network of host factors on HBV and contribute to the development of new antiviral drugs based on this mechanism.Material and MethodsWith ENPP1 overexpression and knockdown by transfection of pcDNA3.1-Flag-ENPP1 plasmid in Huh7 cells and siRNA targeting ENPP1,and pUC19HB-Bj plasmid after 24h,western blot verified the effect of ENPP1 overexpression and knockdown and the effect of ENPP1 on the expression levels of HBsAg and HBcAg,the effects of overexpression and knockdown of ENPP1 on HBV pgRNA were determined by RT-qPCR.A series of HBV promoter and mutant plasmids were constructed,and the effect of norkidney luciferase reporter on the expression of ENPP1 on the activity of HBV promoter and HBV EnhⅡ/BCP was determined by ENPP 1.ZDOCK and PyMOL software were used to predict the interaction between ENPP1 and LMNB1 and key amino acid sites,pcDNA3.1-Flag-LMNB1 plasmid in Huh7 cells,and arrilla luciferase reporter assay tested the effect of LMNB1 overexpression on the activity of HBV promoter and its mutants.Acetylation omics analysis of differential changes in LMNB1 acetylation after HBV infection,then mutants associated with LMNB1 acetylation sites were constructed,and kidney luciferase reporter assay tested the effect of overexpressing LMNB1 and its mutants on the activity of the HBV promoter.ResultsOverexpression of ENPP 1 reduces the expression levels of pgRNA and HBcAg;knockdown of ENPP 1 promotes the expression level of pgRNA and HBcAg;ENPP 1 suppresses the activity of HBV Core promoter and EnhⅡ/BCP,but ENPP1 does not directly target the promoter EnhⅡ/BCP region,There is an obvious interaction between LMNB1 and ENPP1,with the key amino acid sites:Asn98,Arg106,Arg109 of ENPP1,and Glu446,Asp448,Asp450,Arg455,Asp512,and Glu525 of LMNB1.LMNB1 interacted with the promoter EnhⅡ/BCP region to inhibit the promoter activity,and the simultaneous overexpression of ENPP1 and LMNB1 was more inhibitory to the promoter activity than single plasmids.Acetylation omics analysis showed that the acetylation levels at 111K,261K and 483K of LMNB1 were reduced compared with controls,and the promoter activity in the LMNB1 and the 111Q and 532Q groups,and the 111R,261Q,483Q and 483R groups were restored compared with the LMNB1 overexpression group,suggesting that the LMNB1 acetylation site may be the target of LMNB1 acting on the promoter.ConclusionBy interacting with LMNB1,ENPP1 up-regulates the acetylation levels of LMNB1 protein at 111K,261K,and 483K,promoting the recognition of LMNB1 and inhibiting the HBV promoter,and then downregulates the expression of pregenomic RNA and HBcAg.