A micromolar pool of antigenically distinct precursors is required to initiate cooperative assembly of hepatitis B virus capsids in Xenopus oocytes.

Assembly of hepatitis B virus capsid-like (core) particles occurs efficiently in a variety of heterologous systems via aggregation of approximately 180 molecules of a single 21.5-kDa core protein (p21.5), resulting in an icosahedral capsid structure with T = 3 symmetry. Recent studies on the assembly of hepatitis B virus core particles in Xenopus oocytes suggested that dimers of p21.5 represent the major building block from which capsids are generated. Here we determined the concentration dependence of this assembly process. By injecting serially diluted synthetic p21.5 mRNA into Xenopus oocytes, we expressed different levels of intracellular p21.5 and monitored the production of p21.5 dimers and capsids by radiolabeling and immunoprecipitation, by radioimmunoassay, or by quantitative enzyme-linked immunosorbent assay analysis. The data revealed that (i) p21.5 dimers and capsids are antigenically distinct, (ii) capsid assembly is a highly cooperative and concentration-dependent process, and (iii) p21.5 must accumulate to a signature concentration of approximately 0.7 to 0.8 microM before capsid assembly initiates. This assembly process is strikingly similar to the assembly of RNA bacteriophage R17 as defined by in vitro studies.     

A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the viral capsid surface.

Core particles of hepatitis B virus are assembled from dimers of a single 185-residue (subtype adw) viral capsid or core protein (p21.5) which possesses two distinct domains: residues 1 to 144 form a minimal capsid assembly domain, and the arginine-rich, carboxyl-terminal residues 150 to 185 form a protamine-like domain that mediates nucleic acid binding. Little is known about the topography of the p21.5 polypeptide within either the p21.5 capsids or dimers. Here, using site-specific proteases and monoclonal antibodies, we have defined the accessibility of p21.5 residues in dimers and capsids assembled from wild-type and mutant hepatitis B virus core proteins in Xenopus oocytes and in vitro. The data reveal the protamine region to be accessible to external reagents in p21.5 dimers but largely cryptic in wild-type capsids. Strikingly, in capsids the only protease target region was a 9-residue peptide covering p21.5 residues Glu-145 to Asp-153, which falls largely between the two core protein domains. By analogy with protease-sensitive interdomain regions in other proteins, we propose that this peptide constitutes a hinge between the assembly and nucleic acid binding domains of p21.5. We further found that deletion or replacement of the terminal Cys-185 residue greatly increased surface exposure of the protamine tails in capsids, suggesting that a known disulfide linkage involving this residue tethers the protamine region inside the core particles. We propose that disruption of this disulfide linkage allows the protamine region to appear transiently on the surface of the core particle.     

Production of hepatitis B virus nucleocapsidlike core particles in Xenopus oocytes: assembly occurs mainly in the cytoplasm and does not require the nucleus.

The location of hepatitis B virus (HBV) nucleocapsid (core particle) assembly in infected cells remains controversial. Some lines of evidence implicate the nucleus; others favor the cytoplasm. Via injection of a synthetic mRNA encoding the HBV nucleocapsid protein (p21.5), we have expressed both unassembled p21.5 and nucleocapsidlike core particles in Xenopus oocytes. Subcellular fractionation reveals that approximately 91% of the unassembled p21.5 and 95% of the core particles are cytoplasmic, with only 9 and 5%, respectively, in the nucleus. We present evidence showing that unassembled p21.5 equilibrates between nucleus and cytoplasm by passive diffusion and that intact core particles do not enter the nucleus. To examine the role of the nucleus in core particle formation, we expressed p21.5 in surgically anucleate oocytes. We show that anucleate oocytes support efficient core particle formation, indicating that (i) the nucleus is not essential for assembly and (ii) the cytoplasm can assemble most core particles found in oocytes. On the basis of our data, we propose that in oocytes, most core particle assembly (up to 95%) occurs in the cytoplasm, but that at least approximately 5% of the cellular core particles are assembled in the nucleus and remain there. We discuss the implications of these findings for the formation of replication-competent core particles in infected cells.     

Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability.

In the spherical capsid of hepatitis B virus (HBV), intermolecular disulfide bonds cross-link the approximately 180 p21.5 capsid protein subunits into a stable lattice. In this study, we used mutant capsid proteins to investigate the role that disulfide bonds and the four p21.5 Cys residues (positions 48, 61, 107, and 185) play in capsid assembly and/or stabilization. p21.5 Cys residues were either replaced by Ala or removed (Cys-185) by carboxyl-terminal truncation, creating Cys-minus mutants which were expressed in Xenopus oocytes via microinjected synthetic mRNAs. Fractionation of radiolabeled oocyte extracts on 10 to 60% sucrose gradients revealed that Cys-minus core proteins resolved into the nonparticulate and capsid forms seen for wild-type p21.5. On 5 to 30% sucrose gradients, nonparticulate Cys-minus core proteins sedimented as dimers of approximately 40 kDa. We conclude that Cys residues and disulfides are not required for the assembly of either HBV capsids or the dimers that provide the precursors for capsid assembly. Since assembly presumably demands an appropriate p21.5 tertiary structure, it is unlikely that Cys residues are required for proper p21.5 folding. However, Cys residues stabilize isolated p21.5 structures, as evidenced by the marked reduction in stability of Cys-minus dimers and capsids (i) in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and (ii) upon protease digestion. We discuss these results in the context of the HBV life cycle and the role of Cys residues in other proteins.     

Conformational equilibria and rates of localized motion within hepatitis B virus capsids

Functional analysis of hepatitis B virus (HBV) core particles has associated a number of biological roles with the C terminus of the capsid protein. One set of functions require the C terminus to be on the exterior of the capsid, while others place this domain on the interior. According to the crystal structure of the capsid, this segment is strictly internal to the capsid shell and buried at a protein-protein interface. Using kinetic hydrolysis, a form of protease digestion assayed by SDS-PAGE and mass spectrometry, the structurally and biologically important C-terminal region of HBV capsid protein assembly domain (Cp149, residues 1-149) has been shown to be dynamic in both dimer and capsid forms. HBV is an enveloped virus with a T = 4 icosahedral core that is composed of 120 copies of a homodimer capsid protein. Free dimer and assembled capsid forms of the protein are readily hydrolyzed by trypsin and thermolysin, around residues 127-128, indicating that this region is dynamic and exposed to the capsid surface. The measured conformational equilibria have an opposite temperature dependence between free dimer and assembled capsid. This work helps to explain the previously described allosteric regulation of assembly and functional properties of a buried domain. These observations make a critical connection between structure, dynamics, and function: made possible by the first quantitative measurements of conformational equilibria and rates of conversion between protein conformers for a megaDalton complex.     

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.     

Assembly Pathway of Hepatitis B Core Virus-like Particles from Genetically Fused Dimers

Macromolecular complexes are responsible for many key biological processes. However, in most cases details of the assembly/disassembly of such complexes are unknown at the molecular level, as the low abundance and transient nature of assembly intermediates make analysis challenging. The assembly of virus capsids is an example of such a process. The hepatitis B virus capsid (core) can be composed of either 90 or 120 dimers of coat protein. Previous studies have proposed a trimer of dimers as an important intermediate species in assembly, acting to nucleate further assembly by dimer addition. Using novel genetically-fused coat protein dimers, we have been able to trap higher-order assembly intermediates and to demonstrate for the first time that both dimeric and trimeric complexes are on pathway to virus-like particle (capsid) formation.     

Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide.

We have analyzed the translocation of hepatitis B virus (HBV) precore (PC) proteins by using Xenopus oocytes injected with a synthetic PC mRNA. The PC region is a 29-amino-acid sequence that precedes the 21.5-kDa HBV capsid or core (C) protein (p21.5) and directs the secretion of core-related proteins. The first 19 PC amino acids provide a signal peptide that is cleaved with the resultant translocation of a 22.5-kDa species (p22.5), in which the last 10 PC residues precede the complete p21.5 C polypeptide. Most p22.5 is matured to 16-20 kDa species by carboxyl-terminal proteolytic cleavage prior to secretion. Here we show that some four unexpected PC proteins of 24 to 25 kDa are produced in addition to the secretion products described above. Protease protection and membrane cosedimentation experiments reveal that all PC proteins behave as expected for proteins that are translocated into the lumen of the endoplasmic reticulum except for the single largest PC protein (p25), which is not translocated. Like p21.5, p25 is a phosphoprotein that localizes to the oocyte cytosol and nucleus, and protease digestion studies suggest that the two molecules have similar two-domain structures. Radiosequencing of immobilized p25 demonstrates that it contains the intact PC signal peptide and represents the unprocessed translation product of the entire PC/C locus. Thus, while many HBV PC protein molecules are correctly targeted to intracellular membranes and translocated, a significant fraction of these molecules can evade translocation and processing.     

Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein.

As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.     

Generation of filamentous instead of icosahedral particles by repression of African swine fever virus structural protein pB438L

The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.     

Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.     

Purification and characterization of the hepatitis B virus core antigen produced in the yeast Saccharomyces cerevisiae

Hepatitis B virus core antigen gene was expressed in Saccharomyces cerevisiae and the product (yHBcAg) was purified from a crude lysate of the yeast by three steps: sucrose step-gradient ultracentrifugation, hydroxyapatite chromatography and CsCl-isopycnic ultracentrifugation. yHBcAg was synthesized in yeast cells as a particle consisting of polypeptides which have a molecular weight of 21.5 kDa (p21.5). In the CsCl-density gradient, yHBcAg particles synthesized with the expression vector pYG701c (the GAP promoter) had two peaks, at 1.35 g cm⁻³ (HP; high-density particle) and 1.31 g cm⁻³ (LP; low-density particle). On the other hand, the particles synthesized with expression vector pAC701 (the PHO5 promoter) had only one peak at 1.32 g cm⁻³. The isoelectric points of HP and LP were estimated to be 4.05 and 4.07, respectively. Absorption spectrum analysis showed that the HP contains nucleic acids. yHBcAg particles possessed the immunogenicity of HBcAg and its component polypeptide (p21.5) possessed that of HBeAg in addition to HBcAg. Moreover, Western blotting analysis of p21.5 using a monoclonal antibody against yHBcAg or yHBeAg indicated that the hepatitis B virus C-gene-coded protein shares the antigenic sites responsible for both antibodies.     

Zinc ions trigger conformational change and oligomerization of hepatitis B virus capsid protein

Assembly of virus particles in infected cells is likely to be a tightly regulated process. Previously, we found that in vitro assembly of hepatitis B virus (HBV) capsid protein is highly dependent on protein and NaCl concentration. Here we show that micromolar concentrations of Zn2+ are sufficient to initiate assembly of capsid protein, whereas other mono- and divalent cations elicited assembly only at millimolar concentrations, similar to those required for NaCl-induced assembly. Altered intrinsic protein fluorescence and highly cooperative binding of at least four Zn2+ ions (KD approximately 7 microM) indicated that binding induced a conformational change in capsid protein. At 37 degrees C, Zn2+ enhanced the initial rate of assembly and produced normal capsids, but it did not alter the extent of assembly at equilibrium. Assembly mediated by high zinc concentrations (> or =300 microM) yielded few capsids but produced a population of oligomers recognized by capsid-specific antibodies, suggesting a kinetically trapped assembly reaction. Comparison of kinetic simulations to in vitro assembly reactions leads us to suggest that kinetic trapping was due to the enhancement of the nucleation rate relative to the elongation rate. Zinc-induced HBV assembly has hallmarks of an allosterically regulated process: ligand binding at one site influences binding at other sites (cooperativity) indicating that binding is associated with conformational change, and binding of ligand alters the biological activity of assembly. We conclude that zinc binding enhances the kinetics of assembly by promoting formation of an intermediate that is readily consumed in the reaction. Free zinc ions may not be the true in vivo activator of assembly, but they provide a model for regulation of assembly.     

Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential.

Hepadnaviruses encode a single core (C) protein which assembles into a nucleocapsid containing the polymerase (P) protein and pregenomic RNA during viral replication in hepatocytes. We examined the ability of heterologous hepadnavirus C proteins to cross-oligomerize. Using a two-hybrid assay in HepG2 cells, we observed cross-oligomerization among the core proteins from hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus. When expressed in Xenopus oocytes, in which hepadnavirus C proteins form capsids, the C polypeptides from woodchuck hepatitis virus and ground squirrel hepatitis virus, but not duck hepatitis B virus, can efficiently coassemble with an epitope-tagged HBV core polypeptide to form mixed capsids. However, when two different core mRNAs are coexpressed in oocytes the core monomers show a strong preference for forming homodimers rather than heterodimers. This holds true even for coexpression of two HBV C proteins differing only by an epitope tag, suggesting that core monomers are not free to diffuse and associate with other monomers. Thus, mixed capsids result from aggregation of different species of homodimers.     

Folding and processing of the capsid protein precursor P1 is kinetically retarded in neutralization site 3B mutants of poliovirus.

Poliovirus mutants in neutralizing antigenic site 3B were constructed by replacing the glutamic acid residue at amino acid 74 of capsid protein VP2 (VP2074E), using site-specific mutagenesis methods. All viable mutants display small-plaque phenotypes. Characterization of these mutants indicates that capsid assembly is perturbed. Although the defect in capsid assembly reduces the yield of mutant virus particles per cell, the resultant assembled particle is wild-type-like in structure and infectivity. Analyses of capsid assembly intermediates show a transient accumulation of the unprocessed capsid protein precursor, P1, indicating that cleavage of the mutant P1 by the 3CD protease is retarded. The mutant VP0-VP3-VP1 complex generated upon P1 cleavage appears assembly competent, forming pentamer and empty capsid assembly intermediates and infectious virion particles. Although the structure of the infectious mutant virus is virtually identical with that of the wild-type virus, the thermal stability of the mutant virus is dramatically increased over that of the wild-type virus. Thus, mutations at this residue are pleiotropic, altering the kinetics of capsid assembly and generating a virus that is more thermostable and more resistant to neutralization by the site 3B monoclonal antibodies.     

Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids

The icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid-like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C-terminal basic domain by the 17 kDa Staphylococcus aureus nuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild-type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy.     

Expression, purification and characterization of full-length RNA-free hepatitis B core particles

The nucleocapsid or core particle of the hepatitis B virus has become one of the favourite recombinant vaccine carriers for foreign peptides, proteins and stimulatory oligonucleotides. The core protein consists of three regions: an N-terminal, a central and a C-terminal region that can accommodate the addition or insertion of the foreign sequences. The protamine-like C-terminal region that binds host RNA randomly during recombinant particle formation is often truncated. It is commonly thought that these truncations do not affect particle assembly. Recent studies have demonstrated that the C-terminal domains mediate a glycosaminoglycan-dependent attachment of nucleocapsids to the plasma membranes of host cells. This interaction might well contribute to the immunogenicity of nucleocapsids. Testing the hypothesis that full-length particles might be safer and superior for the induction of an immune response against the nucleocapsids and inserted sequences, requires the availability of purified particles. In this report, we detail a novel method for the synthesis and purification of full-length core particles essentially free of RNA from Escherichia coli.