Identification of interactions in the E1E2 heterodimer of hepatitis C virus important for cell entry

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.     

Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes

Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3' untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3' UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21.     

HIV-1 Nef promotes cell proliferation and microRNA dysregulation in lung cells

Non-small cell lung cancer (NSCLC) represents about 85% of all lung cancer cases. Lung cancer is the most frequent non-AIDS-defining malignancies in HIV-infected patients. The mechanism of the increased risk for lung cancer in HIV-1 patients is poorly understood. HIV-1 Nef protein has been suggested to be one of the key players in HIV-related lung disease. In here, we showed the involvement of Nef protein in cell modifications such as fibroblasts (IMR-90) and normal (BEAS-2B) or cancerous (A549) epithelial cells. We demonstrated that Nef protein reprograms initial stages of lung cancer (e.g. changes in the metabolism, improved cell survival and invasion, increase the angiogenesis factor VEGF). Additionally, we showed that Nef is provoking a global decrease of mature miRNA and a decrease of DICER1 and AGO expression in lung cells. MiRNAs play a crucial role in cell signaling and homeostasis, functioning as oncogenes or tumor suppressors, and their dysregulation can contribute to the tumorigenic process. These results showed that HIV-1 Nef protein is directly involved in preventing cell death and contributes to tumor progression.     

Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.Plants have evolved a complex array of defenses when attacked by microbial pathogens. The success of plant resistance first relies on the capacity of the plant to recognize its invader. Among early events, a transient production of reactive oxygen species (ROS), known as the oxidative burst, is characteristic of successful pathogen recognition (Torres, 2010). Perception of pathogen attack then initiates a large array of immune responses, including modification of cell walls, as well as the production of antimicrobial proteins and metabolites like pathogenesis-related (PR) proteins and phytoalexins, respectively (Schwessinger and Ronald, 2012). The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are key players in the signaling networks involved in plant resistance (Bari and Jones, 2009; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate array of defense responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). Whereas SA is considered essential for resistance to (hemi)biotrophic pathogens, it is assumed that JA and ET signaling pathways are important for resistance to necrotrophic pathogens in Arabidopsis (Arabidopsis thaliana; Thomma et al., 2001; Glazebrook, 2005). A successful innate immune response often includes the so-called hypersensitive response (HR), a form of rapid programmed cell death (PCD) occurring in a limited area at the site of infection. This suicide of infected cells is thought to limit the spread of biotrophic pathogens, including viruses, bacteria, fungi, and oomycetes (Mur et al., 2008).During the past decade, significant progress has been made in our understanding of the cellular function of plant sphingolipids. Besides being structural components of cell membranes, sphingolipids are bioactive metabolites that regulate important cellular processes such as cell survival and PCD, occurring during either plant development or plant defense (Dunn et al., 2004; Berkey et al., 2012; Markham et al., 2013). The first evidence of the role of sphingolipids in these processes came from the use of the fungal toxins fumonisin B1 (FB1) and AAL, produced by the necrotrophic agent Alternaria alternata f. sp. lycopersici. These toxins are structural sphingosine (d18:1) analogs and function as ceramide synthase inhibitors. They triggered PCD when exogenously applied to plants. Mutant strains in which the production of such toxins is abrogated failed to infect the host plant, implying that toxin accumulation is required for pathogenicity and that the induction of plant PCD could be considered a virulence tool used by necrotrophic pathogens (Berkey et al., 2012). Moreover, several studies revealed that ceramides (Cers) and long-chain bases (LCBs) are also potent inducers of PCD in plants. For example, exogenously applied Cers and LCBs (d18:0, d18:1, or t18:0) induced PCD either in cell suspension cultures (Liang et al., 2003; Lachaud et al., 2010, 2011; Alden et al., 2011) or in whole seedlings (Shi et al., 2007; Takahashi et al., 2009; Saucedo-García et al., 2011). AAL- and FB1-induced PCD seemed to be due to the accumulation of free sphingoid bases (dihydrosphingosine [d18:0] and phytosphingosine [t18:0]; Abbas et al., 1994; Brandwagt et al., 2000; Shi et al., 2007). Spontaneous cell death in lag one homolog1 or l-myoinositol1-phosphate synthase mutant could be due to trihydroxy-LCB and/or Cer accumulation (Donahue et al., 2010; Ternes et al., 2011). Deciphering of Cer participation in the induction of HR and associated PCD also came from studies on accelerated cell death5 (acd5) and enhancing resistance to powdery mildew8 (RPW8)-mediated hypersensitive response (erh1) mutants, which displayed overaccumulation of Cers. These mutants exhibited spontaneous cell death and resistance to biotrophic pathogens, which seemed to be linked with SA and PR protein accumulation (Liang et al., 2003; Wang et al., 2008).Altogether, these data provide evidence of a link between PCD, defense, and sphingolipid metabolism. However, the fatty acid hydroxylase1/2 (atfah1/atfah2) double mutant that accumulates SA and Cers was more tolerant to the obligate biotrophic fungus Golovinomyces cichoracearum but did not display a PCD-like phenotype, suggesting that Cers alone are not involved in the induction of PCD (König et al., 2012). Moreover, Saucedo-García et al. (2011) postulated that dihydroxy-LCBs, but not trihydroxy-LCBs, might be primary mediators for LCB-induced PCD. The sphingoid base hydroxylase sbh1/sbh2 double mutant completely lacking trihydroxy-LCBs showed enhanced expression of PCD marker genes (Chen et al., 2008). On the contrary, increase in t18:0 was specifically sustained in plant interaction with the avirulent Pseudomonas syringae pv tomato (Pst) strain and correlated with a strong PCD induction in leaves (Peer et al., 2010). Thus, the nature of sphingolipids able to induce PCD is still under debate and may evolve depending on plants and their environment. The phosphorylated form of LCBs (LCB-Ps) could abrogate PCD induced by LCBs, Cers, or heat stress in a dose-dependent manner (Shi et al., 2007; Alden et al., 2011). Furthermore, blocking the conversion of LCBs to LCB-Ps by using specific inhibitors induced PCD in cell suspension culture (Alden et al., 2011). Recently, overexpression of rice (Oryza sativa) LCB kinase in transgenic tobacco (Nicotiana tabacum) plants reduced PCD after treatment with FB1 (Zhang et al., 2013). Genetic mutation on LCB-P lyase encoded by the AtDPL1 gene, modifying the LCB-LCB-P ratio, could impact PCD levels after treatment with FB1 (Tsegaye et al., 2007). Altogether, these data point to the existence of a rheostat between LCBs and their phosphorylated forms that controls plant cell fate toward cell death or survival.Data on plant sphingolipid functions are still fragmentary. Only a few reports have described interconnections between sphingolipids, cell death, and plant defense responses, almost exclusively in response to (hemi)biotrophic pathogens. Knowledge about such relations in response to necrotrophic pathogens is still in its infancy (Rivas-San Vicente et al., 2013; Bi et al., 2014). In this report, the link between sphingolipids, cell death, and plant defense has been explored in response to Botrytis cinerea infection and in comparison with Pst infection. For this purpose, Atdpl1 mutant plants, disturbed in LCB/LCB-P accumulation without displaying any phenotype under standard growth conditions (Tsegaye et al., 2007), have been analyzed after pathogen infection. Our results revealed that modification of sphingolipid contents not only impacted plant tolerance to hemibiotrophs but also greatly affected resistance to necrotrophs. Whereas the SA signaling pathway is globally repressed in Atdpl1-1 compared with wild-type plants, the JA signaling pathway is significantly enhanced. Cell death and ROS accumulation are markedly modified in Atdpl1-1 mutant plants. We further demonstrated that phytosphingosine-1-phosphate (t18:0-P) and d18:0 are key players in pathogen-induced cell death and ROS generation. Here, we thus established a link between JA signaling, PCD, and sphingolipid metabolism.     

Caspases activation in hyperthermia-induced stimulation of TRAIL apoptosis

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.     

Diseases and reproductive success in a wild mammal: example in the alpine chamois

Density-dependent and climatic factors affect reproduction and dynamics of wild ungulates. Parasites can also decrease reproductive success through either a direct abortive effect or a negative impact on host growth and body condition. However, few studies have investigated the effect of parasitism on fecundity of ungulates in natural conditions. We studied three bacterial infections caused by Salmonella enterica serovar Abortusovis, Chlamydophila abortus and Coxiella burnetii. These bacteria are leading causes of reproductive failure in sheep, goat and cattle, which raises the question of their influence on population dynamics of wild ungulates. A long-term study of demography and epidemiology of an alpine chamois (Rupicapra rupicapra, L.) population (Les Bauges Reserve, France) and a generalized linear modeling approach were used to analyze the reproductive success of chamois according to population density, weather conditions and the prevalence of antibodies against the three bacteria in females. This approach enabled us to identify the confounding effect of weather and parasitism on fecundity in a natural population. After accounting for density, the prevalence of antibodies against the three bacteria explained 36% of the annual variation in reproductive success, and weather conditions explained an additional 31%. This study was, to our knowledge, the first to compare the decrease in fecundity due to bacterial infections and weather conditions in a population of wild mountain ungulates.