Capsid Wall Research Articles

Helical organization of DNA-like liquid crystal filaments in cylindrical viral capsids

We study equilibrium configurations of double-stranded DNA in a cylindrical viral capsid. The state of the encapsidated DNA consists of a disordered inner core enclosed by an ordered outer region, next to the capsid wall. The DNA configuration is described by a unit helical vector field, tangent to an associated centre curve, passing through properly selected locations. We postulate an expression for the energy of the encapsulated DNA based on that of columnar chromonic liquid crystals. A thorough analysis of the Euler–Lagrange equations yields multiple solutions. We demonstrate that there is a trivial, non-helical solution, together with two solutions with non-zero helicity of opposite sign. Using bifurcation analysis, we derive the conditions for local stability and determine when the preferred coiling state is helical. The bifurcation parameters are the ratio of the twist versus the bend moduli of DNA and the ratio between the sizes of the ordered and the disordered regions.

Ultrastructure of Meelsvirus: A nuclear virus of arrow worms (phylum Chaetognatha) producing giant "tailed" virions.

Most known giant viruses, i.e., viruses producing giant virions, parasitize amoebae and other unicellular eukaryotes. Although they vary in the level of dependence on host nuclear functions, their virions self-assemble in the host cell’s cytoplasm. Here we report the discovery of a new prototype of giant virus infecting epidermal cells of the marine arrow worm Adhesisagitta hispida. Its 1.25 μm-long virions self-assemble and accumulate in the host cell’s nucleus. Conventional transmission electron microscopy reveals that the virions have a unique bipartite structure. An ovoid nucleocapsid, situated in a broad “head” end of the virion is surrounded by a thin envelope. The latter extends away from the head to form a voluminous conical “tail” filled with electron-dense extracapsidular material. The 31nm-thick capsid wall has a distinctive substructure resulting from a patterned arrangement of subunits; it bears no ultrastructural resemblance to the virion walls of other known giant viruses. The envelope self-assembles coincident with the capsid and remotely from all host membranes. We postulate that transmission to new hosts occurs by rupture of protruding virion-filled nuclei when infected arrow worms mate. Future genomic work is needed to determine the phylogenetic position of this new virus, which we have provisionally named Meelsvirus.

Details of charge distribution in stable viral capsid

We present the results of Molecular Dynamics simulations of a viral capsid with the aim to analyse ion distribution on the capsid's surface that defines its stability. Two systems were modelled, a stable capsid with neutralising number of ions and an unstable capsid with low number of ions. For the ion distribution analysis the capsid's structure was identical and fixed in both simulations. It was then released for the stability analysis. The ion distribution demonstrated two types of the local regions on the inner surface of the capsid's wall: highly occupied with chloride ions in both systems despite a largely uniform electrostatic potential everywhere on the surface, and the regions that lose acid. The latter regions are located close to the cracks that are formed when the capsid is destabilised and thus could initiate the collapse of the capsid.

Forced phage uncorking: viral DNA ejection triggered by a mechanically sensitive switch.

The foremost event of bacteriophage infection is the ejection of genomic material into the host bacterium after virus binding to surface receptor sites. How ejection is triggered is yet unknown. Here we show, in single mature T7 phage particles, that tapping the capsid wall with an oscillating atomic-force-microscope cantilever triggers rapid DNA ejection via the tail complex. The triggering rate increases exponentially as a function of force, following transition-state theory, across an activation barrier of 23 kcal mol-1 at 1.2 nm along the reaction coordinate. The conformation of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This force, arising from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Chemical immobilization of the tail fibers also resulted in enhanced DNA ejection, suggesting that the triggering process might involve a conformational switch that can be mechanically activated either by external forces or via the tail-fiber complex.

Complete virus capsid at all-atom resolution: Simulations using molecular dynamics and hybrid molecular dynamics/hydrodynamics methods reveal semipermeable membrane function

Simulations of complete virus capsid at atomistic details have been performed using standard molecular dynamics as well as original hybrid molecular dynamics/hydrodynamics methodologies. The results show that the capsid is stable in water solution at room temperature and ions composition similar to physiological conditions. Detailed analysis of the flow of water molecules and ions through the capsid's wall is performed. It demonstrates that ions do not cross the capsid shell, while water exhibits substantial flows in both directions. This behaviour can be classified as a semipermeable membrane and may play a role in mechanical properties of the virus particle.

Forced Bacteriophage Uncorking: Viral DNA Ejection Triggered by a Sensitive Mechanical Switch

The foremost event of bacteriophage infection is the ejection of genomic DNA into the host bacterium after virus binding to surface receptor sites. The DNA ejection triggering mechanism is yet largely unknown. We investigated the role of mechanical force in triggering DNA ejection of the T7 bacteriophage. Mature phages containing tightly packed 40 kbp dsDNA were studied. We employed atomic force microscopy (AFM) to visualize the topographical structure and mechanically-induced changes of the phage particles covalently attached to mica surface. The sample was gently tapped so as to prevent the dislodging or fracture of the T7 capsids. We found that tapping the capsid wall with an oscillating AFM cantilever triggered a rapid DNA ejection via the tail complex. The forces employed were calculated to cause a very small (0.1-0.2%) change in the internal pressure of the capsid. Triggering rate increased exponentially as a function of force, following transition-state kinetics with an activation barrier of 23 kcal/mol at 1.2 nm along the reaction coordinate. The configuration of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This propulsive force, which is thought to arise from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Furthermore, the internal pressure likely contributes to mechanically pre-loading the ejection machinery, thereby tilting the energy landscape towards DNA ejection; in this state a small additional, external force is sufficient to trigger the process with an apparently switch-like mechanism.

Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages.

We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages λ and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals. A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage λ) as an experimental model system, we investigated the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.

RNA topology remolds electrostatic stabilization of viruses.

Simple RNA viruses efficiently encapsulate their genome into a nano-sized protein shell: the capsid. Spontaneous coassembly of the genome and the capsid proteins is driven predominantly by electrostatic interactions between the negatively charged RNA and the positively charged inner capsid wall. Using field theoretic formulation we show that the inherently branched RNA secondary structure allows viruses to maximize the amount of encapsulated genome and make assembly more efficient, allowing viral RNAs to out-compete cellular RNAs during replication in infected host cells.

Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle

During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNA-protein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp(38) of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.

Evolution of Type 2 Vaccine Derived Poliovirus Lineages. Evidence for Codon-Specific Positive Selection at Three Distinct Locations on Capsid Wall

Partial sequences of 110 type 2 poliovirus strains isolated from sewage in Slovakia in 2003–2005, and most probably originating from a single dose of oral poliovirus vaccine, were subjected to a detailed genetic analysis. Evolutionary patterns of these vaccine derived poliovirus strains (SVK-aVDPV2) were compared to those of type 1 and type 3 wild poliovirus (WPV) lineages considered to have a single seed strain origin, respectively. The 102 unique SVK-aVDPV VP1 sequences were monophyletic differing from that of the most likely parental poliovirus type 2/Sabin (PV2 Sabin) by 12.5–15.6%. Judging from this difference and from the rate of accumulation of synonymous transversions during the 22 month observation period, the relevant oral poliovirus vaccine dose had been administered to an unknown recipient more than 12 years earlier. The patterns of nucleotide substitution during the observation period differed from those found in the studied lineages of WPV1 or 3, including a lower transition/transversion (Ts/Tv) bias and strikingly lower Ts/Tv rate ratios at the 2nd codon position for both purines and pyrimidines. A relatively low preference of transitions at the 2nd codon position was also found in the large set of VP1 sequences of Nigerian circulating (c)VDPV2, as well as in the smaller sets from the Hispaniola cVDPV1 and Egypt cVDPV2 outbreaks, and among aVDPV1and aVDPV2 strains recently isolated from sewage in Finland. Codon-wise analysis of synonymous versus non-synonymous substitution rates in the VP1 sequences suggested that in five codons, those coding for amino acids at sites 24, 144, 147, 221 and 222, there may have been positive selection during the observation period. We conclude that pattern of poliovirus VP1 evolution in prolonged infection may differ from that found in WPV epidemics. Further studies on sufficiently large independent datasets are needed to confirm this suggestion and to reveal its potential significance.

Effect of capsid confinement on the chromatin organization of the SV40 minichromosome.

Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a ‘molten droplet’ state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement.

How simple can a model of an empty viral capsid be? Charge distributions in viral capsids.

We investigate and quantify salient features of the charge distributions on viral capsids. Our analysis combines the experimentally determined capsid geometry with simple models for ionization of amino acids, thus yielding a detailed description of spatial distribution for positive and negative charges across the capsid wall. The obtained data is processed in order to extract the mean radii of distributions, surface charge densities, as well as dipole moment densities. The results are evaluated and examined in light of previously proposed models of capsid charge distributions, which are shown to have to some extent limited value when applied to real viruses.

Productive Entry Pathways of Human Rhinoviruses

Currently, complete or partial genome sequences of more than 150 human rhinovirus (HRV) isolates are known. Twelve species A use members of the low-density lipoprotein receptor family for cell entry, whereas the remaining HRV-A and all HRV-B bind ICAM-1. HRV-Cs exploit an unknown receptor. At least all A and B type viruses depend on receptor-mediated endocytosis for infection. In HeLa cells, they are internalized mainly by a clathrin- and dynamin-dependent mechanism. Upon uptake into acidic compartments, the icosahedral HRV capsid expands by ~4% and holes open at the 2-fold axes, close to the pseudo-3-fold axes and at the base of the star-shaped dome protruding at the vertices. RNA-protein interactions are broken and new ones are established, the small internal myristoylated capsid protein VP4 is expelled, and amphipathic N-terminal sequences of VP1 become exposed. The now hydrophobic subviral particle attaches to the inner surface of endosomes and transfers its genomic (+) ssRNA into the cytosol. The RNA leaves the virus starting with the poly(A) tail at its 3′-end and passes through a membrane pore contiguous with one of the holes in the capsid wall. Alternatively, the endosome is disrupted and the RNA freely diffuses into the cytoplasm.

Simulations of Viral DNA Packaging and Ejection: Geometrical Order and Topological Disorder

The packing of DNA inside bacteriophages arguably yields the simplest example of genome organisation in living organisms. Cryo-em studies showed that DNA in bacteriophages epsilon-15 and phi-29 is neatly ordered in concentric shells close to the capsid wall, while an increasing level of disorder was measured when moving away from the capsid internal surface. On the other hand the detected spectrum of knots formed by DNA that is circularised inside the P4 viral capsid showed that DNA tends to be knotted with high probability, with a knot spectrum characterized by complex knots and biased towards torus knots and against achiral ones. Existing coarse-grain DNA models, while being capable of reproducing the salient physical aspects of free, unconstrained DNA, are not able to reproduce the experimentally observed features of packaged viral DNA. Here we show, using stochastic simulation techniques, that both the shell ordering and the knot spectrum can be reproduced quantitatively if one accounts for the preference of contacting DNA strands to juxtapose at a small twist angle, as in cholesteric liquid crystals. The DNA knots we observe are strongly delocalized and, intriguingly, this is shown not to interfere with genome ejection out of the phage.

P22 Coat Protein Structures Reveal a Novel Mechanism for Capsid Maturation: Stability without Auxiliary Proteins or Chemical Crosslinks

Viral capsid assembly and stability in tailed, dsDNA phage and Herpesviridae are achieved by various means including chemical crosslinks (unique to HK97), or auxiliary proteins (lambda, T4, phi29, and herpesviruses). All these viruses have coat proteins (CP) with a conserved, HK97-like core structure. We used a combination of trypsin digestion, gold labeling, cryo-electron microscopy, 3D image reconstruction, and comparative modeling to derive two independent, pseudoatomic models of bacteriophage P22 CP: before and after maturation. P22 capsid stabilization results from intersubunit interactions among N-terminal helices and an extensive "P loop," which obviate the need for crosslinks or auxiliary proteins. P22 CP also has a telokin-like Ig domain that likely stabilizes the monomer fold so that assembly may proceed via individual subunit addition rather than via preformed capsomers as occurs in HK97. Hence, the P22 CP structure may be a paradigm for understanding how monomers assemble in viruses like phi29 and HSV-1.

Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg 2+, DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 °C and 96 °C. In other conditions (absence of Mg 2+ and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit—probably via the portal vertex–while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.

Single Particle Force Spectroscopy Reveals Virus DNA Storage Strategies And Structural Properties Of Capsids And Virions

By using Atomic Force Microscopy in buffer conditions we performed nanoindentation experiments on several kinds of viruses. In the spherical virus Minute Virus of Mice we discovered that the DNA molecule exerts an architectural role by increasing the stiffness of the virus on symmetries axis 3 and 2. This mechanical reinforcement is a consequence of the interaction between crystallographically visible, short DNA patches and the inner capsid wall, and apparently does not build internal pressure inside the virus.However, preliminary experiments performed on T7 bacteriophage capsids and virions reveal that DNA storage produces an internal pressure of around 30 atmospheres, comparable to that found in other bacteriophages such us phi-29. We have also visualized for the first time the topography of in situ reversible buckling events in a capsid (T7) which shows the behaviour of the inter-capsomers bonds under stress.We have studied the existence of built in stress in the equatorial zone of phi-29 which can not be account by using continuum mechanics models.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Manipulation of the mechanical properties of a virus by protein engineering

In a previous study, we showed that the DNA molecule within a spherical virus (the minute virus of mice) plays an architectural role by anisotropically increasing the mechanical stiffness of the virus. A finite element model predicted that this mechanical reinforcement is a consequence of the interaction between crystallographically visible, short DNA patches and the inner capsid wall. We have now tested this model by using protein engineering. Selected amino acid side chains have been truncated to specifically remove major interactions between the capsid and the visible DNA patches, and the effect of the mutations on the stiffness of virus particles has been measured using atomic force microscopy. The mutations do not affect the stiffness of the empty capsid; however, they significantly reduce the difference in stiffness between the DNA-filled virion and the empty capsid. The results (i) reveal that intermolecular interactions between individual chemical groups contribute to the mechanical properties of a supramolecular assembly and (ii) identify specific protein-DNA interactions as the origin of the anisotropic increase in the rigidity of a virus. This study also demonstrates that it is possible to control the mechanical properties of a protein nanoparticle by the rational application of protein engineering based on a mechanical model.

A director-field model of DNA packaging in viral capsids

The present work is concerned with the formulation of a continuum theory of viral DNA packaging based on a director field representation of the encapsidated DNA. The point values of the director field give the local direction and density of the DNA. The continuity of the DNA strand requires that the director field be divergence-free and tangent to the capsid wall. The energy of the DNA is defined as a functional of the director field which accounts for bending, torsion, and for electrostatic interactions through a density-dependent cohesive energy. The operative principle which determines the encapsidated DNA conformation is assumed to be energy minimization. We show that torsionless toroidal solenoids, consisting of planar coils contained within meridional planes and wrapped around a spool core, and fine mixtures of the solenoid and spool phase, beat the inverse spool construction.

Structure of L-A virus: a specialized compartment for the transcription and replication of double-stranded RNA.

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment-the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-A resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10-15 A across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40-45 A, compared with 25-30 A in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.