Hepatitis B virus (HBV) remains a major public health concern, with more than 250 million chronically infected people who are at high risk of developing liver diseases, including cirrhosis and hepatocellular carcinoma. Although antiviral treatments efficiently control virus replication and improve liver function, they cannot cure HBV infection. Viral persistence is due to the maintenance of the viral circular episomal DNA, called covalently closed circular DNA (cccDNA), in the nuclei of infected cells. cccDNA not only resists antiviral therapies, but also escapes innate antiviral surveillance. This viral DNA intermediate plays a central role in HBV replication, as cccDNA is the template for the transcription of all viral RNAs, including pregenomic RNA (pgRNA), which in turn feeds the formation of cccDNA through a step of reverse transcription. The establishment and/or expression of cccDNA is thus a prime target for the eradication of HBV. In this review, we provide an update on the current knowledge on the initial steps of HBV infection, from the nuclear import of the nucleocapsid to the formation of the cccDNA.

Viruses that transcribe their DNA within the nucleus have to adapt to the existing cellular mechanisms that govern transcriptional regulation. Recent technological breakthroughs have highlighted the highly hierarchical organization of the cellular genome and its role in the regulation of gene expression. This review provides an updated overview on the current knowledge on how the hepatitis B virus interacts with the cellular 3D genome and its consequences on viral and cellular gene expression. We also briefly discuss the strategies developed by other DNA viruses to co-opt and sometimes subvert cellular genome spatial organization.

Integrative mobilizable elements (IMEs) are widespread but very poorly studied integrated elements that can excise and hijack the transfer apparatus of co-resident conjugative elements to promote their own spreading. Sixty-four putative IMEs, harboring closely related mobilization and recombination modules, were found in 14 Streptococcus species and in Staphylococcus aureus. Fifty-three are integrated into the origin of transfer (oriT) of a host integrative conjugative element (ICE), encoding a MobT relaxase and belonging to three distant families: ICESt3, Tn916, and ICE6013. The others are integrated into an unrelated IME or in chromosomal sites. After labeling by an antibiotic resistance gene, the conjugative transfer of one of these IMEs (named IME_oriTs) and its host ICE was measured. Although the IME is integrated in an ICE, it does not transfer as a part of the host ICE (no cis-mobilization). The IME excises and transfers separately from the ICE (without impacting its transfer rate) using its own relaxase, distantly related to all known MobT relaxases, and integrates in the oriT of the ICE after transfer. Overall, IME_oriTs use MobT-encoding ICEs both as hosts and as helpers for conjugative transfer. As half of them carry lsa(C), they actively participate in the dissemination of lincosamide–streptogramin A–pleuromutilin resistance among Firmicutes.

The transcription of genes and engineered circuits can deeply vary when inserted into different genomic loci. This unpredictable performance, termed context sensitivity, complicates strain development. Although the causes and mechanisms of context sensitivity are emerging, it is poorly known how to engineer circuits and synthetic pathways isolated from it. Using tools of synthetic biology for designing and inserting various reporter cassettes in the Escherichia coli genome and RT-qPCR for directly measuring gene transcription, we first surveyed the genomic landscape for context sensitivity at 214 positions in cells grown in glucose or glycerol. The results show deep variations in cassette transcription with respect to position (up to 160-fold) and growth condition (up to a 30-fold). We then demonstrated that this position-dependent transcription variability is strongly reduced when the reporter cassette is insulated in an artificial protein-bound DNA loop. Finally, we measured the transcription of two loop-insulated genes at different genomic positions. The results show that transcription strongly depends on the relative orientation of the genes, promoter strength, and positive supercoiling. We present a model suggesting that DNA looping is an important cause of context sensitivity and can be used for better controlling the transcription of engineered circuits.

The expression of genes and engineered circuits can deeply vary when inserted into different genomic loci. This unpredictable performance of expression, termed context sensitivity, complicates strain development. Although the causes and mechanisms of context sensitivity are emerging, it is poorly known how to engineer circuits and synthetic pathways isolated from it. Using tools of synthetic biology for designing and inserting various reporter cassettes in the Escherichia coli genome and RT-qPCR for directly measuring gene expression, we first surveyed the genomic landscape for context sensitivity at 209 positions in cells grown in glucose or glycerol. Results showed deep variations in cassette expression with respect to position (up to 150-fold) and growth condition (up to a 1400-fold). We then demonstrated that this position-dependent expression variability is strongly reduced when the reporter cassette is insulated in an artificial protein-bound DNA loop. Finally, we measured expression of two loop-insulated genes at different genomic positions. Results showed that expression strongly depends on the relative orientation of the genes, promoter strength and positive supercoiling. We present a model suggesting that DNA looping is an important cause of context sensitivity and can be used for better controlling expression of engineered circuits.

Background: Chronic hepatitis B virus (HBV) infection remains a major global health burden due to the persistence of covalently closed circular DNA (cccDNA), a stable episomal viral reservoir resistant to current antiviral therapies. The transcriptional activity of cccDNA depends on its chromatin organization, yet the contribution of histone variants to this regulation remains poorly understood. Methods: Using mass spectrometry–based proteomics on native cccDNA purified from infected primary human hepatocytes, we identified the histone variant H2A.Z and its chaperone SRCAP as cccDNA-associated proteins. Functional analyses combining shRNA-mediated silencing, chromatin immunoprecipitation (ChIP), and ATAC-seq were performed in HBV-infected HepG2-NTCP cells and primary human hepatocytes to characterize their roles in cccDNA formation and transcription. Results: Depletion of H2A.Z.1, H2A.Z.2, or SRCAP reduced both HBV RNA levels and cccDNA formation. H2A.Z recruitment to cccDNA correlated with the establishment of active chromatin marks (H3K4me3) and enhanced RNA polymerase II loading, promoting an open and transcriptionally active chromatin state. In addition, we identified BRD2, an H2A.Z-associated transcriptional co-activator, as a positive regulator of HBV transcription. shRNA mediated depletion and pharmacological degradation of BRD2 using the PROTAC ARV-771 reduced HBV RNA levels, supporting its potential as an antiviral target. Conclusion: Our findings uncover a critical role of the H2A.Z variant and SRCAP complex in cccDNA formation and transcriptional activation. By facilitating chromatin accessibility and RNA polymerase II recruitment, H2A.Z establishes an epigenetic environment favorable to HBV persistence. Targeting H2A.Z-associated co-activators such as BRD2 may represent a promising strategy to silence cccDNA transcription and achieve functional HBV cure.