Norman R. Watts

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

Hepatitis B virus (HBV) is a major human pathogen. In addition to its importance in human health, there is growing interest in adapting HBV and other viruses for drug delivery and other nanotechnological applications. In both contexts, precise biophysical characterization of these large macromolecular particles is fundamental. HBV capsids are unusual in that they exhibit two distinct icosahedral geometries, nominally composed of 90 and 120 dimers with masses of ≈3 and ≈4 MDa, respectively. Here, a mass spectrometric approach was used to determine the masses of both capsids to within 0.1%. It follows that both lattices are complete, consisting of exactly 180 and 240 subunits. Nanoindentation experiments by atomic-force microscopy indicate that both capsids have similar stabilities. The data yielded a Youngs modulus of ≈0.4 GPa. This experimental approach, anchored on very precise and accurate mass measurements, appears to hold considerable potential for elucidating the assembly of viruses and other macromolecular particles.

Implications of the HIV-1 Rev Dimer Structure at 3. 2 A Resolution for Multimeric Binding to the Rev Response Element.

HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process Rev oligomerizes in association with a highly structured RNA motif, the Rev response element. Crystallographic studies of Rev have been hampered by the protein’s tendency to aggregate, but Rev has now been found to form a stable soluble equimolar complex with a specifically engineered monoclonal Fab fragment. We have determined the structure of this complex at 3.2 Å resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9–65) contains two coplanar α-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer is likely to promote higher order interactions, suggesting a model for Rev oligomerization onto the viral RNA.

Stability and Shape of Hepatitis B Virus Capsids In Vacuo

The hepatitis B virus (HBV) is a major cause of liver disease in humans[1] and its non-infectious capsid is of interest for nanotechnology, including for drug-delivery applications. A precise biophysical characterization of these particles is of importance not only for these applications, but also because it may provide further insight into the replication cycle and assembly pathway of the virus, and thus contribute to the future development of drugs.[2,3] The HBV capsid protein (cp) forms icosahedral capsids of two sizes in vivo and in vitro (with triangulation numbers of T = 3 and T = 4[4] that contain 180 and 240 subunits, respectively[5–8]).The capsid protein has two domains—a core domain (amino acids 1–140) and a “protamine domain” (amino acids 150–183)—connected by a 10-residue linker;[9] of these, the core domain is necessary and sufficient for assembly of the capsid. The length of the linker and the conditions under which assembly take place determine the ratio of the T = 3 and T = 4 capsids obtained.[10] Capsid protein dimers are stabilized by an intermolecular four-helix bundle[11–13] and a disulfide bond within the bundle (Cys61); these dimers represent the building blocks for the formation of the capsid. However, the disulfide bond is not required for dimerization or assembly, as Cys61 can be replaced with Ala[9, 10] without affecting either process. The interfaces of the dimers display protruding spikes that result in an uneven surface.[6, 13] Although extensive structural studies of the HBV capsid structure have been performed by electron microscopy (EM) and X-ray crystallography,[14] knowledge of its biophysical properties is limited.[15, 16] Here, we present data from macromolecular tandem and ion mobility mass spectrometry (MS)[17–19] that have a bearing on the stability and conformational diversity of HBV capsids in vacuo.

Structure, Assembly, and Antigenicity of Hepatitis B Virus Capsid Proteins

Publisher Summary This chapter reviews current information pertaining to the structure and assembly properties of hepatitis B virus (HBV) capsid protein, as well as the insights into its antigenicity and other interactions. HBV has a small (3.2 kb) DNA genome, although this modest genetic endowment is amplified by a variety of strategies, including alternative expression products of the same gene. In the replication cycle of HBV, the genome is initially incorporated into the assembling virus particle as a single-stranded RNA molecule—the pregenome—that is subsequently retrotranscribed in situ by the viral reverse transcriptase (RT). The DNA-containing nucleocapsid subsequently becomes enveloped by a membrane containing the viral glycoprotein—surface antigen (sAg), of which there are three size variants called S, M, and L, respectively—to yield the completely assembled and infectious virion. The capsid protein of HBV has several unexpected properties. It was found to have a novel fold, rich in a helix, and quite distinct from the eight-stranded b barrel that was common to the first dozen or so capsid proteins to be solved (from plant, animal, and bacterial viruses) and the other capsid protein folds that have been determined more recently.

The morphogenic linker peptide of HBV capsid protein forms a mobile array on the interior surface.

Many capsid proteins have peptides that influence their assembly. In hepatitis B virus capsid protein, the peptide STLPETTVV, linking the shell‐forming ‘core’ domain and the nucleic acid‐binding ‘protamine’ domain, has such a role. We have studied its morphogenic properties by permuting its sequence, substituting it with an extraneous peptide, deleting it to directly fuse the core and protamine domains and assembling core domain dimers with added linker peptides. The peptide was found to be necessary for the assembly of protamine domain‐containing capsids, although its size‐determining effect tolerates some modifications. Although largely invisible in a capsid crystal structure, we could visualize linker peptides by cryo‐EM difference imaging: they emerge on the inner surface and extend from the capsid protein dimer interface towards the adjacent symmetry axis. A closely sequence‐similar peptide in cellobiose dehydrogenase, which has an extended conformation, offers a plausible prototype. We propose that linker peptides are attached to the capsid inner surface as hinged struts, forming a mobile array, an arrangement with implications for morphogenesis and the management of encapsidated nucleic acid.

Squeezing Protein Shells: How Continuum Elastic Models, Molecular Dynamics Simulations, and Experiments Coalesce at the Nanoscale

The current rapid growth in the use of nanosized particles is fueled in part by our increased understanding of their physical properties and ability to manipulate them, which is essential for achieving optimal functionality. Here we report detailed quantitative measurements of the mechanical response of nanosized protein shells (viral capsids) to large-scale physical deformations and compare them with theoretical descriptions from continuum elastic modeling and molecular dynamics (MD). Specifically, we used nanoindentation by atomic force microscopy to investigate the complex elastic behavior of Hepatitis B virus capsids. These capsids are hollow, approximately 30 nm in diameter, and conform to icosahedral (5-3-2) symmetry. First we show that their indentation behavior, which is symmetry-axis-dependent, cannot be reproduced by a simple model based on Föppl-von Kármán thin-shell elasticity with the fivefold vertices acting as prestressed disclinations. However, we can properly describe the measured nonlinear elastic and orientation-dependent force response with a three-dimensional, topographically detailed, finite-element model. Next, we show that coarse-grained MD simulations also yield good agreement with our nanoindentation measurements, even without any fitting of force-field parameters in the MD model. This study demonstrates that the material properties of viral nanoparticles can be correctly described by both modeling approaches. At the same time, we show that even for large deformations, it suffices to approximate the mechanical behavior of nanosized viral shells with a continuum approach, and ignore specific molecular interactions. This experimental validation of continuum elastic theory provides an example of a situation in which rules of macroscopic physics can apply to nanoscale molecular assemblies.

Diversity of core antigen epitopes of hepatitis B virus

Core antigen (cAg), the viral capsid, is one of the three major clinical antigens of hepatitis B virus. cAg has been described as presenting either one or two conformational epitopes involving the “immunodominant loop.” We have investigated cAg antigenicity by cryo-electron microscopy at ≈11-Å resolution of capsids labeled with monoclonal Fabs, combined with molecular modeling, and describe here two conformational epitopes. Both Fabs bind to the dimeric external spikes, and each epitope has contributions from the loops on both subunits, explaining their discontinuous nature: however, their binding aspects and epitopes differ markedly. To date, four cAg epitopes have been characterized: all are distinct. Although only two regions of the capsid surface are accessible to antibodies, local clustering of the limited number of exposed peptide loops generates a potentially extensive set of discontinuous epitopes. This diversity has not been evident from competition experiments because of steric interference effects. These observations suggest an explanation for the distinction between cAg and e-antigen (an unassembled form of capsid protein) and an approach to immunodiagnosis, exploiting the diversity of cAg epitopes.

Characterization of a Conformational Epitope on Hepatitis B Virus Core Antigen and Quasiequivalent Variations in Antibody Binding

ABSTRACT We have characterized a conformational epitope on capsids of hepatitis B virus (HBV) by cryo-electron microscopy and three-dimensional image reconstruction of Fab-labeled capsids to ∼10-Å resolution, combined with molecular modeling. The epitope straddles the interface between two adjacent subunits and is discontinuous, consisting of five peptides—two on one subunit and three on its neighbor. Together, the two icosahedral forms of the HBV capsid—T=3 and T=4 particles—present seven quasiequivalent variants of the epitope. Of these, only three bind this Fab. Occupancy ranges from ∼100 to ∼0%, reflecting conformational variations in the epitope and steric blocking effects. In the former, small shifts of the component peptides have large effects on binding affinity. This approach appears to hold general promise for elucidating conformational epitopes of HBV and other viruses, including those of neutralizing and diagnostic significance.

Intermediate filament structure: hard α-keratin

Abstract Structurally there are four classes of intermediate filaments (IF) with distinct but closely related axial organisations. One of these, hard α-keratin IF, has been studied to clarify several apparently exceptional features which include the number of molecules in the IF cross-section and the mode by which the axial organisation of its constituent molecules is stabilised. Using the dark-field mode of the STEM at the Brookhaven National Laboratory (USA) mass measurements were obtained from unstained IF isolated from hair keratin. The data thus obtained show that the number of chains in cross-section is about 30 (±3: standard deviation) and is very similar to the numbers determined in previous STEM experiments for the dominant filament type in other classes of IF (about 32). Furthermore, re-analysis of the low-angle equatorial X-ray diffraction pattern reveals, in contrast to earlier work, solutions that are compatible with the number of chains in cross-section indicated by the STEM data. The absence of the head-to-tail overlap between parallel molecules characteristic of most IF may be compensated in hard α-keratin by a network of intermolecular disulfide bonds. It is concluded that native IF of hard α-keratin and desmin/vimentin —and probably many other kinds of IF as well— contain about 32 chains in cross-section, and that the axial structures of these various kinds of IF differ in small but significant ways, while generally observing the same basic modes of aggregation.

Subunit exchange rates in Hepatitis B virus capsids are geometry- and temperature-dependent

Native tandem mass spectrometry reveals marked differences in the rates at which the two polymorphic forms of the HBV capsid exchange dimeric subunits with the soluble pool.