Editor's Note: HBV infection is a global public health issue, with its chronic progression leading to severe liver diseases such as chronic hepatitis B (CHB), cirrhosis, and hepatocellular carcinoma (HCC). In recent years, the phenomenon of HBV DNA integrating into the host genome has garnered widespread attention as it unveils new mechanisms of HBV pathogenesis and significantly impacts the diagnosis, treatment, and prognosis of HBV-related diseases. Recently, Dr. Fabien Zoulim from Lyon Hepatology Institute, France, published a comprehensive review article in the Journal of Hepatology (JOH), detailing the mechanisms of HBV DNA integration, its impact on liver disease progression, and its implications for the functional cure of CHB. This review aims to provide new perspectives and methods for the prevention and treatment of CHB.Mechanisms and Dynamics of HBV DNA Integration
HBV is a partially double-stranded DNA virus whose genome integrates into the host genome through reverse transcription, a crucial step in the HBV life cycle. Although integration events do not directly participate in viral replication, their frequent occurrence and early appearance play an indispensable role in HBV pathogenesis. Integration primarily occurs during the production of double-stranded linear DNA (DSL DNA) in the viral DNA replication process, with these DSL DNAs being inserted into the host chromosome breakpoints via non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ).
The accumulation of integration events is dynamic during HBV infection. During acute infection, only a few hepatocytes exhibit HBV DNA integration, but as the infection persists, the integration frequency significantly increases. In chronic infection, especially in patients with prolonged viral replication, integration events are pervasive throughout the liver, detected in up to 90% of hepatocytes. This accumulation reflects the complex interactions between the virus and the host genome and reveals the potential threat of persistent HBV infection to host genomic stability.
Impact of Integrated HBV DNA on Liver Diseases
The impact of integrated HBV DNA on liver diseases is multifaceted. Firstly, integrated HBV DNA can drive the expression of HBsAg and HBx proteins. HBsAg is a primary marker of HBV infection, maintaining the infection state even after viral replication ceases, thereby exerting continuous immune pressure on the host immune system. HBx protein, with its various biological functions such as promoting cell proliferation, inhibiting apoptosis, and inducing DNA damage, plays a crucial role in the development and progression of HBV-related liver cancer.
Secondly, integrated HBV DNA plays a key role in the development of HCC. Integration events promote tumor cell growth and metastasis through mechanisms such as activating known oncogenes, causing chromosomal instability, and inducing epigenetic changes. Identifying integration hotspots like the TERT promoter and MLL4 gene exon further elucidates the specific mechanisms by which integrated HBV DNA contributes to HCC development. These integration events not only affect the regulation of host gene expression but may also promote tumorigenesis by altering cellular signaling pathways and metabolic processes.
Furthermore, integrated HBV DNA is essential for the persistence of HDV infection. HDV, a defective virus, requires HBV envelope proteins to complete its life cycle. In patients co-infected with HBV and HDV, integrated HBV DNA can express HBsAg and other envelope proteins, supporting HDV replication and persistence even when HBV replication is suppressed. This complicates and challenges the treatment of HDV infection.
Detection and Characterization Technologies for Integrated HBV DNA
With the rapid development of high-throughput sequencing technologies, various platforms have been applied to detect and characterize integrated HBV DNA. Long-read sequencing technologies such as PacBio and Oxford Nanopore can generate ultra-long reads covering the entire HBV DNA integration site and its flanking host genomic sequences. This sequence information is crucial for analyzing the distribution of integration sites, structural characteristics of integration events, and the impact of integration on host gene expression.
In addition, probe capture combined with short-read sequencing technologies is widely used in integrated HBV DNA research. By designing specific probes targeting HBV sequences, HBV DNA integration sites can be efficiently enriched and analyzed through high-throughput sequencing in the complex background of the host genome. This method improves detection sensitivity and specificity and can identify integration sites across the entire genome, providing powerful tools for diagnosing and researching HBV-related diseases.
Impact of Integrated HBV DNA on Chronic Delta Hepatitis (CHD)
In CHD patients, integrated HBV DNA significantly impacts disease progression and treatment response. Since HDV replication depends on HBV envelope proteins, the expression of integrated HBV DNA in liver tissues of CHD patients is crucial for HDV persistence. Even when cccDNA levels decline, integrated HBV DNA can maintain HBsAg expression and HDV particle assembly and release. This makes CHD treatment more challenging, requiring simultaneous intervention against both HBV and HDV.
Treatment strategies for CHD include antiviral therapy, immunomodulatory therapy, and the application of direct antiviral agents. However, due to the persistent expression of integrated HBV DNA, conventional antiviral drugs may not completely eliminate HDV infection. Developing novel therapies targeting integrated HBV DNA is essential. For example, small interfering RNA (siRNA) and gene editing technologies like CRISPR/Cas9 hold promise for eliminating HDV infection and achieving a functional cure by targeting integrated HBV DNA.
Integrated HBV DNA in HCC Diagnosis and Treatment
Integrated HBV DNA also has significant implications in HCC diagnosis and treatment. Identifying integration hotspots such as the TERT promoter and MLL4 gene exon aids in HCC classification and treatment strategy development and provides new biomarkers for early HCC diagnosis. By detecting HBV-host gene fusion points in circulating tumor DNA (ctDNA), tumor signals can be identified early in HCC development, enabling early intervention and treatment.
In terms of HCC treatment, integrated HBV DNA presents new challenges and opportunities. Traditional methods such as surgery, radiotherapy, and chemotherapy can control tumor growth to some extent but cannot eradicate the persistent infection and immune evasion caused by integrated HBV DNA. Therefore, novel therapies targeting integrated HBV DNA, such as gene therapy and immunotherapy, are expected to bring breakthroughs in HCC treatment. Further research into the specific mechanisms by which integrated HBV DNA contributes to HCC will provide more precise targets and strategies for HCC prevention and treatment.
Impact of Integrated HBV DNA on Functional Cure of CHB
Achieving a functional cure for CHB, marked by the sustained disappearance of HBsAg and long-term remission of liver inflammation, is a crucial treatment goal. However, the persistent expression of HBsAg from integrated HBV DNA challenges the definition and assessment of functional cure. Since integrated HBV DNA can express HBsAg independently of viral replication, HBsAg levels may remain detectable even after complete cccDNA clearance. This renders traditional HBsAg detection methods unable to accurately reflect the true state of HBV infection.
To overcome this challenge, researchers are developing new non-invasive biomarkers such as circulating HBV RNA and HBcrAg to assess cccDNA transcriptional activity. These biomarkers can more accurately reflect HBV replication status and treatment response. However, biomarkers that can directly quantify the load and expression levels of integrated HBV DNA are still lacking. Further research into the role of integrated HBV DNA in functional cure and the development of new detection methods is key to achieving a functional cure for HBV.
Summary
In conclusion, integrated HBV DNA is a complex and critical phenomenon in the HBV infection process. It not only reveals new mechanisms of HBV pathogenesis but also significantly impacts the diagnosis, treatment, and prognosis evaluation of HBV-related diseases. Future research should further explore the role of integrated HBV DNA in disease progression and treatment, develop new detection methods and therapeutic strategies to achieve a functional cure for CHB.
