The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1ß in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1ß. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

The Inflammasome Pathway is Activated by Dengue Virus Non-structural Protein 1 and is Protective During Dengue Virus Infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1ß in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1ß. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

An improved reference of the grapevine genome reasserts the origin of the PN40024 highly homozygous genotype.

The genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. "Helfensteiner" (cross of cv. "Pinot noir" and "Schiava grossa") instead of a single "Pinot noir". These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome.

SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-ß signaling.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of vascular leak are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to induce barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-ß signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-ß signaling axis are required for S-mediated barrier dysfunction. Notably, we show that SARS-CoV-2 infection caused leak in vivo, which was reduced by inhibiting integrins. Our findings offer mechanistic insight into SARS-CoV-2-triggered vascular leak, providing a starting point for development of therapies targeting COVID-19.

Flavivirus NS1 Triggers Tissue-Specific Disassembly of Intercellular Junctions Leading to Barrier Dysfunction and Vascular Leak in a GSK-3ß-Dependent Manner.

The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins ß-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of ß-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of ß-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3ß (GSK-3ß). Supporting this model, we found that a chemical inhibitor of GSK-3ß reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3ß as a potential therapeutic target for treatment of vascular leakage during severe dengue disease.

Sulfated ß-glucan from Agaricus subrufescens inhibits flavivirus infection and nonstructural protein 1-mediated pathogenesis.

Despite substantial morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika, and the currently available dengue vaccine is only recommended for dengue virus (DENV)-immune individuals. Thus, development of therapeutic and/or preventive drugs is urgently needed. DENV and Zika virus (ZIKV) nonstructural protein 1 (NS1) can directly trigger endothelial barrier dysfunction and induce inflammatory responses, contributing to vascular leak in vivo. Here we evaluated the efficacy of the (1-6,1-3)-ß-D-glucan isolated from Agaricus subrufescens fruiting bodies (FR) and its sulfated derivative (FR-S) against DENV-2 and ZIKV infection and NS1-mediated pathogenesis. FR-S, but not FR, significantly inhibited DENV-2 and ZIKV replication in human monocytic cells (EC50 = 36.5 and 188.7 µg/mL, respectively) when added simultaneously with viral infection. No inhibitory effect was observed when FR or FR-S were added post-infection, suggesting inhibition of viral entry as a mechanism of action. In an in vitro model of endothelial permeability using human pulmonary microvascular endothelial cells (HPMECs), FR and FR-S (0.12 µg/mL) inhibited DENV-2 NS1- and ZIKV NS1-induced hyperpermeability by 50% and 100%, respectively, as measured by Trans-Endothelial Electrical Resistance. Treatment with 0.25 µg/mL of FR and FR-S inhibited DENV-2 NS1 binding to HPMECs. Further, FR-S significantly reduced intradermal hyperpermeability induced by DENV-2 NS1 in C57BL/6 mice and protected against DENV-induced morbidity and mortality in a murine model of dengue vascular leak syndrome. Thus, we demonstrate efficacy of FR-S against DENV and ZIKV infection and NS1-induced endothelial permeability in vitro and in vivo. These findings encourage further exploration of FR-S and other glycan candidates for flavivirus treatment alone or in combination with compounds with different mechanisms of action.

Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection.

Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.

SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-ß signaling.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo , independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-ß signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-ß signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

Introgression reshapes recombination distribution in grapevine interspecific hybrids.

KEY MESSAGE: In grapevine interspecific hybrids, meiotic recombination is suppressed in homeologous regions and enhanced in homologous regions of recombined chromosomes, whereas crossover rate remains unchanged when chromosome pairs are entirely homeologous. Vitis rotundifolia, an American species related to the cultivated European grapevine Vitis vinifera, has a high level of resistance to several grapevine major diseases and is consequently a valuable resource for grape breeding. However, crosses between both species most often lead to very few poorly fertile hybrids. In this context, identifying genetic and genomic features that make cross-breeding between both species difficult is essential. To this end, three mapping populations were generated by pseudo-backcrosses using V. rotundifolia as the donor parent and several V. vinifera cultivars as the recurrent parents. Genotyping-by-sequencing was used to establish high-density genetic linkage maps and to determine the genetic composition of the chromosomes of each individual. A good collinearity of the SNP positions was observed between parental maps, confirming the synteny between both species, except on lower arm of chromosome 7. Interestingly, recombination rate in V. rotundifolia × V. vinifera interspecific hybrids depends on the length of the introgressed region. It is similar to grapevine for chromosome pairs entirely homeologous. Conversely, for chromosome pairs partly homeologous, recombination is suppressed in the homeologous regions, whereas it is enhanced in the homologous ones. This balance leads to the conservation of the total genetic length of each chromosome between V. vinifera and hybrid maps, whatever the backcross level and the proportion of homeologous region. Altogether, these results provide new insight to optimize the use of V. rotundifolia in grape breeding and, more generally, to improve the introgression of gene of interest from wild species related to crops.

Subcellular localization of ENS-1/ERNI in chick embryonic stem cells.

The protein of retroviral origin ENS-1/ERNI plays a major role during neural plate development in chick embryos by controlling the activity of the epigenetic regulator HP1γ, but its function in the earlier developmental stages is still unknown. ENS-1/ERNI promoter activity is down-regulated upon differentiation but the resulting protein expression has never been examined. In this study, we present the results obtained with custom-made antibodies to gain further insights into ENS-1 protein expression in Chicken embryonic stem cells (CES) and during their differentiation. First, we show that ENS-1 controls the activity of HP1γ in CES and we examined the context of its interaction with HP1γ. By combining immunofluorescence and western blot analysis we show that ENS-1 is localized in the cytoplasm and in the nucleus, in agreement with its role on gene's promoter activity. During differentiation, ENS-1 decreases in the cytoplasm but not in the nucleus. More precisely, three distinct forms of the ENS-1 protein co-exist in the nucleus and are differently regulated during differentiation, revealing a new level of control of the protein ENS-1. In silico analysis of the Ens-1 gene copies and the sequence of their corresponding proteins indicate that this pattern is compatible with at least three potential regulation mechanisms, each accounting only partially. The results obtained with the anti-ENS-1 antibodies presented here reveal that the regulation of ENS-1 expression in CES is more complex than expected, providing new tracks to explore the integration of ENS-1 in CES cells regulatory networks.

A reference genetic map of Muscadinia rotundifolia and identification of Ren5, a new major locus for resistance to grapevine powdery mildew.

Muscadinia rotundifolia, a species closely related to cultivated grapevine Vitis vinifera, is a major source of resistance to grapevine downy and powdery mildew, two major threats to cultivated traditional cultivars of V. vinifera respectively caused by the oomycete Plasmopara viticola and the ascomycete Erisyphe necator. The aim of the present work was to develop a reference genetic linkage map based on simple sequence repeat (SSR) markers for M. rotundifolia. This map was created using S1 M. rotundifolia cv. Regale progeny, and covers 948 cM on 20 linkage groups, which corresponds to the expected chromosome number for muscadine. The comparison of the genetic maps of V. vinifera and M. rotundifolia revealed a high macrosynteny between the genomes of both species. The S1 progeny was used to assess the general level of resistance of M. rotundifolia to P. viticola and E. necator, by scoring different parameters of pathogen development. A quantitative trait locus (QTL) analysis allowed us to highlight a major QTL on linkage group 14 controlling resistance to powdery mildew, which explained up to 58 % of the total phenotypic variance. This QTL was named 'Resistance to Erysiphe Necator 5' (Ren5). A microscopic evaluation E. necator mycelium development on resistant and susceptible genotypes of the S1 progeny showed that Ren5 exerts its action after the formation of the first appressorium, and acts by delaying, and then stopping, mycelium development.

The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue.

BACKGROUND: Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized in vitro and in vivo the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells. RESULTS: We show that Ens-1 LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the Ens-1 gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of Ens-1. CONCLUSION: Our results show that Ens-1 LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, Ens-1 LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.

Construction of a reference linkage map of Vitis amurensis and genetic mapping of Rpv8, a locus conferring resistance to grapevine downy mildew.

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the major threats to grapevine. All traditional cultivars of grapevine (Vitis vinifera) are susceptible to downy mildew, the control of which requires regular application of fungicides. In contrast, many sources of resistance to P. viticola have been described in the Vitis wild species, among which is V. amurensis Rupr. (Vitaceae), a species originating from East Asia. A genetic linkage map of V. amurensis, based on 122 simple sequence repeat and 6 resistance gene analogue markers, was established using S1 progeny. This map covers 975 cM on 19 linkage groups, which represent 82% of the physical coverage of the V. vinifera reference genetic map. To measure the general level of resistance, the sporulation of P. viticola and the necrosis produced in response to infection, five quantitative and semi-quantitative parameters were scored 6 days post-inoculation on the S1 progeny. A quantitative trait locus (QTL) analysis allowed us to identify on linkage group 14 a major QTL controlling the resistance to downy mildew found in V. amurensis, which explained up to 86.3% of the total phenotypic variance. This QTL was named 'Resistance to Plasmopara viticola 8' (Rpv8).

Structural variability of Tvv1 grapevine retrotransposons can be caused by illegitimate recombination.

Structural variability of Tvv1, a grapevine retrotransposon Ty1 copia-like family, was investigated within the grape genome and the canonical sequence of Tvv1 determined. Then, two remarkable elements, Tvv1-Delta3001 and Tvv1-Delta3640, which had suffered large deletions 3,001 bp and 3,460 bp in length of their coding sequences were compared to the canonical copy. In both deleted elements, the deletion breakpoint was characterized by a stretch 13 bp-long in Tvv1-Delta3001 and 11 bp-long in Tvv1-Delta3640 found duplicated in the canonical copy at each bound of the deleted regions. Tvv1-Delta3001 and Tvv1-Delta3460 were both shown to be unique copies fixed at a single locus in the grapevine genome. Their presence was very variable in a set of 58 varieties and wild vines. These elements have most likely been dispersed through natural intermixing after their initial insertion whose chronology was estimated. The model that we propose to explain the structure of Tvv1-Delta3001 and Tvv1-Delta3640, implies illegitimate recombination involving template switching between two RNA molecules co-packaged in the VLP prior to the integration of the deleted daughter copy into the host genome.