In the serum of patients chronically infected with hepatitis B virus one does not only find complete DNA-containing virions, but also virions harboring “empty” or RNA-containing capsids. The proportion of these non-canonical forms of virions in chronic carriers varies drastically depending on the intrahepatic viral repli-cation activity and the treatment status. Currently, there is little known about the ultrastructural and biochem-ical foundations of particle morphogenesis giving rise to these non-canonical particles. In earlier research work we identified two morphotypes of hepatitis B virions termed “compact” and “gapped” (Seitz et al., 2007). Compact virions displayed intensive capsid-envelope contacts and an integer bilayer profile of the envelope; in gapped virions the capsid-envelope contacts were lost and the bilayer appeared rearranged. A defined subpopulation of compact particles displayed a unique capsid conformation differing from those known from capsid-like particles expressed in E. coli. Furthermore, circulating virions undergo a distinct maturation process. Virions become released from the host cell in an inactive form and successively devel-op infectivity due to the translocation of the PreS domain of the large envelope protein across the viral membrane (Seitz et al., 2016). Based on these observations, we propose that HBV particles are subject to defined ultrastructural dynamics with a series of thermodynamically driven, temporally and spatially regulat-ed events during particle morphogenesis. This might be key to determining the fate of nucleocapsids in the host cell by deciding between either secretion as enveloped virions or recycling to the nucleus to sustain the pool of viral covalently closed circular DNA, which is the central reservoir for HBV persistence. Moreover, we argue that these ultrastructural dynamics comprise several critical checkpoints pivotal for the varying composition of circulating virions during the course of chronic infection. In the proposed funding period, we will characterize nucleocapsids containing the authentic replication complex by biochemical and structural biology methods (Schütz et al., 2015, 2016, 2018). The nucleocapsids will be arrested in different maturation states and subjected to structure determination by cryo-electron microscopy. We will investigate the impact of any structural changes on the binding competence of nucleocapsids to surface protein segments as prerequisite for capsid envelopment in vitro. Moreover, we will perform thorough biochemical interaction studies to quantitatively describe the binding kinetics of nucleocapsids to the HBV surface glycoproteins. Nuclear magnetic resonance techniques will be applied to resolve the structure of surface protein segments bound to nucleocapsids. The insight gained into structural details and the biochemical parameters of the interactions determining nucleocapsid envelopment will allow us the quantitative characterization and model-ling of the pathways of HBV particle morphogenesis with respect to their variability during chronic infection.