Spike protein VP4 assembly with maturing rotavirus requires a postendoplasmic reticulum event in polarized caco-2 cells

Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.     

Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms,and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.Rotaviruses are a major cause of acute gastroenteritis in infants and young children, producing a high burden of disease worldwide and over 600,000 deaths per annum, mainly in developing countries (43). Recently, two live attenuated rotavirus vaccines (49, 58) have been licensed in various countries, and their widespread use in universal mass vaccination programs is being implemented (55).Rotaviruses form a genus of the Reoviridae family. They contain a genome of 11 segments of double-stranded RNA (dsRNA) encoding six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). After entry into the host cell the outer layer of the triple-layered particles (TLPs; infectious virions) is removed in endocytic vesicles, and the resulting double-layered particles (DLPs) actively transcribe mRNAs from the 11 RNA segments and release them into the cytoplasm. The mRNAs are translated into proteins but also act as templates for dsRNA synthesis (RNA replication). The early stages of viral morphogenesis and viral RNA replication occur in cytoplasmic inclusion bodies termed “viroplasms.” Partially assembled DLPs are released from viroplasms and receive their outer layer in the rough endoplasmic reticulum (RER), forming TLPs (for details, see Estes and Kapikian [20]).The rotavirus nonstructural proteins NSP2 and NSP5 are major components of viroplasms (20, 47). These two proteins alone are sufficient to induce the formation of viroplasm-like structures (VLS) (21). Blocking of either NSP2 or NSP5 in rotavirus-infected cells significantly reduces viroplasm formation and the production of infectious viral progeny (11, 54, 57). Until now, host cell proteins involved in viroplasm formation have not been identified.Morphological similarities between viroplasms and lipid droplets (LDs) prompted us to investigate their relationship. Both structures have phosphoproteins (NSP5 and perilipin A, respectively) inserted on their surface in ringlike shapes (16, 34). LDs are intracellular organelles involved in lipid and carbohydrate metabolism. They consist of a neutral lipid core surrounded by a phospholipid monolayer containing LD-associated proteins; those include proteins of the PAT family consisting of perilipin, adipophilin (adipose differentiation-related protein [ADRP]), and TIP-47 (9, 37). Lipolysis from LDs is regulated by hormones such as catecholamines, which trigger the phosphorylation of hormone-sensitive lipase (HSL) and perilipin A and induce LD fragmentation. Incubating adipocytes with the β-adrenergic agonist isoproterenol and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) activates this pathway (27, 34). LD formation can also be blocked by triacsin C, a specific inhibitor of long chain acyl coenzyme A synthetases (30, 39).We demonstrate here that rotavirus viroplasms colocalize with the LD-associated proteins perilipin A and ADRP and also with the lipids of LDs. These interactions appear to be required for the formation of functional viroplasms and the production of infectious viral progeny, since compounds dispersing LDs or blocking LD formation significantly decrease the number and size of viroplasms and the amount of infectious progeny. Taken together, these findings strongly suggest a critical role of LDs in rotavirus replication.     

Control of the modularity of {Mo2O2S2}-containing polyoxometalates. Synthesis, structure and characterization of tri- and tetra-modular assemblies based on the [β-B-AsW9O33] anion

Reaction at pH = 2.3 of the [Mo₂O₂S₂(OH₂)₆]²⁺ aqua cation with the tetravacant ion [β-B-HAs₂W₈O₃₁]⁷⁻ leads to the formation of a red solid from which three mixed salts have been obtained as single crystals and characterized by X-ray diffraction analysis. Three mixed salts K-5a, RbNa-5b, DMACs-5c exhibit a similar molecular arrangement consisting in three {β-HAs₂W₉O₃₄} subunits mutually linked by three {Mo₂O₂S₂} groups. The triangular arrangement delimits a large open-cavity, lined on the periphery by three outer {As-OH} groups and closed at the bottom by a small hexagonal pocket formed by six terminal oxygen atoms. The central hexagonal cavity is filled either by a potassium, a rubidium or a cesium cation. The outer {As-OH} groups are pointed towards two directions labelled up and down, respectively. In K-6a the three {As-OH} bonds are in up configuration leading to the {up, up, up} isomer. The structure of RbNa-5b is rather consistent with the superposition of the two {up, up, up} and {down, up, up} isomers disordered over the same crystallographic site, while only the {down, up, up} isomer is present in DMACs-5c. In solution, ¹⁸³W NMR characterization of 6a as sodium salt results in a complicated spectrum consistent with the simultaneous presence of the four isomers, {up, up, up}, {down, up, up}, {down, down, up} and {down, down, down}, respectively. 5a reacts with three equivalents of iodine to give CsNa-6 isolated as single crystals. In 6, four β-{AsW₉O₃₃} moieties are located at the corner of a super tetrahedron and are mutually connected by six {Mo₂O₂S₂} linkers. The three outer {As-OH} groups can be selectively removed by iodine, this oxidation reaction consisting in fact in a deprotecting process permitting the extension of the arrangement from triangular to tetrahedral.     

Amnion and ocular surface problems

The amniotic membrane, the most internal placental membrane, has various properties useful in ophthalmology. Collected on delivery by elective Caesarean section, the amnion is prepared under sterile conditions, and, usually, cryopreserved until its use as a biological bandage or as a substrate for epithelial growth in the management of various ocular surface conditions. Specifically, the amnion is used to : (1) limit formation of adhesive bands between eyelids and eyeball (symblepharon) or the progression of a fibrovascular outgrowth towards the cornea (pterygium) or to (2) facilitate the healing of corneal ulcers, bullous keratopathy, and corneal stem cell deficiency. In this last condition, either hereditary or acquired after a thermal or a chemical burn, corneal stem cells, located at a transitional zone between the cornea and conjunctiva, are lost. These cells are essential for renewal of corneal epithelium in normal and in diseased states. The loss of these cells leaves the corneal surface free for invasion by conjunctival epithelium. Not only, does conjunctival epithelium support the development of vascularisation on the normally avascular cornea, but some conjunctival cells differentiate into mucus secreting goblet cells. Such a change in phenotype leads to loss of corneal transparency and visual disability. The removal of this fibro-vascular outgrowth in combination with transplantation of both amniotic membrane and corneal stem cells are used to treat this condition. The amnion stimulates the proliferation of less differentiated cells which have the potential to reconstruct the cornea. This potential is at the origin of the hypothesis that the amnion may provide an alternative niche for limbal stem cells of the corneal epithelium. It abounds in cytokines and has antalgic, anti-bacterial, anti-inflammatory and anti-immunogenic properties, in addition to allowing, like fetal skin does, wound healing with minimal scar formation. These desirable properties are responsible for the increasing use of amniotic membrane in ophthalmology. The complete understanding of the mechanisms of action of amniotic membrane for ocular surface diseases has yet to be understood. Once revealed by research, they may provide new pharmacological avenues to treat ocular surface diseases.     

Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.     

Extracellular matrix deposition by fibroblasts is necessary to promote capillary-like tube formation in vitro

The contribution of the cellular and fibrillar microenvironment to angiogenesis still remains unclear. Our purpose was to evaluate the effect of the extracellular matrix deposited by fibroblasts on the capacity of human endothelial cells to form capillaries in vitro. We have drastically decreased the amount of extracellular matrix surrounding fibroblasts in our model of endothelialized-reconstructed connective tissue (ERCT) by culturing it without ascorbate. Under these conditions, the number of capillary-like tubes (CLT) formed by endothelial cells was reduced by up to 10-fold after 31 days of culture compared to controls. This decrease was due neither to a variation of MMP-2 and MMP-9 secretion, nor to a reduction in the number of fibroblasts and/or endothelial cells, or a diminution of fibroblast growth factor 2 (FGF2) synthesis. The secretion of vascular endothelial growth factor (VEGF) by fibroblasts accounted for 25-70% of the capillary-like tube formation when tissues were cultured in the presence or absence of ascorbate, as demonstrated by VEGF-blocking studies. The culture of endothelial cells on a similar extracellular matrix but in the absence of living fibroblasts did not promote the formation of CLT, even when tissues were fed with fibroblast-conditioned medium. Thus, the deposition of a rich extracellular matrix by living fibroblasts appeared necessary, but not sufficient to promote capillary-like formation. Fibroblasts seem to induce endothelial cells to spontaneously form CLT by secreting and organizing an abundant extracellular matrix, which creates a microenvironment around cells that could in turn trap growth factors produced by fibroblasts and promote three-dimensional cell organization.     

Overcoming original antigenic sin to generate new CD8 T cell IFN-gamma responses in an antigen-experienced host

The failure to mount effective immunity to virus variants in a previously virus-infected host is known as original antigenic sin. We have previously shown that prior immunity to a virus capsid protein inhibits induction by immunization of an IFN-gamma CD8+ T cell response to an epitope linked to the capsid protein. We now demonstrate that capsid protein-primed CD4+ T cells secrete IL-10 in response to capsid protein presented by dendritic cells, and deviate CD8+ T cells responding to a linked MHC class I-restricted epitope to reduce IFN-gamma production. Neutralizing IL-10 while delivering further linked epitope, either in vitro or in vivo, restores induction by immunization of an Ag-specific IFN-gamma response to the epitope. This finding demonstrates a strategy for overcoming inhibition of MHC class I epitopes upon immunization of a host already primed to Ag, which may facilitate immunotherapy for chronic viral infection or cancer.     

Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor alpha, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.