Hepatitis B virus (HBV) infection remains a major global public health challenge. Recent estimates indicate that approximately 260 million people worldwide are living with chronic HBV infection, corresponding to about 3% of the global population, and viral hepatitis causes more than one million deaths each year, mainly due to cirrhosis and hepatocellular carcinoma [1], [2]. In Japan, the number of people living with chronic hepatitis B was estimated at approximately 1.02–1.19 million in 2015 [3], and more recent reports from the Ministry of Health, Labor and Welfare indicate that this figure has remained largely unchanged over the past decade. Among the currently approved therapies, nucleotide analogues (NUCs) remain the mainstay of chronic hepatitis B treatment [4], [5], [6], [7], [8]. Although NUCs potently suppress HBV DNA replication and substantially reduce the risk of cirrhosis and hepatocellular carcinoma, they exert only minimal effects on intrahepatic covalently closed circular DNA (cccDNA) and integrated HBV DNA [4], [7], both of which continue to drive viral antigen production, particularly hepatitis B surface antigen (HBsAg) [5]. As a result, functional cure remains exceedingly rare: even after 8–10 years of continuous NUC monotherapy [6], HBsAg seroclearance is achieved in only approximately 1–2% of patients [8]. This persistent antigenemia reflects the long-term stability of cccDNA and the transcriptional activity of integrated HBV sequences, underscoring the need to elucidate the mechanisms that regulate cccDNA chromatin structure and transcriptional competence. A deeper understanding of these regulatory processes is essential for developing curative strategies that can effectively silence or eliminate the persistent viral reservoir. Recent advances in single-cell RNA sequencing and single-nucleus RNA sequencing have provided high-resolution insights into gene expression in HBV-infected hepatocytes and liver cancer [9], [10], [11], [12]. These techniques allow detailed profiling of hepatocytes and other liver cell populations, facilitating a deeper understanding of HBV pathogenesis and liver disease progression. Therapeutic approaches targeting cccDNA, including RNA interference and genome editing strategies, have also been explored, aiming to directly suppress or disrupt cccDNA [13]. However, most studies have not systematically examined the chromatin accessibility of cccDNA or the host factors regulating its epigenetic state.

Assay for transposase-accessible chromatin sequencing (ATAC-seq) is a widely used method for profiling genome-wide chromatin accessibility [14]. This technique utilizes a hyperactive Tn5 transposase that preferentially inserts sequencing adapters into regions of open chromatin, thereby enabling the selective capture and quantification of accessible genomic sites. As a result, ATAC-seq provides a rapid and sensitive means to map regulatory elements such as promoters and enhancers. Recent advances have enabled its application to more diverse and challenging sample types, including preserved or fixed materials [15]. Recent work has applied ATAC-seq to HBV-infected cells to investigate host chromatin changes, revealing that HBV infection can alter accessibility at host promoters and enhancers, particularly affecting liver metabolic pathways, iron homeostasis, and preneoplastic phenotypes [16], [17], [18]. These findings highlight the importance of host chromatin regulators in shaping the transcriptional landscape of both host and viral genomes. However, despite these advances, no studies have applied single-cell ATAC-seq (scATAC-seq) to HBV-infected hepatocytes or directly examined chromatin accessibility of cccDNA at single-cell resolution, leaving the regulatory landscape of cccDNA largely unexplored.

In this study, we performed scATAC-seq on HBV-infected human hepatocytes to investigate chromatin accessibility of cccDNA. This approach enabled us to identify several candidate host factors that may influence cccDNA accessibility, among which CEP290 (centrosomal protein 290) emerged as a representative example with potential involvement in supporting transcriptionally active cccDNA. We further validated its role by combining bulk ATAC-seq and RNA-seq analyses, providing new insights into the epigenetic regulation of HBV replication and potential avenues for antiviral intervention.