Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins

Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75 degrees C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T=3 or T=4 icosahedral particles. No difference in the ratio between T=3 (78%) and T=4 particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T=3 and T=4 particles successfully separated by using a novel agarose gel electrophoresis procedure.     

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.     

Native hepatitis B virions and capsids visualized by electron cryomicroscopy

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.     

Assembly of bacteriophage Qbeta virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris

Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well.     

乙肝病毒核心蛋白钉突部位基因工程改造对其功能的影响

通过在乙肝病毒核心蛋白钉突部位插入标签蛋白EGFP及小片段多肽,研究各种改造对HBc功能的影响。采用RLIC方法,构建野生型HBc、HBc钉突部位带不同接头的EGFP融合重组体、缩短的EGFP融合重组体,并构建与HBc功能互补的质粒HBV1.1c－,将不同重组体与HBV1.1c－共转染HEK293细胞,通过观察荧光及Southern blotting检测病毒复制中间体,判断相应基因工程改造对重组蛋白中不同结构域功能的影响。RLIC方法可有效地用来进行片段缺失,且缺失片段大小及位置无明显限制。带柔性或刚性接头的重组HBc-EGFP均可产生绿色荧光,但荧光在细胞内分布形态不同,两种重组HBc-EGFP均不能支持正常的HBV复制,各种截短的插入片段以及aa79-80单独缺失体亦不能支持HBV复制。结果表明RLIC方法是一种基因工程改造的有力工具,不同类型接头对重组蛋白的结构和功能有不同影响,aa79-80对维持HBc的主要功能之一——支持HBV复制有重要作用。     

Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

Mutilation of RNA phage Qbeta virus-like particles: from icosahedrons to rods

Icosahedral virus-like particles (VLPs) of RNA phage Qbeta are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between beta-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasi-six-fold symmetry contacts of the VLPs. In order to reduce the stability of Qbeta VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence. It led to production in Escherichia coli cells of aberrant rod-like Qbeta VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4-30 times the diameter of icosahedral Qbeta VLPs.     

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.     

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.     

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.     

Structure-based design and biochemical evaluation of sulfanilamide derivatives as hepatitis B virus capsid assembly inhibitors

Virus capsid structure is essential in virion maturation and durability, so disrupting capsid assembly could be an effective way to reduce virion count and cure viral diseases. However, currently there is no known antiviral which affects capsid inhibition, and only a small number of assembly inhibitors were experimentally successful. In this present study, we aimed to find hepatitis B virus (HBV) capsid assembly inhibitor which binds to the HBV core protein and changes protein conformation. Several candidate molecules were found to bind to certain structure in core protein with high specificity. Furthermore, these molecules significantly changed the protein conformation and reduced assembly affinity of core protein, leading to decrease of the number of assembled capsid or virion, both in vitro and in vivo. In addition, prediction also suggests that improvements in inhibition efficiency could be possible by changing functional groups and ring structures.     

A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid

Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re‐engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive‐pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90‐mers in the disassembly of uncrosslinked HBV capsids. 90‐mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations.     

BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly

Here we report the effect of a heteroaryldihydropyrimidine (HAP) antiviral compound, BAY 41-4109, on Hepatitis B virus (HBV) capsid assembly and on preformed HBV capsids. The HBV capsid is an icosahedral complex of 120 capsid protein dimers. BAY41-4109 inhibits virus production in vivo by a mechanism that targets the viral capsid. We found that BAY 41-4109 was able to both accelerate and misdirect capsid assembly in vitro. As little as one HAP molecule for every five HBV dimers was sufficient to induce formation of non-capsid polymers. Unlike the related molecule HAP-1 (Stray et al., Proc. Natl. Acad. Sci. USA 102:8138-43, 2005), no stable assembly intermediates were observed in assembly reactions with BAY 41-4109, indicating that accelerated assembly by BAY 41-4109 was still kinetically regulated by the nucleation rate. Preformed capsids were stabilized by BAY 41-4109, up to a ratio of one inhibitor molecule per two dimers. However, at BAY 41-4109:dimer ratios of 1:1 and greater, capsids were destabilized to yield very large non-capsid polymers. These data suggest the existence of two functionally distinguishable classes of drug-binding sites on HBV capsids. Occupation of the first class of site stabilizes capsid, while binding at the second class requires or induces structural changes that cannot be tolerated without destabilizing the capsid. Our data suggest that HAP compounds may inhibit virus replication by inducing assembly inappropriately and, when in excess, by misdirecting assembly decreasing the stability of normal capsids.     

Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner

Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release-mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT.     

Synthesis and Assembly of Simian Virus 40 II. Synthesis of the Major Capsid Protein and Its Incorporation into Viral Particles

African green monkey kidney cells infected by simian virus 40 were analyzed for the presence of the major capsid protein (capsid protein I) by immunological and radiolabeling techniques. Antisera with different specificities were prepared by immunization with intact or denatured viral particles. Antisera prepared against intact virus reacted by complement fixation with viral particles and with an 8S subunit containing the capsid protein I. Antisera prepared against denatured viral particles reacted with unassembled capsid protein(s) as well as with viral particles. These antisera were used to detect 8S viral subunits or unassembled viral capsid protein in soluble extracts of infected cells after centrifugation at 100,000 x g to remove viral particles. The soluble antigen pool was found to be small during infection with wild-type virus or a temperature-sensitive mutant deficient in the synthesis of viral particles. Pulse-chase experiments, performed at a high multiplicity of infection, also indicated a small pool of nonparticle capsid protein I. Radioactive lysine was incorporated into capsid protein I of virus particles during a 2-hr pulse. A subsequent chase with excess unlabeled lysine resulted in only a slight increase in the radio-activity found in capsid protein I of viral particles. Furthermore, in the same experiments, capsid protein I was incorporated preferentially into empty shells during the pulse with a shift in radioactivity to intact virions during the chase period, indicating a possible precursor relationship between the two types of virus particles.     

An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.     

Mapping of amino acid side chains on the surface of hepatitis B virus capsids required for envelopment and virion formation

The crystal structure of recombinant hepatitis B virus (HBV) capsids formed by 240 core proteins has recently been published. We wanted to map sites on the surface of the icosahedral 35-nm particle that are important for nucleocapsid envelopment by HBV surface proteins during virion morphogenesis. For this purpose, we individually mutated 52 amino acids (aa) within the N-terminal 140 aa of the 185-aa long core protein displaying their side chains to the external surface of the capsid to alanine residues. The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core gene-negative HBV genome in transiently cotransfected cells, immunoprecipitation of nucleocapsids from cells and secreted virions from culture media, and detection of the particles by radioactive endogenous polymerase reactions. Thirteen point mutations impeded nucleocapsid detection by endogenous polymerase reactions. Twenty-seven mutations were compatible with virion formation. Among these were all capsid-forming mutations in the upper half of the spike protruding from the particle shell and two additional triple mutations at tip of the spike. Eleven mutations (S17, F18, L60, L95, K96, F122, I126, R127, N136, A137, and I139) allowed nucleocapsid formation but blocked particle envelopment and virion formation to undetectable levels. These mutations map to a ring-like groove around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell. These residues are candidate sites for the interaction with envelope proteins during virion morphogenesis.