Poster Presentation 12th Australasian Virology Society Meeting 2024

Evidence of chromosomal translocation, complex integration patterns, and pre-genomic RNA expression in de novo Hepatitis B virus integration (#121)

Harout Ajoyan 1 , Gabriela Wu 1 , Brian Gloss 1 , Rebecca Rockett 2 , Jochen Wettengel 3 4 , Vikki Ho 1 , Dong Li 1 , Sarah Bae 1 , Henrik Zhang 1 , Delgerbat Boldbaatar 1 , Ulrike Protzer 3 4 , Mark W Douglas 1 2 5 , Jacob George 1 2 5 , Thomas Tu 1 2 5
  1. Storr Liver Centre, The Westmead Institute for Medical Research, The University of Sydney , Westmead, NSW, Australia
  2. Centre for Infectious Diseases and Microbiology–Public Health, Westmead Hospital, Westmead, NSW, Australia
  3. Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Bavaria, Germany
  4. German Center for Infection Research (DZIF), Munich Partner Site, Munich, Bavaria, Germany
  5. Sydney Institute for Infectious Diseases, The University of Sydney at Westmead Hospital, Westmead, NSW, Australia

Introduction: Hepatitis B virus (HBV) is the most common cause of liver cancer. Besides its epigenomic replication cycle, HBV can occasionally integrate into the human genome (~1 per 1000 cells) as replication-deficient forms, which are enriched in liver cancers (most tumours contain ≥1 integrated copy). Recently HBV transcripts consistent with viral replication from integrated forms have been detected in human tissues; though it is currently unclear how common this phenomenon is due to the inability to isolate cells with integrations.

 

Methods: We solve this shortcoming using a replication-deficient reporter HBV, encoding zeocin-resistance (replacing the HBsAg and part of Pol ORFs). We infected HepG2-NTCP cells and cells with integrations were isolated after zeocin selection. Viral expression was measured using ELISA, immunofluorescence, and RT-qPCR. Integrations were quantified by digital droplet-PCR. Virus-cell junctions were identified using capture-based enrichment sequencing and a custom bioinformatics pipeline.

 

Results: 118 clones with integrations were isolated, each with 1-2 integrations per cell. Sequence analysis found evidence of HBV genome inversions and chromosomal translocations in ~20% and ~8% of the clones, respectively. This suggests that precancerous molecular lesions occur early after HBV infection, decades prior to tumour formation. Consistent with the new human tissue data, HBeAg expression (a marker of viral replication, sharing the same sequence as pre-genomic RNA) was detected in 18% of the clones by ELISA. We confirmed expression of this transcript through RT-qPCR and immunofluorescence.  

 

Discussion: This study is the first to isolate and characterise integrations from de novo HBV infections. Overturning current paradigms, we show that HBV DNA integrations can commonly express HBeAg and HBcAg, suggesting the potential for replication-competent integrated HBV.

 

Conclusion: Clearance of cccDNA may not be sufficient to achieve a functional cure. Integrated HBV DNA should be targeted by future curative strategies to reduce risk of liver cancer and prevent viral recurrence.