The hypoxic HBV transcriptome is regulated by the demethylase ALKBH5

Chronic hepatitis B is a global health problem with over 290 million cases and 880,000 deaths/year from liver disease. Current treatments only suppress hepatitis B virus (HBV) replication and a greater understanding of the host pathways that regulate infection will aid the identification and screening of new therapeutic targets. N6-methyladenosine (m6A) modifications regulate multiple aspects of HBV RNA including transcript stability, translation and encapsidation. HBV replicates in the liver, a low oxygen environment where hypoxia inducible factors (HIFs) regulate the activity of m6A writer and eraser proteins and may influence HBV replication. We investigated the interplay between m6A methylation and HIF-signalling and their role in the HBV life cycle. m6A modifications have an essential role in regulating the HBV transcriptome and long read sequencing revealed oxygen- and m6A-dependent regulation of canonical and non-canonical viral RNAs. The m6A motif in the 5-prime stem loop of pregenomic RNA regulates transcript half-life in a low-oxygen environment. HIFs activate the RNA demethylase ALKBH5 and we noted reduced levels of m6A-RNAs under low oxygen conditions. Silencing ALKBH5 expression ablated the hypoxic induction of pgRNA in a HIF-1 alpha dependent manner. Our study highlights a previously unrecognized role for ALKBH5 in orchestrating both viral and cellular transcriptional responses to low oxygen.