N6-methyladenosine (m⁶A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m⁶A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m⁶A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m⁶A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m⁶A site in the HBV RNA and found that a conserved m⁶A consensus motif situated within the epsilon stem loop structure, is the site for m⁶A modification. The epsilon stem loop is located in the 3' terminus of all HBV mRNAs and at both the 5' and 3' termini of the pgRNA. Mutational analysis of the identified m⁶A site in the 5' epsilon stem loop of pgRNA revealed that m⁶A at this site is required for efficient reverse transcription of pgRNA, while m⁶A methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m⁶A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m⁶A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8's ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.