Abstract
The hepatitis B virus (HBV) core particle serves as a protective capsid shell for the viral genome and is highly immunogenic. Recombinant capsid-like core particles are used as effective carriers of foreign T and B cell epitopes and as delivery vehicles for oligonucleotides. The core monomer contains an arginine-rich C terminus that directs core particle attachment to cells via membrane heparan sulfate proteoglycans. Here we investigated the mechanism of recombinant core particle uptake and its intracellular fate following heparan sulfate binding. We found that the core particles are internalized in an energy-dependent manner. Core particle uptake is inhibited by chlorpromazine and by cytosol acidification known to block clathrin-mediated endocytosis but not by nystatin, which blocks lipid raft endocytosis. Particle uptake is abolished by expression of dominant negative forms of eps15 and Rab5, adaptors involved in clathrin-mediated endocytosis and early endosome transport, respectively. Endocytosed particles are transported to lysosomes where the core monomer is endoproteolytically cleaved into its distinct domains. Using protease inhibitors, cathepsin B was identified as the enzyme responsible for core monomer cleavage. Finally we found that monomer cleavage promotes particle dissociation within cells. Together, our results show that HBV capsid-like core particles are internalized through clathrin-mediated endocytosis, leading to lysosomal cleavage of the core monomer and particle dissociation.
Highlights
When expressed in various heterologous systems, HBcAg efficiently self-assembles into capsid-like core particles indistinguishable from native capsid shells (6 – 8)
ODN encapsulated in core particles or conjugated to an arginine-rich peptide derived from the C terminus of HBcAg exert enhanced immunostimulatory effects in vivo, and these effects have been suggested to result from their efficient endosomal delivery and activation of toll-like receptor 9 (TLR9) signaling [11,12,13]
We show here that the attachment step is followed by clathrin-mediated endocytosis of the particles, depending on Eps15 and the small GTPase Rab5
Summary
When expressed in various heterologous systems, HBcAg efficiently self-assembles into capsid-like core particles indistinguishable from native capsid shells (6 – 8). Immunofluorescent microscopy analysis showed that cytosol acidification resulted in abolishment of the signal representing internalized core (Fig. 2B), consistent with clathrin-mediated endocytosis of the particles into cells. HBc Core Particles Are Transported to Lysosomes Where HBcAg Is Cleaved into Its Distinct Domains by Cathepsin B—Ligands internalized by clathrin-mediated endocytosis are either recycled to the plasma membrane or targeted to late endosomes and lysosomes.
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