Intracellular Hepadnavirus Nucleocapsids Are Selected for Secretion by Envelope Protein-Independent Membrane Binding

Abstract

Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surfaceexposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBVtransfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.

Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

“…This is consistent with previously published flotation experiments, which showed that only mature nucleocapsids have the intrinsic affinity and ability to interact with intracellular membranes. 32 We provide ultrastructural evidence that electron-dense nucleocapsids dock to the membrane of VCVs, leading to its deformation in the sense of an extrusion into the vesicle lumen (Fig. 4F).…”

“…32 Figure 4E shows particles of different densities measuring 22 to 27 nm in diameter in the cytosol (Fig. 4E, arrowheads).…”

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

“…These mechanisms would also apply to progeny capsids that are redirected to the nucleus for cccDNA amplification. How newly formed DHBV capsids are directed to the secretory pathway remains to be elucidated, however, since Mabit and Schaller (13) have shown that dephosphorylation alone is not sufficient to confer membrane affinity. The suggested model is in line with the previous observation in the duck model that serum-derived nucleocapsids are non-phosphorylated whereas nucleocapsids from liver tissue are highly phosphorylated (14).…”

“…Nuclear translocation of viral rcDNA is also mediated by incoming capsids in newly infected cells. However, the precise mechanisms regulating capsid trafficking and uncoating in both settings are unknown.Earlier studies in the DHBV model have shown that capsids from infected livers are highly phosphorylated while serumderived capsids and particles secreted from cultured cells are dephosphorylated (13,14). Furthermore, binding of in vitro generated HBV capsids to the nucleus of digitonin-permeabilized hepatoma cells was previously found to depend on the phosphorylation status of the core protein (15).…”

“…Earlier studies in the DHBV model have shown that capsids from infected livers are highly phosphorylated while serumderived capsids and particles secreted from cultured cells are dephosphorylated (13,14). Furthermore, binding of in vitro generated HBV capsids to the nucleus of digitonin-permeabilized hepatoma cells was previously found to depend on the phosphorylation status of the core protein (15).…”