Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release.

The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. IMPORTANCE: Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV.

Crystal Structure of the Human Astrovirus Capsid Protein.

UNLABELLED: Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP90(71-415) (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP90(71-415) encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP90(71-415) is comprised of two domains: an S domain, which adopts the typical jelly-roll ß-barrel fold, and a P1 domain, which forms a squashed ß-barrel consisting of six antiparallel ß-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP90(71-415) structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation. IMPORTANCE: Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus, HAstV exhibits an intriguing feature in that its maturation requires extensive proteolytic processing of the astrovirus capsid protein (CP) both inside and outside the host cell. Mature HAstV contains three predominant protein species, but the mechanism for acquired infectivity upon maturation is unclear. We have solved the crystal structure of VP90(71-415) of human astrovirus serotype 8. VP90(71-415) encompasses the conserved N-terminal domain of the viral CP. Fitting of the VP90(71-415) structure into the cryo-EM maps of HAstV produced an atomic model for the T=3 icosahedral capsid. Our model of the HAstV capsid provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation. Such information has potential applications in the development of a VLP vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation.

Hemagglutinin Spatial Distribution Shifts in Response to Cholesterol in the Influenza Viral Envelope.

Influenza virus delivers its genome to the host cytoplasm via a process of membrane fusion mediated by the viral hemagglutinin protein. Optimal fusion likely requires multiple hemagglutinin trimers, so the spatial distribution of hemagglutinin on the viral envelope may influence fusion mechanism. We have previously shown that moderate depletion of cholesterol from the influenza viral envelope accelerates fusion kinetics even though it decreases fusion efficiency, both in a reversible manner. Here, we use electron cryo-microscopy to measure how the hemagglutinin lateral density in the viral envelope changes with cholesterol extraction. We extract this information by measuring the radial distribution function of electron density in >4000 viral images per sample, assigning hemagglutinin density by comparing images with and without anti-HA Fab bound. On average, hemagglutinin trimers move closer together: we estimate that the typical trimer-trimer spacing reduces from 94 to 84 Å when ∼90% of cholesterol is removed from the viral membrane. Upon restoration of viral envelope cholesterol, this spacing once again expands. This finding can qualitatively explain the observed changes to fusion kinetics: contemporary models from single-virus microscopy are that fusion requires the engagement of several hemagglutinin trimers in close proximity. If removing cholesterol increases the lateral density of hemagglutinin, this should result in an increase in the rate of fusion.

Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry.

Microparticles (MPs) are submicron vesicles released from cell membranes in response to activation, cell injury, or apoptosis. The clinical importance of MPs has become increasingly recognized, although no standardized method exists for their measurement. Flow cytometry (FCM) is the most commonly used technique, however, because of the small size of MPs, and the limitations of current FCM instrumentation, accurate identification is compromised by this methodology. We decided to investigate whether the use of FCM combined with imaging, such as is possible with the ImagestreamX imaging FC (ISX), would be a more sensitive approach to characterizing MPs. Combining FCM with imaging eliminates some of the limitations demonstrated by conventional FCM, whereas also providing morphological confirmation and the ability to distinguish true single events from aggregates and cell debris. The detection limit of standard nonspecialized FCM is suboptimal when compared to ISX. Evaluating MPs below 0.200 µm and sizing remain a challenge as some MPs remain below the detection limit of ISX. Standardized calibrators, that more closely reflect the physical characteristics of MPs, need further development.

Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture.

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine.

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.

Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids.

TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids.

Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examples.

Electron microscopy provides an efficient method for rapidly assessing whether a solution of macromolecules is homogeneous and monodisperse. If the macromolecules can be induced to form two-dimensional crystals that are a single layer in thickness, then electron crystallography of frozen-hydrated crystals has the potential of achieving three-dimensional density maps at sub-nanometer or even atomic resolution. Here we describe the lipid monolayer and sparse matrix screening methods for growing two-dimensional crystals and present successful applications to soluble macromolecular complexes: carboxysome shell proteins and HIV CA, respectively. Since it is common to express recombinant proteins with poly-His tags for purification by metal affinity chromatography, the monolayer technique using bulk lipids doped with Ni(2+) lipids has the potential for broad application. Likewise, the sparse matrix method uses screening conditions for three-dimensional crystallization and is therefore of broad applicability.

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis E virus.

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T=3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~350Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~440Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. & Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA108, 12681-12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.

Two-dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three-dimensional crystals.

Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two-dimensional (2D) layers within the context of three-dimensional (3D) crystals. Here, we show that well-ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His-tagged proteins bind to a monolayer of nickelated lipid molecules at an air-water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells.

Ab initio random model method facilitates 3D reconstruction of icosahedral particles.

Model-based, three-dimensional (3D) image reconstruction procedures require a starting model to initiate data analysis. We have designed an ab initio method, which we call the random model (RM) method, that automatically generates models to initiate structural analysis of icosahedral viruses imaged by cryo-electron microscopy. The robustness of the RM procedure was demonstrated on experimental sets of images for five representative viruses. The RM method also provides a straightforward way to generate unbiased starting models to derive independent 3D reconstructions and obtain a more reliable assessment of resolution. The fundamental scheme embodied in the RM method should be relatively easy to integrate into other icosahedral software packages.

The role of subunit hinges and molecular "switches" in the control of viral capsid polymorphism.

The coat protein (CP) of cowpea chlorotic mottle virus assembles exclusively into a T=3 capsid in vivo and, under proper conditions, in vitro. The N-terminal domain of CP has been implicated in proper assembly and was viewed as a required switch for mediating hexamer and pentamer formation in T=3 assembly. We observed that a mutant CP lacking most of the N-terminal domain, NDelta34, assembles, in vitro, into statistically predictable numbers of: native-like T=3 capsids of 90 dimers; "T=2" capsids of 60 dimers; T=1 capsids of 30 dimers. We generated cryo-EM image reconstructions of each form and built pseudo-atomic models based on the subunits from the crystal structure of plant-derived T=3 virus allowing a detailed comparison of stabilizing interactions in the three assemblies. The statistical nature of the distribution of assembly products and the observed structures indicates that the N-terminus of CP is not a switch that is required to form the proper ratio of hexamers and pentamers for T=3 assembly; rather, it biases the direction of assembly to T=3 particles from the possibilities available to NDelta34 through flexible dimer hinges and variations in subunit contacts. Our results are consistent with a pentamer of dimers (PODs) nucleating assembly in all cases but subunit dimers can be added with different trajectories that favor specific T=3 or T=1 global particle geometries. Formation of the "T=2" particles appears to be fundamentally different in that they not only nucleate with PODs, but assembly propagates by the addition of mostly, if not exclusively PODs generating an entirely new subunit interface in the process. These results show that capsid geometry is flexible and may readily adapt to new requirements as the virus evolves.

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.

An archaeal antioxidant: characterization of a Dps-like protein from Sulfolobus solfataricus.

Evolution of an oxygenic atmosphere required primordial life to accommodate the toxicity associated with reactive oxygen species. We have characterized an archaeal antioxidant from the hyperthermophilic acidophile Sulfolobus solfataricus. The amino acid sequence of this approximately 22-kDa protein shares little sequence similarity with proteins with known function. However, the protein shares high sequence similarity with hypothetical proteins in other archaeal and bacterial genomes. Nine of these hypothetical proteins form a monophyletic cluster within the broad superfamily of ferritin-like diiron-carboxylate proteins. Higher order structural predictions and image reconstructions indicate that the S. solfataricus protein is structurally related to a class of DNA-binding protein from starved cells (Dps). The recombinant protein self assembles into a hollow dodecameric protein cage having tetrahedral symmetry (SsDps). The outer shell diameter is approximately 10 nm, and the interior diameter is approximately 5 nm. Dps proteins have been shown to protect nucleic acids by physically shielding DNA against oxidative damage and by consuming constituents involved in Fenton chemistry. In vitro, the assembled archaeal protein efficiently uses H2O2 to oxidize Fe(II) to Fe(III) and stores the oxide as a mineral core on the interior surface of the protein cage. The ssdps gene is up-regulated in S. solfataricus cultures grown in iron-depleted media and upon H2O2 stress, but is not induced by other stresses. SsDps-mediated reduction of hydrogen peroxide and possible DNA-binding capabilities of this archaeal Dps protein are mechanisms by which S. solfataricus mitigates oxidative damage.

Nodavirus coat protein imposes dodecahedral RNA structure independent of nucleotide sequence and length.

The nodavirus Flock house virus (FHV) has a bipartite, positive-sense RNA genome that is packaged into an icosahedral particle displaying T=3 symmetry. The high-resolution X-ray structure of FHV has shown that 10 bp of well-ordered, double-stranded RNA are located at each of the 30 twofold axes of the virion, but it is not known which portions of the genome form these duplex regions. The regular distribution of double-stranded RNA in the interior of the virus particle indicates that large regions of the encapsidated genome are engaged in secondary structure interactions. Moreover, the RNA is restricted to a topology that is unlikely to exist during translation or replication. We used electron cryomicroscopy and image reconstruction to determine the structure of four types of FHV particles that differed in RNA and protein content. RNA-capsid interactions were primarily mediated via the N and C termini, which are essential for RNA recognition and particle assembly. A substantial fraction of the packaged nucleic acid, either viral or heterologous, was organized as a dodecahedral cage of duplex RNA. The similarity in tertiary structure suggests that RNA folding is independent of sequence and length. Computational modeling indicated that RNA duplex formation involves both short-range and long-range interactions. We propose that the capsid protein is able to exploit the plasticity of the RNA secondary structures, capturing those that are compatible with the geometry of the dodecahedral cage.