LINC01431 Promotes Histone H4R3 Methylation To Impede HBV Covalently Closed Circular DNA Transcription By Stabilizing PRMT1

Chronic hepatitis B virus(HBV)infection greatly increases the risk of end-stage liver diseases including hepatocellular carcinoma(HCC).Effective control of HBV infection is the first step to prevent and treat HCC.Covalently closed circular DNA(cccDNA),the transcriptional template of HBV,interacts with both host and viral proteins to form minichromosome in the nucleus,and serves as the fundamental obstacle for the complete cure of chronic hepatitis B(CHB).The transcriptional activity of cccDNA is regulated by many factors,among which the epigenetic modifications of cccDNA-bound histones play a crucial role.Meanwhile,HBV-coded proteins,especially HBx,could transactivate the expression of multiple host factors to maintain HBV replication.It has been reported that long noncoding RNAs(lncRNAs)could participate in the replication of a variety of viruses through epigenetic manners,however,whether lncRNAs could epigenetically modulate the transcription of HBV cccDNA remains unclear.This study aims to explore the transcriptional regulation of HBV cccDNA by lncRNAs,as well as,to explore the molecular mechanism of HBV evading host antiviral response through modulating lncRNAs expression,both of them provide novel strategies for HBV treatment.In order to achieve the purposes above,HBV-infected hepatocytes from different sources including HepaRGNTCP,HepG2NTCP,Huh7NTCP and HLCZ01 cells,as well as HBV expression plasmids pHBV1.3,pHBV1.1 and MC-HBV-transfected Huh7 and HepG2 cells were used in this study.And The HBV carried mice were constructed by hydrodynamically injecting with pAAV-HBV1.2 expression plasmid.The main research methods and results are as follows:Part Ⅰ LINC01431 is a novel lncRNA which is involved in regulating HBV replication1.LncRNA-seq identifies HBV infection downregulates LINC01431 expressionTo identify differentially expressed lncRNAs during HBV infection,lncRNA sequencing was performed in HLCZ01 cells with or without HBV infection.Data analysis showed that transcriptional regulation and RNA biosynthesis-related biological processes were significantly enriched in HBV-infected HLCZ01 cells,among these,11 unannotated lncRNAs were significantly dysregulated in HBV-infected cells.The expression of these lncRNAs were detected in HBV-infected and HBx-overexpressed hepatocytes.The results showed that LINC01431 expression was significantly inhibited in HBx-overexpressed hepatocytes and also showed a dynamic decrease in HBV-infected cells.Concurrently,we further confirmed the downregulation of LINC01431 induced by HBV in multiple HBV models and HBV inhibited LINC01431 expression in HBx-dependent manner.2.LINC01431 inhibits the transcription of HBV cccDNATo explore the role of LINC01431 on HBV replication,LINC01431 overexpression and knockdown were performed in various HBV replication models,the level of HBV antigens,HBV RNAs,HBV-DNA and cccDNA were detected.The results showed that,LINC01431 significantly inhibited HBV replication,displaying as decreased levels of viral antigens,HBV RNAs,and HBV-DNA,while the level of cccDNA remained unchanged.Furthermore,dualluciferase reporter experiments showed that LINC01431 inhibited the transcriptional activity of different HBV promoters.ChIP assays showed that LINC01431 inhibited the enrichments of active epigenetic modifications on cccDNA.Collectively,these results suggest that LINC01431 might inhibit cccDNA transcription and HBV replication through epigenetic manners.Part Ⅱ LINC01431 inhibits HBV cccDNA transcription through interacting with PRMT11.LINC01431 is mainly localized in the nucleusThe subcellular localization of lncRNAs is the primary determinant of their molecular functions.To confirm the subcellular localization of LINC01431,nucleoplasmic RNA isolation experiments and RNA in situ hybridization(FISH)assays were performed,and the results showed that LINC01431 was mainly localized in the nucleus.2.PRMT1 is the interacting protein of LINC01431In order to explore the molecular mechanism by which LINC01431 inhibited HBV cccDNA transcription,RNA pull-down coupled with mass spectrometry analysis was performed,and the results showed that PRMT1 is a potential target of LINC01431.Meanwhile,RNA immunoprecipitation(RIP),RNA pull-down and immunofluorescence(IFA)assays further confirmed the interaction between PRMT1 and LINC01431,and the 178-265 domain of PRMT1 was necessary for interacting with LINC01431.3.LINC01431 inhibits HBV cccDNA transcription in PRMT1-dependent mannerTo confirm whether PRMT1 is involved in the inhibitory role of LINC01431 on HBV transcription,PRMT1 knockdown were performed in HBV-infected cells harboring LINC01431.The results showed that,knockdown of PRMT1 markedly rescued the antiviral effect of LINC01431 in HBV-infected HepaRGNTCP and Huh7NTCP cells,and PRMT1 mediated the inhibitory role of LINC01431 on HBV replication via its methyltransferase activity.Collectively,PRMT1 mediates the transcriptional inhibition of LINC01431 on HBV cccDNA.Part Ⅲ LINC01431 promotes PRMT1-mediated H4R3me2a on cccDNA and transcriptional repression on HBV replication1.LINC01431 enhances the protein stability of PRMT1LncRNAs modulating the stability of interacted proteins is one of the dominant manners to exert its function.Cell transfection and PRMT1 expression analysis showed that,ectopic LINC01431 expression increased PRMT1 protein abundance and in a dose-dependent manner,while,the level of PRMT1 mRNA remained unchanged either with LINC01431 overexpression or knockdown.Protein stability assays showed that LINC01431 overexpression significantly prolonged the half-life of PRMT1 protein,while knockdown of LINC01431 accelerated PRMT1 degradation,suggesting that LINC01431 enhanced the protein stability of PRMT12.LINC01431 inhibits the ubiquitination of PRMT1 proteinUbiquitination modification plays a crucial role in regulating protein stability.Protein ubiquitination experiments showed that LINC01431 overexpression significantly reduced the ubiquitination level of PRMT1,and knockdown of LINC01431 promoted the ubiquitination degradation of PRMT1,indicating that LINC01431 enhanced the protein stability of PRMT1 through repressing PRMT1 ubiquitination.3.LINC01431 inhibits HBx-mediated ubiquitination degradation of PRMT1It has been reported that HBx could bind with and promote the ubiquitination degradation of PRMT1 to facilitate HBV replication.Co-immunoprecipitation assay(Co-IP)and ubiquitination assays showed that,LINC01431 inhibited the interaction between HBx and the 178-265 domain of PRMT1 and inhibited the HBx-mediated ubiquitination degradation of PRMT1 in HBV-transfected and HBx-overexpressed hepatocytes,indicating that LINC01431 competitively inhibited the interaction of HBx and PRMT1 to protect PRMTl from the HBxmediated ubiquitination degradation.4.LINC01431 increases the enrichment of PRMT1 and H4R3me2a on cccDNATo further explore whether LINC01431 could affect the occupancy of PRMT1 on HBV cccDNA,LINC01431 overexpression and knockdown were performed in different HBVinfected hepatocytes.ChIP assays showed that LINC01431 increased the enrichments of PRMT1 and H4R3me2a on HBV cccDNA.5.LINC01431 promotes PRMT1-induced cccDNA transcriptional repressionTo explore whether the LINC01431-PRMT1-H4R3me2a axis could epigenetically regulate cccDNA transcription,LINC01431 overexpression were performed in multiple HBV-infected cells.ChIP assays showed that LINC01431 significantly decreased the occupancies of Ac-H3,Ac-H4,H3K27ac,H4K8/K12ac,and RNA Pol Ⅱ on cccDNA.Furthermore,rescue assays showed that,knockdown of PRMT1 or treatment with the PRMT1 inhibitor significantly rescued the reduced enrichments of histones acetylation and RNA PolⅡ on cccDNA.Taken together,LINC01431 epigenetically regulates transcriptional repression of cccDNA through interacting with PRMT1.Part Ⅳ HBx transcriptionally represses LINC01431 expression to evade the transcriptional repression of host cell through HBx1.HBV,especially HBx inhibits the transcription of LINC01431In order to explore the crucial viral protein that inhibited the transcription of LINC01431,LINC01431 promoter reporter assay and RT-qPCR were performed using the expressing plasmids coding different HBV proteins.The results showed that,except for HBV Polymerase,HBV and viral proteins inhibited the transcription of LINC01431,among which HBx showed the predominant effect and repressed the transcriptional activity of LINC01431 promoter in a dose-dependent manner.2.The transcription factor ZHX2 mediates the transcriptional repression of HBx on LINC01431To further investigate the molecular mechanism by which HBx inhibited the transcription of LINC01431,the potential transcription factors binding sites in the promoter region of LINC01431 were predicted using online databases,and the common enriched transcription factors were compared with the reported HBx-regulated transcription factors in the literatures.The results showed that transcription factor ZHX2 could interact with HBx and potentially modulate LINC01431 transcription.ChIP and RT-qPCR assays showed that ZHX2 promoted LINC01431 transcription by directly binding to the promoter region of LINC01431.Furthermore,knockdown of ZHX2 was performed in HBx-transfected cells,and the result showed that knockdown of ZHX2 significantly rescued the HBx-mediated repression on LINC01431.Collectively,these results confirmed that HBx repressed the transcription of LINC01431 via manipulating transcription factor ZHX2.Conclusion and SignificanceIn this study,we identified a novel lncRNA LINC01431 involved in regulating the transcriptional activity of cccDNA.LINC01431 binds with and stabilizes PRMT1,leading to altered methylation and acetylation statues on cccDNA-bound histones,which results in epigenetically repressing the transcription of cccDNA.On the other hand,HBV,especially HBx inhibits the transcription of LINC01431 to promote HBV replication.Our study revealed a novel mechanism by which LINC01431 mediates the interaction of host cells and HBV cccDNA,providing a new target for HBV therapy.