| Hepatitis B virus, a member of the hepadnaviridae, remains a major public health problem, with 350 million chronic carriers worldwide and one million associated deaths per year. Hepadnaviruses package their pregenomic RNA (pgRNA) into cytoplasmic nucleocapsids, which subsequently undergo a maturation phase characterized by the reverse transcription of the pgRNA into the mature double-stranded (DS) DNA genome. In natural infections, only mature nucleocapsids containing DS DNA are enveloped and secreted as virions; the immature nucleocapsids containing packaged pgRNA or immature reverse transcription intermediates are excluded from virion formation. The mechanism underlying selective virion formation has been examined in this study, using a synchronized duck hepatitis B virus replication system. This system allowed the determination of the intrinsic secretion competence of nucleocapsids at each stage of reverse transcription, in the absence of ongoing viral DNA synthesis or potential competition from mature nucleocapsids. Results showed that secretion competence is an intrinsic property of mature nucleocapsids, and thereby supported the maturation signal hypothesis which states that a signal for envelopment emerges on the nucleocapsids as reverse transcription proceeds. To define the maturation signal, changes in post-translational modifications of the core protein during the course of nucleocapsid maturation have been characterized. Immature, mature, and virion-derived nucleocapsids were analyzed by Western blotting and a variety of mass spectrometric techniques to identify and localize core protein modifications. These studies identified two novel sites of core C-terminal phosphorylation (S230 and S232), confirmed four previously known phosphorylation sites (T239, S245, S257, and S259), and revealed N-terminal methionine acetylation of the core protein. Moreover, all six phosphorylation sites were shown to be dephosphorylated during nucleocapsid maturation, while N-terminal acetylation remained unchanged. Mutational analyses indicated that phosphorylation at the novel sites plays a role in nucleocapsid assembly. Together, these results demonstrate that viral DNA synthesis is associated with a structural change in the hepadnaviral nucleocapsids which may constitute part of the maturation signal. |
