An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus

The genome of influenza A viruses (IAV) is split into eight viral RNAs (vRNAs) that are encapsidated as viral ribonucleoproteins. The existence of a segment-specific packaging mechanism is well established, but the molecular basis of this mechanism remains to be deciphered. Selective packaging could be mediated by direct interaction between the vRNA packaging regions, but such interactions have never been demonstrated in virions. Recently, we showed that the eight vRNAs of a human H3N2 IAV form a single interaction network in vitro that involves regions of the vRNAs known to contain packaging signals in the case of H1N1 IAV strains. Here, we show that the eight vRNAs of an avian H5N2 IAV also form a single network of interactions in vitro, but, interestingly, the interactions and the regions of the vRNAs they involve differ from those described for the human H3N2 virus. We identified the vRNA sequences involved in five of these interactions at the nucleotide level, and in two cases, we validated the existence of the interaction using compensatory mutations in the interacting sequences. Electron tomography also revealed significant differences in the interactions taking place between viral ribonucleoproteins in H5N2 and H3N2 virions, despite their canonical ‘7 + 1’ arrangement.     

Does variation in host plant association and symbiont infection of pea aphid populations induce genetic and behaviour differentiation of its main parasitoid, Aphidius ervi?

The host-associated differentiation (HAD) hypothesis states that higher trophic levels in parasitic associations should exhibit similar divergence in case of host sympatric speciation. We tested HAD on populations of Aphidius ervi the main parasitoid of the pea aphid Acyrthosiphon pisum, emerging from host populations specialized on either alfalfa or red clover. Host and parasitoid populations were assessed for genetic variation and structure, while considering geography, host plant and host aphid protective symbionts Regiella insecticola and Hamiltonella defensa as potential covariables. Cluster and hierarchical analyses were used to assess the contribution of these variables to population structure, based on genotyping pea aphids and associated A. ervi with microsatellites, and host aphid facultative symbionts with 16S rDNA markers. Pea aphid genotypes were clearly distributed in two groups closely corresponding with their plant origins, confirming strong plant associated differentiation of this aphid in North America. Overall parasitism by A. ervi averaged 21.5 % across samples, and many parasitized aphids producing a wasp hosted defensive bacteria, indicating partial or ineffective protective efficacy of these symbionts in the field. The A. ervi population genetic data failed to support differentiation according to the host plant association of their pea aphid host. Potential for parasitoid specialization was also explored in experiments where wasps from alfalfa and clover aphids were reciprocally transplanted on alternate hosts, the hypothesis being that wasp behaviour and parasitic stages should be most adapted to their host of origin. Results revealed higher probability of oviposition on the alfalfa aphids, but higher adult emergence success on red clover aphids, with no interaction as expected under HAD. We conclude that our study provides no support for the HAD in this system. We discuss factors that might impair A. ervi specialization on its divergent aphid hosts on alfalfa and clover.     

Inhibition of ATR kinase with the selective inhibitor VE-821 results in radiosensitization of cells of promyelocytic leukaemia (HL-60)

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 μM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 μM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 μM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 μM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.     

Conformations Consistent with Charge Migration Observed in DNA and RNA X-ray Structures

Abstract

Electron holes are known to migrate along the DNA or RNA duplexes and to localize preferentially on successive guanines. The stationary point conformations of Gua pairs that can trap or let pass these holes have been characterized by quantum chemistry calculations. Here we show their recurrent occurrence in DNA and RNA X-ray structures, often in quadruplex conformations or in interaction with proteins, ligands or metal ions. These findings give support to the biological, possibly regulatory, roles of charge migration in cell functioning.     

Can plant sugars mediate the effect of nitrogen fertilization on lettuce susceptibility to two necrotrophic pathogens: Botrytis cinerea and Sclerotinia sclerotiorum?

Aims

Nitrogen (N) fertilization is known to modify a plant’s susceptibility to necrotrophic diseases. However, the effect of N nutrition on defence is not well known. It was hypothesized that not only molecules related to the N metabolism but also main sugars could mediate the effect of plant fertilization on its susceptibility to pathogens.

Methods

Two necrotrophic fungi, Botrytis cinerea and Sclerotinia sclerotiorum were inoculated on leaves of lettuce plants grown at five nitrate (NO₃ ^?) fertilization levels, in three independent experiments. Variations in plant composition at the time of inoculation were linked to the size of lesions observed after 5–6 days.

Results

Both diseases were favoured by high NO₃ ^? fertilization. However, the highest disease levels were not found in the same experiment for B. cinerea and S. sclerotiorum. Among the components measured, NO₃ ^? and sucrose (SUC) were positively and negatively correlated to the two diseases in the three experiments, but the relationship between SUC and lesion size was more significant for S. sclerotiorum. Water content, N and total carbon (C) were also significantly correlated to the diseases, but the relationships were less straightforward. The ratios of SUC over total sugars and fructose (FRU) over total sugars fitted, very closely for S. sclerotiorum, a negative and positive exponential relationship respectively with lesion size. Absolute or relative glucose levels were not linked to the diseases.

Conclusions

Plant metabolic modifications induced by variations of N availability conferred the plant variable defence ability, which seemed, at least for S. sclerotiorum, mainly mediated by variations in host SUC and FRU levels. The generalization of these findings to other species would be of interest.     

The Amidohydrolases IAR3 and ILL6 Contribute to Jasmonoyl-Isoleucine Hormone Turnover and Generate 12-Hydroxyjasmonic Acid Upon Wounding in Arabidopsis Leaves

Jasmonates (JAs) are a class of signaling compounds that mediate complex developmental and adaptative responses in plants. JAs derive from jasmonic acid (JA) through various enzymatic modifications, including conjugation to amino acids or oxidation, yielding an array of derivatives. The main hormonal signal, jasmonoyl-l-isoleucine (JA-Ile), has been found recently to undergo catabolic inactivation by cytochrome P450-mediated oxidation. We characterize here two amidohydrolases, IAR3 and ILL6, that define a second pathway for JA-Ile turnover during the wound response in Arabidopsis leaves. Biochemical and genetic evidence indicates that these two enzymes cleave the JA-Ile signal, but act also on the 12OH-JA-Ile conjugate. We also show that unexpectedly, the abundant accumulation of tuberonic acid (12OH-JA) after wounding originates partly through a sequential pathway involving (i) conjugation of JA to Ile, (ii) oxidation of the JA-Ile conjugate, and (iii) cleavage under the action of the amidohydrolases. The coordinated actions of oxidative and hydrolytic branches in the jasmonate pathway highlight novel mechanisms of JA-Ile hormone turnover and redefine the dynamic metabolic grid of jasmonate conversion in the wound response.     

Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes

Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2–1.6 μm size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 10⁴–10⁵ genomes ml⁻¹ for the samples from the photic zone and 10²–10³ genomes ml⁻¹ for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts.     

Combined MRI and 31P-MRS Investigations of the ACTA1(H40Y) Mouse Model of Nemaline Myopathy Show Impaired Muscle Function and Altered Energy Metabolism

Nemaline myopathy (NM) is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. Mutations in the skeletal muscle α-actin gene (ACTA1) account for ∼25% of all NM cases and are the most frequent cause of severe forms of NM. So far, the mechanisms underlying muscle weakness in NM patients remain unclear. Additionally, recent Magnetic Resonance Imaging (MRI) studies reported a progressive fatty infiltration of skeletal muscle with a specific muscle involvement in patients with ACTA1 mutations. We investigated strictly noninvasively the gastrocnemius muscle function of a mouse model carrying a mutation in the ACTA1 gene (H40Y). Skeletal muscle anatomy (hindlimb muscles and fat volumes) and energy metabolism were studied using MRI and ³¹Phosphorus magnetic resonance spectroscopy. Skeletal muscle contractile performance was investigated while applying a force-frequency protocol (from 1–150 Hz) and a fatigue protocol (80 stimuli at 40 Hz). H40Y mice showed a reduction of both absolute (−40%) and specific (−25%) maximal force production as compared to controls. Interestingly, muscle weakness was associated with an improved resistance to fatigue (+40%) and an increased energy cost. On the contrary, the force frequency relationship was not modified in H40Y mice and the extent of fatty infiltration was minor and not different from the WT group. We concluded that the H40Y mouse model does not reproduce human MRI findings but shows a severe muscle weakness which might be related to an alteration of intrinsic muscular properties. The increased energy cost in H40Y mice might be related to either an impaired mitochondrial function or an alteration at the cross-bridges level. Overall, we provided a unique set of anatomic, metabolic and functional biomarkers that might be relevant for monitoring the progression of NM disease but also for assessing the efficacy of potential therapeutic interventions at a preclinical level.