A DNA launch method for HBV infection in human hepatocyte chimeric mice enables isogenic studies of basal-core promoter and precore mutants

Background & Aims: Studying isogenic infectious hepatitis B virus (HBV) mutations in human hepatocyte chimeric mice (huFNRGs) has remained challenging due to the low efficiency of infection with cell culture-derived virus and because clinical isolates contain mixed virus populations. We sought to develop an efficient method to launch HBV infection from recombinant HBV-DNA, thereby facilitating research on isogenic mutants, including basal-core promoter (A1762T/G1764A, BCPM) and precore mutant (G1896A, PCM) viruses.

Methods: We engineered silent mutant barcodes in and mixed these in serial dilution (ratios ranging from 1:10 to 1:10,000) with wild-type (WT) HBV-DNA to compare the efficiency of two delivery methods in huFNRGs: ex-vivo human hepatocyte transfection and re-transplantation (TRT) and direct intrahepatic injection of recombinant-cccDNA (IHI). Next, we generated isogenic virus stocks from genotype C2-3 WT, BCPM and PCM and performed infection experiments in huFNRGs.

Results: IHI is superior to TRT, initiating the launch of at least 10,000 rcccDNA molecules and yielding viremia in 90.2% of huFNRGs across genotypes A-F (TRT: 40.6%). We defined the unique transcriptomic profiles attributable to isogenic genotype C2-3 variants (WT, BCPM, PCM) using RNA sequencing. We furthermore found variant-specific effects on the proteomic landscape in liver tissue and, using isolated human hepatocytes from infected huFNRGs, observed variant-specific responses to interferon treatment in the proteome.

Conclusion: The rcccDNA IHI-launch technique efficiently initiates HBV infection, allowing the study of isogenic variants across genotypes. Using this approach in genotype C2-3, we were able describe distinct transcriptomic and proteomic differences that are directly attributable to BCPM and PCM.

Publikationsverlauf

Artikel online veröffentlicht:
20. Januar 2025