Structural characterization of the PCV2d virus-like particle at 3.3 Šresolution reveals differences to PCV2a and PCV2b capsids, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes.

Porcine circovirus 2 (PCV2) has a major impact on the swine industry. Eight PCV2 genotypes (a-h) have been identified using capsid sequence analysis. PCV2d has been designated as the emerging genotype. The cryo-electron microscopy molecular envelope of PCV2d virus-like particles identifies differences between PCV2a, b and d genotypes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. These differences indicate that sequence analysis of genotypes is insufficient, and that it is important to determine the PCV2 capsid structure as the virus evolves. Structure-based sequence comparison demonstrate that each genotype possesses a unique combination of amino acids located on the surface of the capsid that undergo substitution. We also demonstrate that the capsid N-terminus moves in response to increasing amount of nucleic acid packaged into the capsid. Furthermore, we model a tetranucleotide between the 5- and 2-fold axes of symmetry that appears to be responsible for capsid stability.

Heteroaggregation of an enveloped bacteriophage with colloidal sediments and effect on virus viability.

Four sediments in the colloidal size range: goethite, montmorillonite, illite, and kaolinite, were suspended with the bacteriophage φ6, a model enveloped virus, to determine relative rates of heteroaggregation and the effect of aggregation on virus viability. Turbidity was measured on combinations of virus and each sediment type at low concentration to determine aggregation rates. Aggregation of sediment with virus occurred regardless of mineral type, and larger fraction of virus is expected to aggregate with increasing sediment concentration leading to higher deposition rates. The negatively charged sediments, aggregated with φ6 (also negatively charged at neutral pH) at a faster rate than the positively charged sediments, yielding turbidity slopes of 4.94 × 10-3 s-1 and 7.50 × 10-4 s-1 for φ6-montmorillonite and φ6-illite aggregates, respectively, and 2.98 × 10-5 s-1 and 2.84 × 10-5 s-1, for φ6-goethite and φ6-kaolinite, respectively. This indicates that the interaction between sediments and virus is hydrophobic, rather than electrostatic. Large numbers of virions remained viable post-aggregation, despite the fragility of the viral envelope, indicating that small-sized aggregates, which may travel more readily through porous media, may pose an infection risk. The fraction of φ6 that remained viable varied with sediment type, with montmorillonite-φ6 aggregates experiencing the greatest reduction in infectivity at 35%. TEM analyses reveal that in all sediment-φ6 combinations, infectivity loss was likely due to disassembly of the viral envelope as a result of aggregation.

Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions.

The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.

Zika virus-like particle (VLP) based vaccine.

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development.

Exfoliation and intercalation of montmorillonite by small peptides.

Understanding structural changes in clay minerals induced by complexation with organic matter is relevant to soil science and agricultural applications. In this study, the effect of peptide storage in montmorillonite and the thermal stability of peptide-clay complexes was examined through characterization by X-ray diffraction (XRD), electron microscopy, UV absorption, and thermogravimetric analysis (TGA). XRD analysis of small peptide-montmorillonite clay complexes produced profiles consisting of reflections associated with the smectite 001 reflection and related peaks similar to that produced by a mixed layer clay mineral structure. Shifts in higher order diffraction maxima were attributed to disorder caused by the intercalation with the peptides. Increasing peptide concentrations resulted in greater shifts towards smaller 2θ from 6.37° (1.39 nm) to 5.45° (1.62 nm) as the interlayer space expanded. The expansion was accompanied by broadening of the 001 reflection (FWHM increases from 0.51 to 1.22° 2θ). The XRD line broadening was interpreted as caused by poorer crystallinity resulting from intercalation and tactoid exfoliation. SEM images revealed montmorillonite platelets with upwardly rolled edges that tend toward cylindrical structures with the production of tubules. High-resolution TEM images revealed bending of montmorillonite platelets, confirming exfoliation. The distribution of basal spacings in the micrographs was determined from the spatial frequencies obtained by Fourier analysis of density profiles. The distribution indicated the presence of discrete coherent crystallite domains. XRD and TGA results indicated that higher peptide concentrations resulted in a greater fraction of intercalated peptides and that surface adsorption of peptides mediated intercalation. Therefore, higher peptide concentration led to more stable organoclay complexes. However, UV absorption and TGA found that peptide adsorption onto montmorillonite had a finite limit at approximately 16% by weight.

The ϕ6 cystovirus protein P7 becomes accessible to antibodies in the transcribing nucleocapsid: a probe for viral structural elements.

Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein's antigenic sites.

Morphology of influenza B/Lee/40 determined by cryo-electron microscopy.

Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (∼83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ∼460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.

Disassembly of the cystovirus ϕ6 envelope by montmorillonite clay.

Prior studies of clay-virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT-φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.

Protein P7 of the cystovirus φ6 is located at the three-fold axis of the unexpanded procapsid.

The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase), and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.

Detection of glutamate in the eye by Raman spectroscopy.

Raman spectroscopy is used to detect glutamate in the eye. Glutamate, a by-product of nerve cell death, is an indicator of glaucoma and diabetic retinopathy. The Raman spectra of ex vivo whole porcine eyes and individual components (lens, cornea, vitreous) are measured and characterized. Monosodium glutamate is injected into the eyes to simulate disease conditions, and the contribution to the Raman spectrum due to the presence of glutamate is identified. The Raman spectra from the native eye is dominated by vibrational modes from proteins in the lens. An optical system is designed to optimize collection of signal from the vitreous, where the glutamate is located, and reduce the Raman from the lens. Two vibrational fingerprints of monosodium glutamate are detected at 1369 and 1422 cm(-1), although the concentrations are much above physiological concentrations.