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.
Highlights
Viral capsids facilitate multiple functions in the viral life cycle
We demonstrated that E. coli expressed capsids dissociate and reassociate
To confirm the immune reactivity of the capsids, dimers and other capsid subassemblies, we investigated the light and the heavy fractions derived from Nycodenz gradient centrifugation by immune precipitation
Summary
Viral capsids facilitate multiple functions in the viral life cycle. Outside the cell, they protect the enclosed viral genome against nucleases, and in case of non-enveloped viruses they mediate attachment and entry. They protect the enclosed viral genome against nucleases, and in case of non-enveloped viruses they mediate attachment and entry For both enveloped and non-enveloped viruses, they carry the viral genome to the site of replication where they have to release the genome in order to allow access of transcription and/or replication factors. After replication new capsids have to be assembled for encapsidation of the progeny genomes and subsequent release of mature virions. Capsids are assigned to be metastable: early in infection they have to open, later they have to assemble and close. Most data on stability of capsids and kinetics of their formation and dissociation are obtained in vitro allowing analysis by biophysical or electron microscopical techniques (e.g. [1,2,3,4,5])
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