Differential promoter activity in episomal and integrated hepatitis B viral genomes results in heterogenous viral transcript patterns and drug-resistant lineages

Hepatitis B virus (HBV) is a small DNA virus encoding 6 major RNAs that are regulated by 4 distinct promoters, which must act in a coordinated manner to facilitate viral replication. Integrated genomes are not essential stage for viral replication, but their activity is implication in immune cell exhaustion and oncogenesis. However, our understanding of the differential activity of viral promoters in episomal and integrated genomes has been hampered by methodological limitations. Using targeted-long read sequencing, we interrogated the natural HBV transcriptome in n=11 patients, in the absence of therapeutic intervention. Analysing the transcription start site (TSS) of HBV RNAs, revealed that up to 40% started outside of defined TSS boundaries, leading us to identify new, unreported viral transcripts. We identified rare genomic length transcripts, and abortive RNAs that terminate at a new cryptic polyadenylation signal. Evaluating the Sp1 and Sp2 promoters responsible for producing surface antigen, revealed that integrated genomes transcribe vastly heterogenous RNAs, producing thousands of virus-host chimeric reads. Using in house bioinformatic pipelines we identified a repertoire of previously uncharacterised HBV RNAs, which are spliced, truncated and chimeric, presenting hurdles to state-of-the-art novel antivirals.