Dimorphism of Hepatitis B Virus Capsids Is Strongly Influenced by the C-Terminus of the Capsid Protein

Abstract

Hepatitis B virus (HBV) is an enveloped virus with an icosahedral capsid. Its homodimeric capsid protein ("core antigen") assembles into particles of two sizes, one with T = 3 icosahedral symmetry (90 dimers) and the other with T = 4 symmetry (120 dimers). We have investigated this assembly process in vitro, using a variety of purified, bacterially expressed, capsid proteins. All of our constructs lacked the predominantly basic C-terminal 34 amino acids of the full-length capsid protein (183 amino acids) and were further truncated to terminate at specific points between residues 138 and 149. While the smallest construct (138 residues) did not assemble into capsids, those terminating at residue 140, and beyond, assembled into mixtures of T = 3 and T = 4 particles. The two kinds of capsids could be separated on sucrose gradients and did not interconvert upon protracted storage. The proportion of T = 3 capsids, assayed by sucrose gradient fractionation, analytical ultracentrifugation, and cryoelectron microscopy, was found to increase systematically with larger deletions from the C-terminus. The variant terminating at residue 149 formed approximately 5% of T = 3 capsids, while the 140-residue protein produced approximately 85% of this isomorph. For the 147-residue capsid protein, the structures of both capsids were determined to 17 A resolution by three-dimensional reconstruction of cryoelectron micrographs. In these density maps, the boundaries of the constituent dimers can be clearly seen and the quaternary structures of the two capsids compared. The arrangement of dimers around their icosahedral five-fold axes is almost identical, whereas the quasi-six-fold arrangements of dimers are distinctly different.

High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids

“…The protamine domain binds RNA, whereas the core domain is necessary and sufficient for capsid assembly. The ratio of T ϭ 3 to T ϭ 4 capsids produced depends on the length of the linker and the conditions of assembly: The smaller T ϭ 3 capsid becomes progressively more abundant as the linker is shortened (12). The building-block for capsid formation is a dimer stabilized via an intermolecular four-helix bundle (13)(14)(15) and a disulfide bond within the bundle (Cys61).…”

Controlling viral capsid assembly with templating

“…Thus, in addition to their biomedical and technological applications, studying viral capsids has revealed fundamental principles of assembly. Although specific assembly mechanisms are poorly understood for most viruses, a general mechanism has emerged for the spontaneous assembly of empty capsids [10][11][12][12][13][14][15]21,26,28,[47][48][49][50][51][52][53]. Assembly occurs through a sequential addition process in which individual subunits or larger intermediates [26,52] bind to a growing capsid.…”

A novel system for efficient gene transfer into primary human hepatocytes via cell‐permeable hepatitis B virus–like particle†

“…42 Efficient in vivo packaging requires the arginine-rich C-terminal domain of HBcAg, [32][33][34] an ⑀-motive on the RNA in cis, and the presence of HBV polymerase in trans. 35,36 To this end, Sf9 cells were triple-infected with recombinant baculovirus encoding for wt-or TLM-core protein, for HBV-polymerase and for an RNA harboring ⑀-encapsidation signals close to its ends. This RNA contained the sequence coding for SHBs or green fluorescence protein (eGFP) that served as marker genes.…”

“…The amount of hepatitis B surface antigen (HBsAg) in supernatant of cells was quantified using a commercial ELISA (Dade Behring, Liederbach, Germany). To analyze the effect of the presence of TLMnucleocapsids on the viability of the cells, 35 SMet and BrdU-incorporation and aminotransferase release was determined. In all assays, no negative effect on cell viability was observed.…”