The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Masculinization of the X Chromosome in the Pea Aphid

Evolutionary theory predicts that sexually antagonistic mutations accumulate differentially on the X chromosome and autosomes in species with an XY sex-determination system, with effects (masculinization or feminization of the X) depending on the dominance of mutations. Organisms with alternative modes of inheritance of sex chromosomes offer interesting opportunities for studying sexual conflicts and their resolution, because expectations for the preferred genomic location of sexually antagonistic alleles may differ from standard systems. Aphids display an XX/X0 system and combine an unusual inheritance of the X chromosome with the alternation of sexual and asexual reproduction. In this study, we first investigated theoretically the accumulation of sexually antagonistic mutations on the aphid X chromosome. Our results show that i) the X is always more favourable to the spread of male-beneficial alleles than autosomes, and should thus be enriched in sexually antagonistic alleles beneficial for males, ii) sexually antagonistic mutations beneficial for asexual females accumulate preferentially on autosomes, iii) in contrast to predictions for standard systems, these qualitative results are not affected by the dominance of mutations. Under the assumption that sex-biased gene expression evolves to solve conflicts raised by the spread of sexually antagonistic alleles, one expects that male-biased genes should be enriched on the X while asexual female-biased genes should be enriched on autosomes. Using gene expression data (RNA-Seq) in males, sexual females and asexual females of the pea aphid, we confirm these theoretical predictions. Although other mechanisms than the resolution of sexual antagonism may lead to sex-biased gene expression, we argue that they could hardly explain the observed difference between X and autosomes. On top of reporting a strong masculinization of the aphid X chromosome, our study highlights the relevance of organisms displaying an alternative mode of sex chromosome inheritance to understanding the forces shaping chromosome evolution.     

Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4⁺ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4⁺ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.     

Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria

Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.     

Metabolomics identifies a biological response to chronic low-dose natural uranium contamination in urine samples

Because uranium is a natural element present in the earth’s crust, the population may be chronically exposed to low doses of it through drinking water. Additionally, the military and civil uses of uranium can also lead to environmental dispersion that can result in high or low doses of acute or chronic exposure. Recent experimental data suggest this might lead to relatively innocuous biological reactions. The aim of this study was to assess the biological changes in rats caused by ingestion of natural uranium in drinking water with a mean daily intake of 2.7 mg/kg for 9 months and to identify potential biomarkers related to such a contamination. Subsequently, we observed no pathology and standard clinical tests were unable to distinguish between treated and untreated animals. Conversely, LC–MS metabolomics identified urine as an appropriate biofluid for discriminating the experimental groups. Of the 1,376 features detected in urine, the most discriminant were metabolites involved in tryptophan, nicotinate, and nicotinamide metabolic pathways. In particular, N-methylnicotinamide, which was found at a level seven times higher in untreated than in contaminated rats, had the greatest discriminating power. These novel results establish a proof of principle for using metabolomics to address chronic low-dose uranium contamination. They open interesting perspectives for understanding the underlying biological mechanisms and designing a diagnostic test of exposure.     

The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.     

Dynamics of Intrinsic Dendritic Calcium Signaling during Tonic Firing of Thalamic Reticular Neurons

The GABAergic neurons of the nucleus reticularis thalami that control the communication between thalamus and cortex are interconnected not only through axo-dendritic synapses but also through gap junctions and dendro-dendritic synapses. It is still unknown whether these dendritic communication processes may be triggered both by the tonic and the T-type Ca²⁺ channel-dependent high frequency burst firing of action potentials displayed by nucleus reticularis neurons during wakefulness and sleep, respectively. Indeed, while it is known that activation of T-type Ca²⁺ channels actively propagates throughout the dendritic tree, it is still unclear whether tonic action potential firing can also invade the dendritic arborization. Here, using two-photon microscopy, we demonstrated that dendritic Ca²⁺ responses following somatically evoked action potentials that mimic wake-related tonic firing are detected throughout the dendritic arborization. Calcium influx temporally summates to produce dendritic Ca²⁺ accumulations that are linearly related to the duration of the action potential trains. Increasing the firing frequency facilitates Ca²⁺ influx in the proximal but not in the distal dendritic compartments suggesting that the dendritic arborization acts as a low-pass filter in respect to the back-propagating action potentials. In the more distal compartment of the dendritic tree, T-type Ca²⁺ channels play a crucial role in the action potential triggered Ca²⁺ influx suggesting that this Ca²⁺ influx may be controlled by slight changes in the local dendritic membrane potential that determine the T-type channels’ availability. We conclude that by mediating Ca²⁺ dynamic in the whole dendritic arborization, both tonic and burst firing of the nucleus reticularis thalami neurons might control their dendro-dendritic and electrical communications.     

Adenosine-Mediated Enteric Neuromuscular Function Is Affected during Herpes Simplex Virus Type 1 Infection of Rat Enteric Nervous System

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A₁ or A_2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A₁ and A_2B receptors were confined to the muscle layers whereas A_2A and A₃ receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.