Tumor suppressive role of the antimicrobial lectin REG3A targeting the O-GlcNAc glycosylation pathway.

BACKGROUND AND AIMS: Antimicrobial proteins of the REG3 family provide a first line of protection against infections and transformed cells. Their expression is inducible by inflammation, which makes their role in cancer biology less clear, since an immune- inflammatory context may preexist or coexist with cancer, as occurs in hepatocellular carcinoma (HCC). The aim of this study is to clarify the role of REG3A in liver carcinogenesis and to determine whether carbohydrate-binding functions are involved. APPROACH AND RESULTS: This study provides evidence of the suppressive role of REG3A in HCC by reducing O-GlcNAcylation in two mouse models of HCC, in vitro cell studies, and in clinical samples. REG3A expression in hepatocytes significantly reduces global O- GlcNAcylation and O-GlcNAcylation of c-MYC in preneoplastic and tumor livers and markedly inhibits HCC development in REG3A-c-MYC double transgenic mice and in mice exposed to diethylnitrosamine (DEN). REG3A modifies O-GlcNAcylation without altering the expression or activity of OGT, OGA, or GFAT. Reduced O-GlcNAcylation was consistent with decreased levels of UDP-GlcNAc in pre-cancerous and cancerous livers. This effect is linked to the ability of REG3A to bind Glc and Glc-6P, suggested by a REG3A mutant unable to bind Glc and Glc- 6P and alter O-GlcNAcylation. Importantly, cirrhotic patients with high hepatic REG3A expression had lower levels of O-GlcNAcylation and longer cancer-free survival than REG3A- negative cirrhotic livers. CONCLUSION: REG3A helps fight liver cancer by reducing O-GlcNAcylation. This study suggests a new paradigm for the regulation of O-GlcNAc signalling in cancer-related pathways through interactions with the carbohydrate-binding function of REG3A.

A combined clinical and biological risk classification improves prediction of outcome in hepatoblastoma patients.

AIM: Stratification of hepatoblastoma (HB) patients is based on clinical and imaging characteristics obtained at the time of diagnosis. We aim to integrate biomarkers into a tool that accurately predicts survival of HB patients. METHODS: We retrospectively analysed 174 HB patients for the presence of four biomarkers and explored their prognostic potential by correlating with overall survival (OS) and event-free survival (EFS). RESULTS: Mutations of CTNNB1, NFE2L2 and TERT were found in 135 (78%), 10 (6%) and 10 (6%) patients, respectively, and the adverse C2 subtype of the 16-gene signature in 63 (36%) patients. C2-patients had more frequent metastatic disease, higher alpha-fetoprotein levels, non-fetal histology and significantly worse 3-year OS (68% versus 95%) and EFS (63% versus 87%) than C1-patients. Patients carrying a NFE2L2 mutation had a significantly worse 3-year OS (57% versus 88%) than NFE2L2 wild-type patients and were more likely to have vessel invasive growth and non-fetal histology. TERT mutations were almost exclusively found in older patients, whereas CTNNB1 mutations showed no association with any clinical feature or outcome. In a multivariable analysis, the C2 subtype remained a significant predictor of poor outcome with hazard ratios of 6.202 and 3.611 for OS and EFS, respectively. When added to the Children's Hepatic tumors International Collaboration risk stratification, the presence of the C2 subtype identified a group of high-risk patients with a very poor outcome. CONCLUSION: We propose a new stratification system based on the combination of clinical factors and the 16-gene signature, which may facilitate a risk-adapted management of HB patients.

Hepatitis B virus replicating in hepatocellular carcinoma encodes HBx variants with preserved ability to antagonize restriction by Smc5/6.

Hepatitis B virus infection is a major cause of liver diseases including hepatocellular carcinoma (HCC). The viral regulatory protein HBx is essential for viral replication and has been involved in the development of HCC. Recently, we characterized a subset of HCCs that replicate HBV. Our aim was to characterize HBx encoded by the full-length HBV DNA (cccDNA) in HCC and non-HCC liver. HBx genes were amplified and sequenced from eight paired HCC and non-HCC tissues in which HBV cccDNA and pgRNA were both present. Sequence analyses identified twelve amino acid positions mutated between HCC and non-HCC liver, and detected in at least three cases. We next assessed the impact of these mutations on HBx function by testing their transcriptional activity. We examined their ability to rescue the transcription of HBV virus deficient for HBx in differentiated HepaRG cells and to induce Smc5/6 degradation, which is mandatory for viral replication. We assessed their capacity to activate a CREB-dependent reporter. Finally we analyzed their growth suppressive activity using colony formation assays. Our results showed that most HBx variants isolated from HCC retain their ability to support HBV cccDNA transcription and to degrade Smc5/6. Strikingly, HCC specific HBx variants are impaired in their antiproliferative activity, which may be detrimental for tumor growth. In conclusion, in contrast to previous observations that tumor HBx variants lack transcriptional activity, we showed here that HBx variants have retained their ability to counteract Smc5/6 and thus to activate cccDNA transcription although they tend to lose antiproliferative activity.

microRNA 193a-5p Regulates Levels of Nucleolar- and Spindle-Associated Protein 1 to Suppress Hepatocarcinogenesis.

BACKGROUND & AIMS: We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. METHODS: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay. RESULTS: Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice. CONCLUSIONS: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).

Hepatitis B Virus Pregenomic RNA in Hepatocellular Carcinoma: A Nosological and Prognostic Determinant.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). However, very little is known about the replication of HBV in HCC tissues. We analyzed viral and cellular parameters in HCC (T) and nontumor liver (NT) samples from 99 hepatitis B surface antigen (HBsAg)-positive, virologically suppressed patients treated by tumor resection or liver transplantation. We examined total HBV DNA and RNA as well as covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA), which are considered as markers of active HBV replication. Total HBV DNA and RNA were detected in both T and NT samples in a majority of cases, but only a subset of tumors harbored detectable levels of HBV cccDNA and pgRNA (39% and 67%) compared to NT livers (66% and 90%; P < 0.01). Further evidence for HBV replication in tumor tissues was provided by sequencing of the X gene derived from episomal forms, showing that HBV genotypes differed between T and matched NT samples in 11 cases. The detection of pgRNA and cccDNA in tumors was correlated to the absence of tumorous microvascular invasion and to better patient survival. Analysis of gene expression profiles by Agilent microarrays revealed that pgRNA-positive HCCs were characterized by low levels of cell cycle and DNA repair markers and expression of the HBV receptor, sodium taurocholate cotransporting polypeptide, indicating well-differentiated tumors. CONCLUSION: HCC replicating HBV represents a subtype of weakly invasive HCC with a transcriptomic signature. pgRNA originating from nonintegrated, complete HBV genomes is a sensitive marker for viral replication and prognosis. (Hepatology 2018;67:86-96).

New insights into diagnosis and therapeutic options for proliferative hepatoblastoma.

Surgery and cisplatin-based treatment of hepatoblastoma (HB) currently guarantee the survival of 70%-80% of patients. However, some important challenges remain in diagnosing high-risk tumors and identifying relevant targetable pathways offering new therapeutic avenues. Previously, two molecular subclasses of HB tumors have been described, C1 and C2, with C2 being the subgroup with the poorest prognosis, a more advanced tumor stage, and the worst overall survival rate. An associated 16-gene signature to discriminate the two tumoral subgroups was proposed, but it has not been transferred into clinical routine. To address these issues, we performed RNA sequencing of 25 tumors and matched normal liver samples from patients. The transcript profiling separated HB into three distinct subgroups named C1, C2A, and C2B, identifiable by a concise four-gene signature: hydroxysteroid 17-beta dehydrogenase 6, integrin alpha 6, topoisomerase 2-alpha, and vimentin, with topoisomerase 2-alpha being characteristic for the proliferative C2A tumors. Differential expression of these genes was confirmed by quantitative RT-PCR on an expanded cohort and by immunohistochemistry. We also revealed significant overexpression of genes involved in the Fanconi anemia (FA) pathway in the C2A subgroup. We then investigated the ability of several described FA inhibitors to block growth of HB cells in vitro and in vivo. We demonstrated that bortezomib, a Food and Drug Administration-approved proteasome inhibitor, strongly impairs the proliferation and survival of HB cell lines in vitro, blocks FA pathway-associated double-strand DNA repair, and significantly impedes HB growth in vivo. CONCLUSION: The highly proliferating C2A subtype is characterized by topoisomerase 2-alpha gene up-regulation and FA pathway activation, and the HB therapeutic arsenal could include bortezomib for the treatment of patients with the most aggressive tumors. (Hepatology 2018;68:89-102).

LIM-Only Protein FHL2 Is a Negative Regulator of Transforming Growth Factor ß1 Expression.

Transforming growth factor ß1 (TGF-ß1) is a master cytokine in many biological processes, including tissue homeostasis, epithelial-to-mesenchymal transition, and wound repair. Here, we report that four and a half LIM-only protein 2 (FHL2) is a critical regulator of TGF-ß1 expression. Devoid of a DNA-binding domain, FHL2 is a transcriptional cofactor that plays the role of coactivator or corepressor, depending on the cell and promoter contexts. We detected association of FHL2 with the TGF-ß1 promoter, which showed higher activity in Fhl2-/- cells than in wild-type (WT) cells in a reporter assay. Overexpression of FHL2 abrogates the activation of the TGF-ß1 promoter, whereas the upregulation of TGF-ß1 gene transcription correlates with reduced occupancy of FHL2 on the promoter. Moreover, ablation of FHL2 facilitates recruitment of RNA polymerase II on the TGF-ß1 promoter, suggesting that FHL2 may be involved in chromatin remodeling in the control of TGF-ß1 gene transcription. Enhanced expression of TGF-ß1 mRNA and cytokine was evidenced in the livers of Fhl2-/- mice. We tested the in vivo impact of Fhl2 loss on hepatic fibrogenesis that involves TGF-ß1 activation. Fhl2-/- mice developed more severe fibrosis than their WT counterparts. These results demonstrate the repressive function of FHL2 on TGF-ß1 expression and contribute to the understanding of the TGF-ß-mediated fibrogenic response.

MicroRNA therapy inhibits hepatoblastoma growth in vivo by targeting ß-catenin and Wnt signaling.

Hepatoblastoma (HBL) is the most common pediatric liver cancer. In this malignant neoplasm, beta-catenin protein accumulates and increases Wnt signaling due to recurrent activating mutations in the catenin-beta 1 (CTNNB1) gene. Therefore, beta-catenin is a key therapeutic target in HBL. However, controlling beta-catenin production with therapeutic molecules has been challenging. New biological studies could provide alternative therapeutic solutions for the treatment of HBL, especially for advanced tumors and metastatic disease. In this study, we identified microRNAs (miRNAs) that target beta-catenin and block HBL cell proliferation in vitro and tumor growth in vivo. Using our dual-fluorescence-FunREG system, we screened a library of 1,712 miRNA mimics and selected candidates inhibiting CTNNB1 expression through interaction with its untranslated regions. After validating the regulatory effect of nine miRNAs on beta-catenin in HBL cells, we measured their expression in patient samples. Let-7i-3p, miR-449b-3p, miR-624-5p, and miR-885-5p were decreased in tumors compared to normal livers. Moreover, they inhibited HBL cell growth and Wnt signaling activity in vitro partly through beta-catenin down-regulation. Additionally, miR-624-5p induced cell senescence in vitro, blocked experimental HBL growth in vivo, and directly targeted the beta-catenin 3'-untranslated region. Conclusion: Our results shed light on how beta-catenin-regulating miRNAs control HBL progression through Wnt signaling inactivation. In particular, miR-624-5p may constitute a promising candidate for miRNA replacement therapy for HBL patients. (Hepatology Communications 2017;1:168-183).

Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma.

Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5' ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers. IMPORTANCE: Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5' ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.

CAGE profiling of ncRNAs in hepatocellular carcinoma reveals widespread activation of retroviral LTR promoters in virus-induced tumors.

An increasing number of noncoding RNAs (ncRNAs) have been implicated in various human diseases including cancer; however, the ncRNA transcriptome of hepatocellular carcinoma (HCC) is largely unexplored. We used CAGE to map transcription start sites across various types of human and mouse HCCs with emphasis on ncRNAs distant from protein-coding genes. Here, we report that retroviral LTR promoters, expressed in healthy tissues such as testis and placenta but not liver, are widely activated in liver tumors. Despite HCC heterogeneity, a subset of LTR-derived ncRNAs were more than 10-fold up-regulated in the vast majority of samples. HCCs with a high LTR activity mostly had a viral etiology, were less differentiated, and showed higher risk of recurrence. ChIP-seq data show that MYC and MAX are associated with ncRNA deregulation. Globally, CAGE enabled us to build a mammalian promoter map for HCC, which uncovers a new layer of complexity in HCC genomics.

HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase.

BACKGROUND & AIMS: Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription. METHODS: We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression. RESULTS: In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA. CONCLUSION: Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin.

Hepatocellular carcinoma.

The hepatitis B virus (HBV) is a widespread human pathogen that causes liver inflammation, cirrhosis, and hepatocellular carcinoma (HCC). Recent sequencing technologies have refined our knowledge of the genomic landscape and pathogenesis of HCC, but the mechanisms by which HBV exerts its oncogenic role remain controversial. In a prevailing view, inflammation, liver damage, and regeneration may foster the accumulation of genetic and epigenetic defects leading to cancer onset. However, a more direct and specific contribution of the virus is supported by clinical and biological observations. Among genetically heterogeneous HCCs, HBV-related tumors display high genomic instability, which may be attributed to the ability of HBV to integrate its DNA into the host cell genome, provoking chromosomal alterations and insertional mutagenesis of cancer genes. The viral transactivator HBx may also participate in transformation by deregulating diverse cellular machineries. A better understanding of the complex mechanisms linking HBV to HCC will improve prevention and treatment strategies.

Massive gene amplification drives paediatric hepatocellular carcinoma caused by bile salt export pump deficiency.

Hepatocellular carcinoma (HCC) is almost invariably associated with an underlying inflammatory state, whose direct contribution to the acquisition of critical genomic changes is unclear. Here we map acquired genomic alterations in human and mouse HCCs induced by defects in hepatocyte biliary transporters, which expose hepatocytes to bile salts and cause chronic inflammation that develops into cancer. In both human and mouse cancer genomes, we find few somatic point mutations with no impairment of cancer genes, but massive gene amplification and rearrangements. This genomic landscape differs from that of virus- and alcohol-associated liver cancer. Copy-number gains preferentially occur at late stages of cancer development and frequently target the MAPK signalling pathway, and in particular direct regulators of JNK. The pharmacological inhibition of JNK retards cancer progression in the mouse. Our study demonstrates that intrahepatic cholestasis leading to hepatocyte exposure to bile acids and inflammation promotes cancer through genomic modifications that can be distinguished from those determined by other aetiological factors.

The oncogenic role of hepatitis B virus.

The hepatitis B virus (HBV) is a small enveloped DNA virus that causes acute and chronic hepatitis. HBV infection is a world health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). HBV has been classified among human tumor viruses by virtue of a robust epidemiologic association between chronic HBV carriage and HCC occurrence. In the absence of cytopathic effect in infected hepatocytes, the oncogenic role of HBV might involve a combination of direct and indirect effects of the virus during the multistep process of liver carcinogenesis. Liver inflammation and hepatocyte proliferation driven by host immune responses are recognized driving forces of liver cell transformation. Genetic and epigenetic alterations can also result from viral DNA integration into host chromosomes and from prolonged expression of viral gene products. Notably, the transcriptional regulatory protein HBx encoded by the X gene is endowed with tumor promoter activity. HBx has pleiotropic activities and plays a major role in HBV pathogenesis and in liver carcinogenesis. Because hepatic tumors carry a dismal prognosis, there is urgent need to develop early diagnostic markers of HCC and effective therapies against chronic hepatitis B. Deciphering the oncogenic mechanisms that underlie HBV-related tumorigenesis might help developing adapted therapeutic strategies.

LIM-only protein FHL2 activates NF-κB signaling in the control of liver regeneration and hepatocarcinogenesis.

Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor-NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2(-/-) macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2(-/-) mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

Methyltransferase PRMT1 is a binding partner of HBx and a negative regulator of hepatitis B virus transcription.

The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.