The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism.

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-Å-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.

Induction of Rod-Shaped Structures by Herpes Simplex Virus Glycoprotein I.

The envelope glycoprotein I (gI) of herpes simplex virus 1 (HSV-1) is a critical mediator of virus-induced cell-to-cell spread and cell-cell fusion. Here, we report a previously unrecognized property of this molecule. In transfected cells, the HSV-1 gI was discovered to induce rod-shaped structures that were uniform in width but variable in length. Moreover, the gI within these structures was conformationally different from the typical form of gI, as a previously used monoclonal antibody mAb3104 and a newly made peptide antibody to the gI extracellular domain (ECD) (amino acids [aa] 110 to 202) both failed to stain the long rod-shaped structures, suggesting the formation of a higher-order form. Consistent with this observation, we found that gI could self-interact and that the rod-shaped structures failed to recognize glycoprotein E, the well-known binding partner of gI. Further analyses by deletion mutagenesis and construction of chimeric mutants between gI and gD revealed that the gI ECD is the critical determinant, whereas the transmembrane domain served merely as an anchor. The critical amino acids were subsequently mapped to proline residues 184 and 188 within a conserved PXXXP motif. Reverse genetics analyses showed that the ability to induce a rod-shaped structure was not required for viral replication and spread in cell culture but rather correlated positively with the capability of the virus to induce cell fusion in the UL24syn background. Together, this work discovered a novel feature of HSV-1 gI that may have important implications in understanding gI function in viral spread and pathogenesis.IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but the molecular mechanisms of how gI exactly works have remained poorly understood. Here, we report a novel property of this molecule, namely, induction of rod-shaped structures, which appeared to represent a higher-order form of gI. We further mapped the critical residues and showed that the ability of gI to induce rod-shaped structures correlated well with the capability of HSV-1 to induce cell fusion in the UL24syn background, suggesting that the two events may have an intrinsic link. Our results shed light on the biological properties of HSV-1 gI and may have important implications in understanding viral pathogenesis.

Dynamic organization of Herpesvirus glycoproteins on the viral envelope revealed by super-resolution microscopy.

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion.

Molecular architecture and modifications of full-length myocilin.

Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the myocilin olfactomedin domain, the structure and function of full-length myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible. We systematically characterized disulfide-dependent and -independent oligomerization as well as confirmed glycosylation and susceptibility to proteolysis. We identified oligomeric states with glycosylation sites that are inaccessible to enzymatic removal. Low-resolution single particle 2D class averaging from conventional transmission electron microscopy imaging confirms an extended arrangement of tetramers, truncated products consistent with dimers, and a higher-ordered state consistent with octamer. Taken together, our study reveals new myocilin misfolded states and layers of intrinsic heterogeneity, expands our knowledge of olfactomedin-family proteins and lays the foundation for a better molecular understanding of myocilin structure and its still enigmatic biological function.

The subcommissural organ and the Reissner fiber: old friends revisited.

The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35S-cysteine and 3H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.

Asymmetrically Branched Precision Glycooligomers Targeting Langerin.

Asymmetrically branched precision glycooligomers are synthesized by solid-phase polymer synthesis for studying multivalent carbohydrate-protein interactions. Through the stepwise assembly of Fmoc-protected oligo(amidoamine) building blocks and Fmoc/Dde-protected lysine, straightforward variation of structural parameters such as the number and length of arms, as well as the number and position of carbohydrate ligands, is achieved. Binding of 1-arm and 3-arm glycooligomers toward lectin receptors langerin and concanavalin A (ConA) was evaluated where the smallest 3-arm glycooligomer shows the highest binding toward langerin, and stepwise elongation of one, two, or all three arms leads to decreased binding. When directly comparing binding toward langerin and ConA, we find that structural variation of the scaffold affects glycomimetic ligand binding differently for the different targets, indicating the potential to tune such ligands not only for their avidity but also for their selectivity toward different lectins.

Characterization of the physical, microbiological, and chemical properties of sonicated raw bovine milk.

Innovative processing technologies, such as ultrasonication, can change the properties of milk, allowing for the improvement or development of dairy foods. Yet taking bench-scale equipment to pilot plant scale has been challenging. Raw milk, standardized to 3% fat and warmed to inlet temperatures of 42 or 54°C, was exposed to continuous, high-intensity, low-frequency ultrasonication (16/20 kHz, 1.36 kW/pass) at flow rates of 0.15, 0.30, and 0.45 L/min that resulted in resident times within the reaction cell of 6, 3, and 2 min per pass, respectively. Multiple passes (3, 5, and 7, respectively) were required to obtain a total exposure time of 14 to 18 min. Evaluation of fat droplet sizes, enzyme coagulation properties, and microstructure of milk and milk gels, as well as determining compositional and lipid properties, were conducted to determine the potential of the ultrasound system to effectively modify milk. Laser scanning particle sizing and confocal microscopy showed that the largest droplets (2.26 ± 0.13 µm) found in raw milk were selectively reduced in size with a concomitant increase in the number of submicron droplets (0.37 ± 0.06 µm), which occurred sooner when exposed to shorter bursts of ultrasonication (0.45 L/min flow rates) and at an inlet temperature of 54°C. Ultrasound processing with milk entering at 42°C resulted in faster gelling times and firmer curds at 30 min; however, extended processing at inlet temperature of 54°C reduced curd firmness and lengthened coagulation time. This showed that ultrasonication altered protein-protein and protein-lipid interactions, thus the strength of the enzyme-set curds. Scanning electron microscopy revealed a denser curd matrix with less continuous and more irregular shaped and clustered strands, whereas transmission electron microscopy showed submicron lipid droplets embedded within the protein strands of the curd matrix. Processing at inlet temperature of 54°C with flow rates of 0.30 and 0.45 L/min also reduced the total aerobic bacterial count by more than 1 log cfu/mL, and the number of psychrophiles below the limit of detection (10 cfu/mL) for this study. Ultrasonication exposures of 14 to 18 min had minimal effect on the milk composition, fatty acid profiles, and lipid heat capacity and enthalpy. The findings show that this continuous ultrasound system, which is conducive to commercial scale-up, modifies the physical and functional properties of milk under the parameters used in this study and has potential use in dairy processing.

Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.

Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.

Concerted regulation of npc2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin.

Niemann-Pick Protein C2 (npc2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, npc2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of npc2-membrane interactions has remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of npc2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic detail two competitively favorable membrane binding orientations of npc2 with a low interconversion barrier. The first binding mode (Prone) places the cholesterol binding pocket in direct contact with the membrane and is characterized by membrane insertion of a loop (V59-M60-G61-I62-P63-V64-P65). This mode is associated with cholesterol uptake and release. On the other hand, the second mode (Supine) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero)phosphate (bmp) is specifically required for strong membrane binding in Prone mode, and that it cannot be substituted by other anionic lipids. Meanwhile, sphingomyelin counteracts bmp by hindering Prone mode without affecting Supine mode. Our results provide concrete evidence that lipids modulate npc2-mediated cholesterol transport either by favoring or disfavoring Prone mode and that they impose this by modulating the accessibility of bmp for interacting with npc2. Overall, we provide a mechanism by which npc2-mediated cholesterol transport is controlled by the membrane composition and how npc2-lipid interactions can regulate the transport rate.

Effects of new n-3 fatty acid sources on milk fatty acid profile and milk fat properties in dairy cows.

Feeding livestock with n-3 fatty acid (FA) sources (linseed, for example) is a common strategy to improve lipid quality of meat and milk products. However, in monogastric animals, linseed tegument decreases digestibility and alphalinolenic acid (ALA) uptake, while the whole linseed is well used by ruminants. In a context of increasing sustainability of feeding systems, providing monogastric animals and ruminants with linseed products adapted to their digestive systems is an important issue. This research paper addresses the hypotheses: (i) sieved extruded linseed (SEL) specific for ruminants is as or more effective than standard extruded linseed (ii) microalgae DHA Gold® is an interesting source of docosahexaenoic acid (DHA) in feedstuff and (iii) the effects of SEL and microalgae on milk characteristics are complementary and additive. Thirty-two cows were divided into 4 groups with different dietary n-3 fatty acid sources using a continuous design. All the diets were fed as mixed rations based on maize silage, energy concentrate and soybean meal. The first group received a control diet (CTRL) with no additional fat. The 3 other groups received SEL, microalgae DHA Gold® (ALG) and a mixture of microalgae DHA Gold® and SEL (SEL/ALG). Milk was collected from morning milkings after six weeks of dietary treatment. In SEL and SEL/ALG, ALA increased (+0·32 and +0·26% unit, respectively), and DHA increased in ALG and SEL/ALG (+0·43 and +0·15% unit, respectively) compared to CTRL, as a consequence of the initial composition of the n-3 FA sources. In SEL, milk yield, fat and protein contents, milk fat globule size and spontaneous lipolysis (measured to evaluate suitability for milk processing) were not different compared with CTRL. In ALG and SEL/ALG, milk yield decreased (-2·8 and -6·0 kg/d, respectively), fat content was halved, and fat globule size was reduced (-1·46 and -1·31 µm, respectively) compared to CTRL. Spontaneous lipolysis increased in ALG (+0·12 mEq/kg of milk) compared to CTRL. Protected microalgae and the doses of microalgae in the diet need further investigation to prevent FA modification in the rumen and the consequent deleterious effects on milk fat.

The temperature-dependent physical state of polar lipids and their miscibility impact the topography and mechanical properties of bilayer models of the milk fat globule membrane.

The polar lipid assembly and biophysical properties of the biological membrane enveloping the milk fat globules (the MFGM) are yet poorly known, especially in connection with the temperature history that milk can experience after its secretion. However, bioactive mechanisms depend on biological structure, which itself highly depend on temperature. The objectives of this study were to investigate polar lipid packing in hydrated bilayers, models of the MFGM, and to follow at intermolecular level temperature-induced changes in the range 60-6°C, using the combination of differential scanning calorimetry, X-ray diffraction, atomic force microscopy (AFM) imaging and force spectroscopy. MFGM polar lipids, especially sphingomyelin, contain long chain saturated fatty acids with high phase transition temperatures. On cooling, the liquid disordered ld to solid ordered so (gel) phase transition of MFGM polar lipids started at about 40°C, leading to phase separation and formation of so phase domains protruding by about 1nm from the ld phase. Indentation measurements using AFM revealed that the resistance of the so phase domains to rupture was significantly higher than that of the ld phase and that it increased for both the domain and fluid phases with decreasing temperature. However, packing and stability of the bilayers were adversely affected by fast cooling to 6°C or by cooling-rewarming cycle. This study showed that MFGM polar lipid bilayers are dynamic systems. Heterogeneity in the structure and mechanical properties of the membrane was induced by temperature-dependent so/ld phase immiscibility of the lipid components. This could have consequences on the MFGM technological and biological functions (e.g. immunity and milk lipid digestion).

Nanoscale observation of the natural structure of milk-fat globules and casein micelles in the liquid condition using a scanning electron assisted dielectric microscopy.

Recently, aqueous nanoparticles have been used in drug-delivery systems for new type medicines. In particular, milk-casein micelles have been used as drug nanocarriers for targeting cancer cells. Therefore, nanostructure observation of particles and micelles in their native liquid condition is indispensable for analysing their function and mechanisms. However, traditional optical and scanning electron microscopy have difficulty observing the nanostructures of aqueous micelles. Recently, we developed a novel imaging technique called scanning electron-assisted dielectric microscopy (SE-ADM) that enables observation of various biological specimens in water with very little radiation damage and high-contrast imaging without staining or fixation at an 8-nm spatial resolution. In this study, for the first time, we show that the SE-ADM system is capable of high-resolution observation of whole-milk specimens in their natural state. Moreover, we successfully observe the casein micelles and milk-fat globules in an intact liquid condition. Our SE-ADM system can be applied to various biological particles and micelles in a native liquid state.

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.

Ultrastructural and immunocytochemical evidence for the reorganisation of the milk fat globule membrane after secretion.

This paper reports a detailed ultrastructural and immunocytochemical investigation of the structure of the milk fat globule membrane (MFGM) in a variety of species. The process follows the same pattern in all mammals so far investigated. The initial (or primary) MFGM immediately on release from the mammary cell is a continuous unit membrane with a thin underlying layer of cytoplasmic origin and a monolayer of phospholipid separating it from the core lipid. This structure changes rapidly as the milk fat globule (MFG) moves into the alveolar lumen. The unit membrane plus the underlying layer of cytoplasm modifies drastically into discontinuous patches and networks. These are superimposed upon a continuous apparently structureless sheet of electron dense material stabilising the MFG and similar to that which bounded the lipid in the cell. The underlying layer of the patches increases in electron density and immunocytochemistry demonstrates localisation of MFGM proteins in this layer. In four species, the dense material shows ordered paracrystalline molecular arrays in section and en face views. All the arrays show the same basic pattern and unit size as determined by optical diffraction. Similar patches, networks and arrays are present on the surface of expressed MFG. Negative staining of lipid-extracted expressed MFGs shows similar patches and networks of membrane. These also occasionally show the crystalline arrays and label with MFGM protein antibodies. Similar networks and strands of plasma membrane on the MFG surface are shown by our CLSM examination of unfixed expressed MFG from mice genetically modified to express a fluorescent molecule as a normal plasma membrane constituent.

Nanoscale structure of the BMP antagonist chordin supports cooperative BMP binding.

Bone morphogenetic proteins (BMPs) orchestrate key cellular events, such as proliferation and differentiation, in development and homeostasis. Extracellular antagonists, such as chordin, are essential regulators of BMP signaling. Chordin binds to BMPs blocking interaction with receptors, and cleavage by tolloid proteinases is thought to relieve this inhibition. A model has been previously proposed where chordin adopts a horseshoe-like arrangement enabling BMP binding cooperatively by terminal domains (1). Here, we present the nanoscale structure of human chordin using electron microscopy, small angle X-ray scattering, and solution-based biophysical techniques, which together show that chordin indeed has a compact horseshoe-shaped structure. Chordin variants were used to map domain locations within the chordin molecule. The terminal BMP-binding domains protrude as prongs from the main body of the chordin structure, where they are well positioned to interact with the growth factor. The spacing provided by the chordin domains supports the principle of a cooperative BMP-binding arrangement that the original model implied in which growth factors bind to both an N- and C-terminal von Willebrand factor C domain of chordin. Using binding and bioactivity assays, we compared full-length chordin with two truncated chordin variants, such as those produced by partial tolloid cleavage. Cleavage of either terminal domain has little effect on the affinity of chordin for BMP-4 and BMP-7 but C-terminal cleavage increases the efficacy of chordin as a BMP-4 inhibitor. Together these data suggest that partial tolloid cleavage is insufficient to ablate BMP inhibition and the C-terminal chordin domains play an important role in BMP regulation.

Cholesterol strongly affects the organization of lipid monolayers studied as models of the milk fat globule membrane: Condensing effect and change in the lipid domain morphology.

The biological membrane that surrounds the milk fat globules exhibits phase separation of polar lipids that is poorly known. The objective of this study was to investigate the role played by cholesterol in the organization of monolayers prepared as models of the milk fat globule membrane (MFGM). Differential scanning calorimetry and X-ray diffraction experiments allowed characterization of the gel to liquid crystalline phase transition temperature of lipids, Tm ~35°C, in vesicles prepared with a MFGM lipid extract. For temperature below Tm, atomic force microscopy revealed phase separation of lipids at 30 mN·m(-1) in Langmuir-Blodgett monolayers of the MFGM lipid extract. The high Tm lipids form liquid condensed (LC) domains that protrude by about 1.5 nm from the continuous liquid expanded (LE) phase. Cholesterol was added to the MFGM extract up to 30% of polar lipids (cholesterol/milk sphingomyelin (MSM) molar ratio of 50/50). Compression isotherms evidenced the condensing effect of the cholesterol onto the MFGM lipid monolayers. Topography of the monolayers showed a decrease in the area of the LC domains and in the height difference H between the LC domains and the continuous LE phase, as the cholesterol content increased in the MFGM lipid monolayers. These results were interpreted in terms of nucleation effects of cholesterol and decrease of the line tension between LC domains and LE phase in the MFGM lipid monolayers. This study revealed the major structural role of cholesterol in the MFGM that could be involved in biological functions of this interface (e.g. mechanisms of milk fat globule digestion).

Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.

The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

Disparate epitopes mediating protective heterologous immunity to unrelated viruses share peptide-MHC structural features recognized by cross-reactive T cells.

Closely related peptide epitopes can be recognized by the same T cells and contribute to the immune response against pathogens encoding those epitopes, but sometimes cross-reactive epitopes share little homology. The degree of structural homology required for such disparate ligands to be recognized by cross-reactive TCRs remains unclear. In this study, we examined the mechanistic basis for cross-reactive T cell responses between epitopes from unrelated and pathogenic viruses, lymphocytic choriomeningitis virus (LCMV) and vaccinia virus. Our results show that the LCMV cross-reactive T cell response toward vaccinia virus is dominated by a shared asparagine residue, together with other shared structural elements conserved in the crystal structures of K(b)-VV-A11R and K(b)-LCMV-gp34. Based on analysis of the crystal structures and the specificity determinants for the cross-reactive T cell response, we were able to manipulate the degree of cross-reactivity of the T cell response, and to predict and generate a LCMV cross-reactive response toward a variant of a null OVA-derived peptide. These results indicate that protective heterologous immune responses can occur for disparate epitopes from unrelated viruses.

Allergens, sources, particles, and molecules: Why do we make IgE responses?

Allergens are foreign proteins or glycoproteins that are the target of IgE antibody responses in humans. The relationship between subsequent exposure and the allergic symptoms is often or usually obvious; however, there is increasing evidence that in asthma, atopic dermatitis and some forms of food allergy the induction of symptoms is delayed or chronic. The primary exposure to inhaled allergens is to the particles, which are capable of carrying allergens in the air. Thus, the response reflects not only the properties of the proteins, but also the biological properties of the other constituents of the particle. This is best understood in relation to the mite fecal particles in which the contents include many different immunologically active substances. Allergic disease first became a major problem over 100 years ago, and for many years sensitization to pollens was the dominant form of these diseases. The rise in pediatric asthma correlates best with the move of children indoors, which started in 1960 and was primarily driven by indoor entertainment for children. While the causes of the increase are not simple they include both a major increase in sensitization to indoor allergens and the complex consequences of inactivity. Most recently, there has also been an increase in food allergy. Understanding this has required a reappraisal of the importance of the skin as a route for sensitization. Overall, understanding allergic diseases requires knowing about the sources, the particles and the routes of exposure as well as the properties of the individual allergens.

Collagen-like glycoprotein BclS is involved in the formation of filamentous structures of the Lysinibacillus sphaericus exosporium.

Lysinibacillus sphaericus produces mosquitocidal binary toxins (Bin toxins) deposited within a balloon-like exosporium during sporulation. Unlike Bacillus cereus group strains, the exosporium of L. sphaericus is usually devoid of the hair-like nap, an external filamentous structure formed by a collagen-like protein, BclA. In this study, a new collagen-like exosporium protein encoded by Bsph_0411 (BclS) from L. sphaericus C3-41 was characterized. Thin-section electron microscopy revealed that deletion of bclS resulted in the loss of the filamentous structures that attach to the exosporium basal layer and spread through the interspace of spores. In vivo visualization of BclS-green fluorescent protein (GFP)/mCherry fusion proteins revealed a dynamic pattern of fluorescence that encased the spore from the mother cell-distal (MCD) pole of the forespore, and the BclS-GFP fusions were found to be located in the interspace of the spore, as confirmed by three-dimensional (3D) superresolution fluorescence microscopy. Further studies demonstrated that the bclS mutant spores were more sensitive to wet-heat treatment and germinated at a lower rate than wild-type spores and that these phenotypes were significantly restored in the bclS-complemented strain. These results suggested novel roles of collagen-like protein in exosporium assembly and spore germination, providing a hint for a further understanding of the genetic basis of the high level of persistence of Bin toxins in nature.