Hepatitis B virus genome packaging and replication are coordinated by a polymerase-responsive RNA switch in the RNA...

Hepatitis B virus (HBV), a major human pathogen, replicates its DNA genome by protein-primed reverse transcription of a pregenomic RNA (pgRNA). This process is directed by the pgRNA-borne epsilon (ε) element, which provides the origin for minus-strand DNA synthesis and mediates coencapsidation of pgRNA with the viral polymerase (P protein) into nucleocapsids. ε adopts a thermodynamically stable hairpin structure that is remodeled upon formation of functional ε-P complexes, but the nature of the rearranged RNA structure and its implication for pgRNA encapsidation has remained elusive. Guided by in silico analyses of ε-like elements from distantly related nackednaviruses, we identify a distinct conformation of HBVε whose defining feature is a cryptic stem-loop (cSL), masked within the upper stem of ε. The P-dependent cSL conformation reorganizes key sequences into a compact structural unit that enables initiation of DNA synthesis and packaging of the viral pgRNA-P complex. RNAs engineered to favor cSL formation exhibit increased P protein affinity and strongly enhanced priming activity in vitro while maintaining replication competence in cells. Mutational analyses identify the cSL and its immediate vicinity, but not the remaining upper stem sequence, as the dominant determinants of ε function. Genetic variation in cSL-forming potential across hepadnaviruses links in vitro priming competence to the energetic accessibility of this alternative fold. Together, our findings reveal ε as a P protein-dependent RNA switch that tightly couples pregenome encapsidation to reverse transcription competence. This regulatory mechanism advances our understanding of HBV replication and could be exploited for antiviral intervention.