Potential Molecular Mechanisms of Rare Anti-Tumor Immune Response by SARS-CoV-2 in Isolated Cases of Lymphomas.

Recently, two cases of complete remission of classical Hodgkin lymphoma (cHL) and follicular lymphoma (FL) after SARS-CoV-2 infection were reported. However, the precise molecular mechanism of this rare event is yet to be understood. Here, we hypothesize a potential anti-tumor immune response of SARS-CoV-2 and based on a computational approach show that: (i) SARS-CoV-2 Spike-RBD may bind to the extracellular domains of CD15, CD27, CD45, and CD152 receptors of cHL or FL and may directly inhibit cell proliferation. (ii) Alternately, upon internalization after binding to these CD molecules, the SARS-CoV-2 membrane (M) protein and ORF3a may bind to gamma-tubulin complex component 3 (GCP3) at its tubulin gamma-1 chain (TUBG1) binding site. (iii) The M protein may also interact with TUBG1, blocking its binding to GCP3. (iv) Both the M and ORF3a proteins may render the GCP2-GCP3 lateral binding where the M protein possibly interacts with GCP2 at its GCP3 binding site and the ORF3a protein to GCP3 at its GCP2 interacting residues. (v) Interactions of the M and ORF3a proteins with these gamma-tubulin ring complex components potentially block the initial process of microtubule nucleation, leading to cell-cycle arrest and apoptosis. (vi) The Spike-RBD may also interact with and block PD-1 signaling similar to pembrolizumab and nivolumab- like monoclonal antibodies and may induce B-cell apoptosis and remission. (vii) Finally, the TRADD interacting "PVQLSY" motif of Epstein-Barr virus LMP-1, that is responsible for NF-kB mediated oncogenesis, potentially interacts with SARS-CoV-2 Mpro, NSP7, NSP10, and spike (S) proteins, and may inhibit the LMP-1 mediated cell proliferation. Taken together, our results suggest a possible therapeutic potential of SARS-CoV-2 in lymphoproliferative disorders.

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.

Vulnerabilities in coronavirus glycan shields despite extensive glycosylation.

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.

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.

A protein scaffold, engineered SPINK2, for generation of inhibitors with high affinity and specificity against target proteases.

Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.

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.

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.

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.

Inactivation, aggregation, secondary and tertiary structural changes of germin-like protein in Satsuma mandarine with high polyphenol oxidase activity induced by ultrasonic processing.

The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity.

Structure of glycoproteins from Acacia gum: an assembly of ring-like glycoproteins modules.

The glycoprotein (GP) molecular fraction structure of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using high-performance size exclusion chromatography-multi angle laser light scattering (HPSEC-MALLS), small angle X-ray scattering (SAXS), synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) observations. In solution, GP would be a mixture of spheroidal monomers and more anisotropic oligomers as suggested by the two exponent values found in the Rg vs. Mw relationship and TEM observations. The GP conformation probed by SAXS was ascribed to a thin object with a triaxial ellipsoid morphology, certainly attributed to GP oligomers. A 9 nm diameter particle was also identified by SAXS in agreement with the dimensions identified by TEM on single isolated ring-like structures. The GP oligomerization process, as probed by TEM, would be the result of ring-like subunits self-association. This self-association would lead to more linear or, sometimes, cyclised assembly. At the molecular level, GP fraction was found to have secondary structures mainly made of ß-sheets and turns (64%) but also, to a lesser extent, made of polyproline II (PPII) and α-helices (19%). These features were characteristic of hydroxyprolin-rich glycoproteins with arabinosylated and arabinogalactan polysaccharide side chains grafted to the polypeptide backbone. The GP molecular fraction structure from Acacia gum would be an assembly of ring-like glycoproteins modules. These ring-like structures were certainly due to hydroxyproline (Hyp)-arabinogalactan (AG) subunits.

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.

A new amyloidosis caused by fibrillar aggregates of mutated corneodesmosin.

Heterozygous nonsense mutations in the CDSN gene encoding corneodesmosin (CDSN), an adhesive protein expressed in cornified epithelia and hair follicles, cause hypotrichosis simplex of the scalp (HSS), a nonsyndromic form of alopecia. Truncated mutants of CDSN ((mut)CDSN), which bear the N-terminal adhesive Gly/Ser-rich domain (GS domain) of the protein, abnormally accumulate as amorphous deposits at the periphery of hair follicles and in the papillary dermis of the patient skin. Here, we present evidence that the (mut)CDSN deposits display an affinity for amyloidophilic dyes, namely Congo red and thioflavin T. We also detected the serum amyloid protein component in the dermis of HSS patients. We demonstrated that recombinant forms of (mut)CDSN and of the GS domain assemble in vitro into ring-shaped oligomeric structures and fibrils. The amyloid-like nature of the fibrils was demonstrated by dye binding and Fourier transform infrared spectrometry measurements. We showed that the ring-shaped oligomers of (mut)CDSN, but not the fibrillar forms, are toxic to cultured keratinocytes. Finally, online algorithms predicted the GS domain to be a particularly disordered region of CDSN in agreement with circular dichroism measurements. This identifies HSS as a human amyloidosis related to the aggregation of natively unfolded (mut)CDSN polypeptides into amyloid fibrils.

Single-particle cryo-electron microscopy of Rift Valley fever virus.

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human and veterinary pathogen causing acute hepatitis in ruminants and has the potential to cause hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T=12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit vaccines.

Potential mechanism for recruitment and migration of CD133 positive cells to areas of vascular inflammation.

OBJECTIVE: Mast cells are found in large numbers in atherosclerotic plaques. The present study was conducted to determine whether tryptase stimulation of human coronary artery endothelial cells (HCAEC) would lead to an increase in transmigration of CD133 positive cells (CD133+). In vitro these cells can differentiate into mast cells under the influence of specific cytokines and growth factors. METHODS AND RESULTS: CD133+ cells were isolated from umbilical cord blood. They express mRNA for several adhesion molecules that are also utilized in neutrophil migration and can migrate across an HCAEC monolayer. Migration increased significantly when HCAEC were stimulated with tryptase and decreased when CD133+ cells were pretreated with CV3988, a platelet activating factor receptor (PTAFR) antagonist. Following long-term cell culture, these cells stained positively for the presence of tryptase, a mast cell enzyme. CONCLUSION: CD133+ cells can be utilized as a mast cell precursor population. The transendothelial migration is facilitated by the presence of tryptase and may utilize the PAF/PTAFR interaction in a manner similar to that involved in neutrophil transmigration. Following transmigration, a subset of these progenitor cells may mature into mast cells in the subendothelial space and play a role in propagation of the inflammatory process in atherosclerosis.

Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain.

The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.

[Nuclear organization and expression of milk protein genes]. / Organisation nucléaire et expression des gènes des protéines du lait.

Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products.

Essential functions of the unique N-terminal region of the varicella-zoster virus glycoprotein E ectodomain in viral replication and in the pathogenesis of skin infection.

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.