Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2_∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2_∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2_∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.     

Short-term changes in polysaccharide utilization mechanisms of marine bacterioplankton during a spring phytoplankton bloom

Spring phytoplankton blooms in temperate environments contribute disproportionately to global marine productivity. Bloom-derived organic matter, much of it occurring as polysaccharides, fuels biogeochemical cycles driven by interacting autotrophic and heterotrophic communities. We tracked changes in the mode of polysaccharide utilization by heterotrophic bacteria during the course of a diatom-dominated bloom in the German Bight, North Sea. Polysaccharides can be taken up in a ‘selfish’ mode, where initial hydrolysis is coupled to transport into the periplasm, such that little to no low-molecular weight (LMW) products are externally released to the environment. Alternatively, polysaccharides hydrolyzed by cell-surface attached or free extracellular enzymes (external hydrolysis) yield LMW products available to the wider bacterioplankton community. In the early bloom phase, selfish activity was accompanied by low extracellular hydrolysis rates of a few polysaccharides. As the bloom progressed, selfish uptake increased markedly, and external hydrolysis rates increased, but only for a limited range of substrates. The late bloom phase was characterized by high external hydrolysis rates of a broad range of polysaccharides and reduced selfish uptake of polysaccharides, except for laminarin. Substrate utilization mode is related both to substrate structural complexity and to the bloom-stage dependent composition of the heterotrophic bacterial community.     

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.     

Partitioning net carbon dioxide fluxes into photosynthesis and respiration using neural networks

The eddy covariance (EC) technique is used to measure the net ecosystem exchange (NEE) of CO₂ between ecosystems and the atmosphere, offering a unique opportunity to study ecosystem responses to climate change. NEE is the difference between the total CO₂ release due to all respiration processes (RECO), and the gross carbon uptake by photosynthesis (GPP). These two gross CO₂ fluxes are derived from EC measurements by applying partitioning methods that rely on physiologically based functional relationships with a limited number of environmental drivers. However, the partitioning methods applied in the global FLUXNET network of EC observations do not account for the multiple co‐acting factors that modulate GPP and RECO flux dynamics. To overcome this limitation, we developed a hybrid data‐driven approach based on combined neural networks (NN_C‐part). NN_C‐part incorporates process knowledge by introducing a photosynthetic response based on the light‐use efficiency (LUE) concept, and uses a comprehensive dataset of soil and micrometeorological variables as fluxes drivers. We applied the method to 36 sites from the FLUXNET2015 dataset and found a high consistency in the results with those derived from other standard partitioning methods for both GPP (R² > .94) and RECO (R² > .8). High consistency was also found for (a) the diurnal and seasonal patterns of fluxes and (b) the ecosystem functional responses. NN_C‐part performed more realistic than the traditional methods for predicting additional patterns of gross CO₂ fluxes, such as: (a) the GPP response to VPD, (b) direct effects of air temperature on GPP dynamics, (c) hysteresis in the diel cycle of gross CO₂ fluxes, (d) the sensitivity of LUE to the diffuse to direct radiation ratio, and (e) the post rain respiration pulse after a long dry period. In conclusion, NN_C‐part is a valid data‐driven approach to provide GPP and RECO estimates and complementary to the existing partitioning methods.     

The KDEL receptor: New functions for an old protein

The KDEL receptor is a seven-transmembrane-domain protein that was first described about 20 years ago. Its well-known function is to retrotransport chaperones from the Golgi complex to the endoplasmic reticulum. Recent studies, however, have suggested that the KDEL receptor has additional functions. Indeed, we have demonstrated that chaperone-bound KDEL receptor triggers the activation of Src family kinases on the Golgi complex. This activity is essential in the regulation of Golgi-to-plasma membrane transport. However, the identification of different KDEL receptor interactors that are inconsistent with these established functions opens the possibility of further receptor activities.     

Gem1 and ERMES do not directly affect phosphatidylserine transport from ER to mitochondria or mitochondrial inheritance

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.     

Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion

NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.     

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics.