Nuclear Entry of Hepatitis B Virus Capsids Involves Disintegration to Protein Dimers followed by Nuclear Reassociation to Capsids

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Abstract
Assembly and disassembly of viral capsids are essential steps in the viral life cycle. Studies on their kinetics are mostly performed in vitro, allowing application of biochemical, biophysical and visualizing techniques. In vivo kinetics are poorly understood and the transferability of the in vitro models to the cellular environment remains speculative. We analyzed capsid disassembly of the hepatitis B virus in digitonin-permeabilized cells which support nuclear capsid entry and subsequent genome release. Using gradient centrifugation, size exclusion chromatography and immune fluorescence microscopy of digitonin-permeabilized cells, we showed that capsids open and close reversibly. In the absence of RNA, capsid re-assembly slows down; the capsids remain disintegrated and enter the nucleus as protein dimers or irregular polymers. Upon the presence of cellular RNA, capsids re-assemble in the nucleus. We conclude that reversible genome release from hepatitis B virus capsids is a unique strategy different from that of other viruses, which employs irreversible capsid destruction for genome release. The results allowed us to propose a model of HBV genome release in which the unique environment of the nuclear pore favors HBV capsid disassembly reaction, while both cytoplasm and nucleus favor capsid assembly. Viral capsids facilitate protection of the enclosed viral genome and participate in the intracellular transport of the genome. At the site of replication capsids have to release the genome, but after replication new capsids have to be assembled for encapsidation of the progeny genomes. Detailed data on stability of capsids and kinetics of their formation and dissociation are obtained for several viruses in vitro, allowing biophysical or electron microscopical techniques. These approaches, however, do not consider the impact of cellular interaction partners. Using digitonin-permeabilized cells which support hepadnaviral genome release actively, we analyzed the disassembly kinetic of the hepatitis B virus (HBV) capsid. Using different analytical methods we found that HBV capsids disintegrate to protein dimers which reassemble to capsids inside the nucleus. The study provides a link between in vitro and in vivo data showing that HBV uses a unique strategy. We propose a model in which the unique environment of the nuclear pore favors the disassembly reaction, while both cytoplasm and nucleus favor assembly.