Hepatitis B Virus Integration Research Articles

Unraveling the role of hepatitis B virus DNA integration in B-cell lymphomagenesis.

Studies have shown that hepatitis B virus (HBV)-associated B-cell non-Hodgkin lymphoma (NHL) constitutes a unique subgroup with distinct clinical features. It still leaves open the question of whether the integration of HBV DNA into the B-cell genome is a causal mechanism in the development of lymphoma. Using the hybridisation capture-based next generation sequencing and RNA sequencing, we characterised the HBV integration pattern in 45 HBV-associated B-cell NHL tumour tissues. A total of 354 HBV integration sites were identified in 13 (28.9%) samples, indicating the relatively low integration frequency in B-cell NHLs. High plasma HBV DNA loads were not associated with the existence of HBV integration. The insertion sites distributed randomly across all the lymphoma genome without any preferential hotspot neither at the chromosomal level nor at the genetic level. Intriguingly, most HBV integrations were nonclonal in B-cell NHLs, implying that they did not confer a survival advantage. Analysis of the paired diagnosis-relapse samples showed the unstable status of HBV integrations during disease progression. Furthermore, transcriptomic analysis revealed the limited biological impact of HBV integration. Our study provides an unbiased HBV integration map in B-cell NHLs, revealing the insignificant role of HBV DNA integration in B-cell lymphomagenesis.

The role of the hepatitis B virus genome and its integration in the hepatocellular carcinoma.

The integration of Hepatitis B Virus (HBV) is now known to be closely associated with the occurrence of liver cancer and can impact the functionality of liver cells through multiple dimensions. However, despite the detailed understanding of the characteristics of HBV integration and the mechanisms involved, the subsequent effects on cellular function are still poorly understood in current research. This study first systematically discusses the relationship between HBV integration and the occurrence of liver cancer, and then analyzes the status of the viral genome produced by HBV replication, highlighting the close relationship and structure between double-stranded linear (DSL)-HBV DNA and the occurrence of viral integration. The integration of DSL-HBV DNA leads to a certain preference for HBV integration itself. Additionally, exploration of HBV integration hotspots reveals obvious hotspot areas of HBV integration on the human genome. Virus integration in these hotspot areas is often associated with the occurrence and development of liver cancer, and it has been determined that HBV integration can promote the occurrence of cancer by inducing genome instability and other aspects. Furthermore, a comprehensive study of viral integration explored the mechanisms of viral integration and the internal integration mode, discovering that HBV integration may form extrachromosomal DNA (ecDNA), which exists outside the chromosome and can integrate into the chromosome under certain conditions. The prospect of HBV integration as a biomarker was also probed, with the expectation that combining HBV integration research with CRISPR technology will vigorously promote the progress of HBV integration research in the future. In summary, exploring the characteristics and mechanisms in HBV integration holds significant importance for an in-depth comprehension of viral integration.

Hepatitis B virus enhancer 1 activates preS1 and preS2 promoters of integrated HBV DNA impairing HBsAg secretion

The expression of HBsAg from integrated HBV DNA limits the achievement of functional cure for chronic hepatitis B. Thus, characterising the unique expression and secretion of HBsAg derived from integrated HBV DNA is of clinical significance. A total of 563 treatment-naive patients and 62 functionally cured patients were enrolled, and HBsAg and HBcAg immunohistochemistry of their liver biopsy tissues was conducted followed by semi-quantitative analysis. Then, based on stratified analysis of HBeAg-positive and -negative patients, long-read RNA sequencing analysis, as well as an invitro HBV integration model, we explored the HBsAg secretion characteristics of integrated HBV DNA and underlying mechanisms. In contrast to the significantly lower serum HBsAg levels, no significant decrease of intrahepatic HBsAg protein was observed in HBeAg-negative patients, as compared with HBeAg-positive patients. The results of long-read RNA sequencing of liver tissues from patients with chronic HBV infection and invitro studies using integrated HBV DNA mimicking dslDNA plasmid revealed that, the lower HBsAg secretion efficiency seen in HBeAg-negative patients might be attributed to an increased proportion of preS1 mRNA derived from integrated HBV DNA instead of covalently closed circular DNA. The latter resulted in an increased L-HBsAg proportion and impaired HBsAg secretion. Enhancer 1 (EnhI) in integrated HBV DNA could retarget preS1 (SP1) and preS2 (SP2) promoters to disrupt their transcriptional activity balance. The secretion of HBsAg originating from integrated HBV DNA was impaired. Mechanistically, functional deficiency of core promoter leads to retargeting of EnhI and thus uneven activation of the SP1 over the SP2 promoter, resulting in an increase in the proportion of L-HBsAg. Integrated hepatitis B virus (HBV) DNA can serve as an important reservoir for HBV surface antigen (HBsAg) expression, and this limits the achievement of a functional cure. This study revealed that secretion efficiency is lower for HBsAg derived from integrated HBV DNA than HBsAg derived from covalently closed circular DNA, as determined by the unique sequence features of integrated HBV DNA. This study can broaden our understanding of the role of HBV integration and shed new light on antiviral strategies to facilitate a functional cure. We believe our results are of great general interest to a broad audience, including patients and patient organisations, the medical community, academia, the life science industry and the public.

Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy

The landscape of hepatitis B virus (HBV) integration in the plasma cell-free DNA (cfDNA) of HBV-infected patients with different stages of liver diseases [chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC)] remains unclear. In this study, we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA, based on DNA probe capture and next-generation sequencing. Using this optimized strategy, we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection, with high sensitivity and accuracy. The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals (CHB, LC, and HCC) were further investigated. A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25 (88%) clinical HBV-infected samples, respectively. In vivo analysis showed that the normalized number of support unique sequences (nnsus) in HCC was significantly higher than in CHB or LC patients (P values ​< ​0.05). All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800. The majority of integration breakpoints (61.86%) are located in the gene-coding region. Both non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) interactions occurred during HBV integration across the three different stages of liver diseases. Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients, including those with CHB, LC, or HCC, using this optimized strategy.

Long-term antiviral therapy is associated with changes in the profile of transcriptionally active HBV integration in the livers of patients with CHB.

Hepatitis B virus (HBV) integration exists throughout the clinical course of chronic hepatitis B (CHB). This study investigated the effects of long-term antiviral therapy on the level and profiles of transcriptionally active HBV integration. Serial liver biopsies and paired blood samples were obtained from 16, 16, and 22 patients with CHB at baseline, 78, and 260 weeks of entecavir monotherapy or combined with pegylated interferon alfa, respectively. Serum HBV biomarkers were longitudinally assessed. RNA-seq and HIVID2 program was used to identify HBV-host chimeric RNAs transcribed from integrated DNA. The counts of HBV integration reads were positively related to both serum HBV DNA levels (r = 0.695, p = 0.004) and HBeAg titers (r = 0.724, p = 0.021) at baseline, but the positive correlation exited only to the serum HBsAg levels after 260 weeks of antiviral therapy (r = 0.662, p = 0.001). After 78 weeks of antiviral therapy, the levels of HBV integration expression decreased by 12.25 folds from baseline. The viral junction points were enriched at the S and HBx genes after the long-term antiviral therapy. HBs-FN1 became one of the main transcripts, with the mean proportion of HBs-FN1 in all integrated expression increased from 2.79% at baseline to 10.54% at Week 260 of antiviral treatment. Antiviral therapy may reduce but not eliminate the HBV integration events and integration expression. Certain integration events, such as HBs-FN1 can persist in long-term antiviral treatment.

The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice

Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.

Abstract 1481: Disparity landscapes of HBV integration in hepatocellular carcinoma: Mechanistic characterization and functional implications

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a common malignancy worldwide. About half of all new liver cancers worldwide each year occur in China due to a high prevalence of hepatitis B virus (HBV) infection. HBV DNA integrates into the human genome, disrupting the endogenous tumor suppressors/regulatory genes or enhancing the activity of proto-oncogenes. It would be useful to examine the different NGS-based databases to provide a more unbiased and comprehensive survey of HBV integration. We aimed to take advantage of publicly available datasets of different regional cohorts to determine the disparity landscapes of integration events among sample cohorts, tissue types, chromosomal positions, individual host and viral genes, and genic locations. By comparing HCC tumor with non-tumorous liver, landscape of HBV integration was delineated at gene-independent and gene-dependent manners. Moreover, we performed mechanistic investigations on how HBV-TERT integration led to TERT activation and derived a score to predict patients’ prognostication according to their clonal disparity landscape of HBV integration. We revealed a global geographical disparity of HBV integration that the landscape of HBV integration between HCC and non-tumorous liver varied in regional cohorts, suggesting the different degrees of clonal enrichment. In the Mainland China, Hong Kong and Mongolia cohorts, HCC tumors had higher numbers of events than the non-tumorous livers, whereas in the French and US cohorts, the reverse was true. Moreover, most HBV integrations were positionally enriched at the telomeres and centromeres, and this highlighted the novel co-involvement of HBV integration at these regions, which likely introduces greater genomic instability. Furthermore, we constructed a large meta-cohort of multiple ethnicities to refine the landscape of HBV integration. This enabled the identification of events at key HCC-related genes (e.g., TERT, KMT2B, and CCNA2) and gene families (e.g., cyclin, cadherin, and contactin). As TERT is the most frequently integrated gene by HBV, we further investigated the mechanistic modulation of TERT transcription activation by combining and analyzing our in-house sample cohort with a previous report and luciferase reporter assays. This revealed the positive concurrent influence by both the orientation and relative distance of HBV integration (closer to transcription start site) from the TERT gene. Additionally, we observed clonal disparity of HBV integration among patients and the higher level of the clonal disparity score correlated with poor prognostication of HCC patients. Our study uncovered the different levels of clonal enrichment of HBV integration and identified mechanistic insights and prognostic biomarker. This strengthens our understanding of HBV-associated hepatocarcinogenesis. Citation Format: Xueying Lyu, Karen Man-Fong Sze, Joyce Man-Fong Lee, Abdullah Husain, Irene Oi-Lin Ng, Daniel Wai-Hung Ho. Disparity landscapes of HBV integration in hepatocellular carcinoma: Mechanistic characterization and functional implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1481.

Deep whole-genome analysis of 494 hepatocellular carcinomas.

Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribedmutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.

Genome-wide loss-of-function genetic screen identifies INSIG2 as the vulnerability of hepatitis B virus-integrated hepatoma cells.

There are approximately 250 million people chronically infected with hepatitis B virus (HBV) worldwide. Although HBV is often integrated into the host genome and promotes hepatocarcinogenesis, vulnerability of HBV integration in liver cancer cells has not been clarified. The aim of our study is to identify vulnerability factors for HBV-associated hepatocarcinoma. Loss-of-function screening was undertaken in HepG2 and HBV-integrated HepG2.2.15 cells expressing SpCas9 using a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library. Genes whose guide RNA (gRNA) abundance significantly decreased in HepG2.2.15 cells but not in HepG2 cells were extracted using the MAGeCK algorithm. We identified four genes (BCL2L1, VPS37A, INSIG2, and CFLAR) that showed significant reductions of gRNA abundance and thus potentially involved in the vulnerability of HBV-integrated cancer cells. Among them, siRNA-mediated mRNA inhibition or CRISPR-mediated genetic deletion of INSIG2 significantly impaired cell proliferation in HepG2.2.15 cells but not in HepG2 cells. Its inhibitory effect was alleviated by cotransfection of siRNAs targeting HBV. INSIG2 inhibition suppressed the pathways related to cell cycle and DNA replication, downregulated cyclin-dependent kinase 2 (CDK2) levels, and delayed the G1 -to-S transition in HepG2.2.15 cells. CDK2 inhibitor suppressed cell cycle progression in HepG2.2.15 cells and INSIG2 inhibition did not suppress cell proliferation in the presence of CDK2 inhibitor. In conclusion, INSIG2 inhibition induced cell cycle arrest in HBV-integrated hepatoma cells in a CDK2-dependent manner, and thus INSIG2 might be a vulnerability factor for HBV-associated liver cancer.

Enrichment Reveals Extensive Integration of Hepatitis B Virus DNA in Hepatitis Delta Virus-Infected Patients.

Hepatitis B virus (HBV) DNA may become integrated into the human genome of infected human hepatocytes. Expression of integrations can produce the surface antigen (HBsAg) that is required for synthesis of hepatitis D virus (HDV) particles and the abundant subviral particles in the blood of HBV- and HDV-infected subjects. Knowledge about the extent and variation of HBV integrations and impact on chronic HDV is still limited. We investigated 50 pieces of liver explant tissue from 5 patients with hepatitis D-induced cirrhosis, using a deep-sequencing strategy targeting HBV RNA. We found that integrations were abundant and highly expressed, with large variation in the number of integration-derived (HBV/human chimeric) reads, both between and within patients. The median number of unique integrations for each patient correlated with serum levels of HBsAg. However, most of the HBV reads represented a few predominant integrations. The results suggest that HBV DNA integrates in a large proportion of hepatocytes, and that the HBsAg output from these integrations vary >100-fold depending on clone size and expression rate. A small proportion of the integrations seems to determine the serum levels of HBsAg and HDV RNA in HBV/HDV coinfected patients with liver cirrhosis.

Impact of HBV Integration on Hepatocellular Carcinoma After Long-Term Antiviral Therapy.

Few studies have reported the integrated characteristics of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after long-term antiviral therapy. This study aimed to investigate the HBV integration features in HBV-HCC patients who had undergone long-term antiviral therapy, evaluate their impact on clinical indicators, and analyze the potential mechanisms involved. We utilized genome-wide association study (GWAS) to analyze liver cancer tissues and detect the presence of HBV integration. Seventeen patients with HBV integration were included in the integration (Int) group, while the remaining five patients were included in the non-integration (N-int) group. Clinical indicators were regularly monitored and compared between the two groups. The characteristics of HBV integration patterns were analyzed, and differences between the groups were explored at the chromosome and genomic levels. After long-term antiviral therapy, although the frequency of HBV integration in HBV-HCC was reduced, residual HBV integration still accelerated the development of HCC. It affected the diagnosis, treatment, and prognosis of patients. HBV integration events led to changes in chromosome structure, which were closely related to HCC. Novel fusion genes were detected at a high frequency and had the potential to be specific detection sites for HBV-HCC. HBV integration events are synergistically involved in the human genome and HBV, which can lead to chromosome structural instability, gene rearrangement events closely related to HCC production, and the formation of new specific fusion genes.

Comprehensive Review of Hepatocellular Carcinoma: Epidemiological Trends, Risk Factors, and Mechanisms of Carcinogenesis

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, often arising in the context of chronic liver diseases like cirrhosis, with significant contributions from hepatitis B virus (HBV), hepatitis C virus (HCV), and metabolic dysfunction-associated fatty liver disease (MAFLD). The disease’s high mortality rate and complex etiology underscore the need for improved diagnostic and therapeutic strategies. Methods: This review synthesizes current epidemiological data and research on HCC. We analyzed global and regional incidence and mortality rates, assessed the role of major risk factors, including HBV, HCV, and MAFLD, and evaluated recent advancements in diagnostic and therapeutic approaches. Data were sourced from recent literature, including epidemiological studies and clinical trials. Results: The incidence of HCC continues to rise globally, with significant regional variations. HBV and HCV infections remain major contributors to HCC, with HBV's integration into the host genome playing a critical role in carcinogenesis. MAFLD, now a prominent risk factor in high-income countries, is expected to impact global HCC rates increasingly. Despite advances in diagnostic techniques such as precision medicine and liquid biopsies, early detection remains challenging, and recurrence rates after treatment are high. Conclusion: The growing burden of HCC highlights the urgent need for enhanced screening methods and novel treatments. Continued research into the disease’s etiology and improved therapeutic approaches are essential to reducing incidence and improving patient outcomes. Enhanced early detection and personalized treatment strategies are critical to addressing the ongoing challenges in HCC management.

Temporal and structural patterns of hepatitis B virus integrations in hepatocellular carcinoma.

Chronic infection of hepatitis B virus (HBV) is the major cause of hepatocellular carcinoma (HCC). Notably, 90% of HBV-positive HCC cases exhibit detectable HBV integrations, hinting at the potential early entanglement of these viral integrations in tumorigenesis and their subsequent oncogenic implications. Nevertheless, the precise chronology of integration events during HCC tumorigenesis, alongside their sequential structural patterns, has remained elusive thus far. In this study, we applied whole-genome sequencing to multiple biopsies extracted from six HBV-positive HCC cases. Through this approach, we identified point mutations and viral integrations, offering a blueprint for the intricate tumor phylogeny of these samples. The emergent narrative paints a rich tapestry of diverse evolutionary trajectories characterizing the analyzed tumors. We uncovered oncogenic integration events in some samples that appear to happen before and during the initiation stage of tumor development based on their locations in reconstituted trajectories. Furthermore, we conducted additional long-read sequencing of selected samples and unveiled integration-bridged chromosome rearrangements and tandem repeats of the HBV sequence within integrations. In summary, this study revealed premalignant oncogenic and sequential complex integrations and highlighted the contributions of HBV integrations to HCC development and genome instability.

Integrated HBV DNA and cccDNA maintain transcriptional activity in intrahepatic HBsAg-positive patients with functional cure following PEG-IFN-based therapy.

Hepatitis B surface antigen (HBsAg) seroclearance marks regression of hepatitis B virus (HBV) infection. However, more than one-fifth of patients with functional cure following pegylated interferon-based therapy may experience HBsAg seroreversion. The mechanisms causing the HBV relapse remain unclear. To investigate the level and origin of HBV transcripts in patients with functional cure and their role in predicting relapse. Liver tissue obtained from patients with functional cure, as well as uncured and treatment-naïve HBeAg-negative patients with chronic hepatitis B (CHB) were analysed for intrahepatic HBV markers. HBV capture and RNA sequencing were used to detect HBV integration and chimeric transcripts. Covalently closed circular DNA (cccDNA) levels and the proportion of HBsAg-positive hepatocytes in functionally cured patients were significantly lower than those in uncured and treatment-naïve HBeAg-negative patients. Integrated HBV DNA and chimeric transcripts declined in functionally cured patients compared to uncured patients. HBsAg-positive hepatocytes present in 25.5% of functionally cured patients, while intrahepatic HBV RNA remained in 72.2%. The levels of intrahepatic HBV RNA, integrated HBV DNA, and chimeric transcripts were higher in functionally cured patients with intrahepatic HBsAg than in those without. The residual intrahepatic HBsAg in functionally cured patients was mainly derived from transcriptionally active integrated HBV DNA; meanwhile, trace transcriptional activity of cccDNA could also remain. Two out of four functionally cured patients with intrahepatic HBsAg and trace active cccDNA experienced HBV relapse. Integrated HBV DNA and cccDNA maintain transcriptional activity and maybe involved in HBsAg seroreversion in intrahepatic HBsAg-positive patients with functional cure and linked to virological relapse.

Multiomics analyses reveal pathological mechanisms of HBV infection and integration in liver cancer.

Hepatitis B virus (HBV) infection and integration are important for hepatocellular carcinoma (HCC) initiation and progression, while disease mechanisms are still largely elusive. Here, we combined bulk and single-cell sequencing technologies to tackle the disease mechanisms of HBV-related HCC. We observed high HBV mutation rate and diversity only in tumors without HBV integration. We identified human somatic risk loci for HBV integration (VIMs). Transcription factors (TFs) enriched in VIMs were involved in DNA repair and androgen receptor (AR) signaling. Aberration of AR signaling was further observed by single-cell regulon analysis in HBV-infected hepatocytes, which showed remarkable interactions between AR and the complement system that, together with the X-linked ZXDB regulon that contains albumin (ALB), probably contribute to HCC male predominance. Complement system dysregulation caused by HBV infection was further confirmed by analyses of single-cell copy numbers and cell-cell communications. Finally, HBV infection-associated immune cells presented critical defects, including TXNIP in T cells, TYROBP in NK cells, and the X-linked TIMP1 in monocytes. We further experimentally validated our findings in multiple independent patient cohorts. Collectively, our work shed light on the pathogenesis of HBV-related HCC and other liver diseases that affect billions of people worldwide.

Mitochondrial DNA is a target of HBV integration

Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development.

Re-analysis of hepatitis B virus integration sites reveals potential new loci associated with oncogenesis in hepatocellular carcinoma.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). HBV DNA can get integrated into the hepatocyte genome to promote carcinogenesis. However, the precise mechanism by which the integrated HBV genome promotes HCC has not been elucidated. To analyze the features of HBV integration in HCC using a new reference database and integration detection method. Published data, consisting of 426 Liver tumor samples and 426 paired adjacent non-tumor samples, were re-analyzed to identify the integration sites. Genome Reference Consortium Human Build 38 (GRCh38) and Telomere-to-Telomere Consortium CHM13 (T2T-CHM13 (v2.0)) were used as the human reference genomes. In contrast, human genome 19 (hg19) was used in the original study. In addition, GRIDSS VIRUSBreakend was used to detect HBV integration sites, whereas high-throughput viral integration detection (HIVID) was applied in the original study (HIVID-hg19). A total of 5361 integration sites were detected using T2T-CHM13. In the tumor samples, integration hotspots in the cancer driver genes, such as TERT and KMT2B, were consistent with those in the original study. GRIDSS VIRUSBreakend detected integrations in more samples than by HIVID-hg19. Enrichment of integration was observed at chromosome 11q13.3, including the CCND1 pro-moter, in tumor samples. Recurrent integration sites were observed in mitochondrial genes. GRIDSS VIRUSBreakend using T2T-CHM13 is accurate and sensitive in detecting HBV integration. Re-analysis provides new insights into the regions of HBV integration and their potential roles in HCC development.

Characteristics of Hepatitis B virus integration and mechanism of inducing chromosome translocation

Hepatitis B virus (HBV) integration is closely associated with the onset and progression of tumors. This study utilized the DNA of 27 liver cancer samples for high-throughput Viral Integration Detection (HIVID), with the overarching goal of detecting HBV integration. KEGG pathway analysis of breakpoints was performed using the ClusterProfiler software. The breakpoints were annotated using the latest ANNOVAR software. We identified 775 integration sites and detected two new hotspot genes for virus integration, N4BP1 and WASHP, along with 331 new genes. Furthermore, we conducted a comprehensive analysis to determine the critical impact pathways of virus integration by combining our findings with the results of three major global studies on HBV integration. Meanwhile, we found common characteristics of virus integration hotspots among different ethnic groups. To specify the direct impact of virus integration on genomic instability, we explained the causes of inversion and the frequent occurrence of translocation due to HBV integration. This study detected a series of hotspot integration genes and specified common characteristics of critical hotspot integration genes. These hotspot genes are universal across different ethnic groups, providing an effective target for better research on the pathogenic mechanism. We also demonstrated more comprehensive key pathways affected by HBV integration and elucidated the mechanism for inversion and frequent translocation events due to virus integration. Apart from the great significance of the rule of HBV integration, the current study also provides valuable insights into the mechanism of virus integration.

Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

Development of hepatocellular carcinoma in treated and untreated patients with chronic hepatitis B virus infection.

Hepatitis B virus (HBV) is responsible for more than 50% of hepatocellular carcinoma (HCC) in HBV hyperendemic areas, such as the Asia-Pacific region. Several hepatitis B viral factors are involved in HBV-related hepatocarcinogenesis. Hepatitis B viral load is the most important risk factor of HCC development. In addition, HBV integration, HBV genotype C, and core-promoter mutations are also associated with a risk of HCC development. For untreated chronic hepatitis B (CHB) patients, the estimated HCC incidence rates per 100 patient-years were 0.03-0.17 in inactive carriers, 0.07-0.42 in asymptomatic carriers, 0.12-0.49 in chronic hepatitis, and 2.03-3.37 in cirrhosis. Complementary to HBV DNA, serum levels of the hepatitis B surface antigen and hepatitis B core-related antigen (HBcrAg) can predict the occurrence of HCC for untreated patients with low and intermediate viral loads, respectively. For patients receiving antiviral therapy, the risks of HCC occurrence 40-60% lower than those for untreated patients. Patients treated with residual detectable HBV DNA or intrahepatic cccDNA still have a risk of HCC. Serum levels of HBcrAg, M2BPGi and fibrosis-4 are predictive of the risk of HCC development in treated patients. Several well-developed HCC risk scores can help clinicians identify high-risk CHB patients for HCC surveillance, regardless of treatment status. These strategies can help minimize the threat of HCC and prolong survival in CHB patients.