Glucosylceramide in bunyavirus particles is essential for virus binding to host cells.

Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.

Δ40p53 isoform up-regulates netrin-1/UNC5B expression and potentiates netrin-1 pro-oncogenic activity.

Netrin-1, a secreted protein recently characterized as a relevant cancer therapeutic target, is the antiapoptotic ligand of the dependence receptors deleted in colorectal carcinoma and members of the UNC5H family. Netrin-1 is overexpressed in several aggressive cancers where it promotes cancer progression by inhibiting cell death induced by its receptors. Interference of its binding to its receptors has been shown, through the development of a monoclonal neutralizing antinetrin-1 antibody (currently in phase II of clinical trial), to actively induce apoptosis and tumor growth inhibition. The transcription factor p53 was shown to positively regulate netrin-1 gene expression. We show here that netrin-1 could be a target gene of the N-terminal p53 isoform Δ40p53, independent of full-length p53 activity. Using stable cell lines, harboring wild-type or null-p53, in which Δ40p53 expression could be finely tuned, we prove that Δ40p53 binds to and activates the netrin-1 promoter. In addition, we show that forcing immortalized human skeletal myoblasts to produce the Δ40p53 isoform, instead of full-length p53, leads to the up-regulation of netrin-1 and its receptor UNC5B and promotes cell survival. Indeed, we demonstrate that netrin-1 interference, in the presence of Δ40p53, triggers apoptosis in cancer and primary cells, leading to tumor growth inhibition in preclinical in vivo models. Finally, we show a positive correlation between netrin-1 and Δ40p53 gene expression in human melanoma and colorectal cancer biopsies. Hence, we propose that inhibition of netrin-1 binding to its receptors should be a promising therapeutic strategy in human tumors expressing high levels of Δ40p53.

Infection of Mammals and Mosquitoes by Alphaviruses: Involvement of Cell Death.

Alphaviruses, such as the chikungunya virus, are emerging and re-emerging viruses that pose a global public health threat. They are transmitted by blood-feeding arthropods, mainly mosquitoes, to humans and animals. Although alphaviruses cause debilitating diseases in mammalian hosts, it appears that they have no pathological effect on the mosquito vector. Alphavirus/host interactions are increasingly studied at cellular and molecular levels. While it seems clear that apoptosis plays a key role in some human pathologies, the role of cell death in determining the outcome of infections in mosquitoes remains to be fully understood. Here, we review the current knowledge on alphavirus-induced regulated cell death in hosts and vectors and the possible role they play in determining tolerance or resistance of mosquitoes.

Activation of the mTOR/ Akt pathway in thymic epithelial cells derived from thymomas.

The pathogenesis of thymic epithelial tumors remains poorly elucidated. The PIK3/Akt/mTOR pathway plays a key role in various cancers; interestingly, several phase I/II studies have reported a positive effect of mTOR inhibitors in disease control in thymoma patients. A major limit for deciphering cellular and molecular events leading to the transformation of thymic epithelial cells or for testing drug candidates is the lack of reliable in vitro cell system. We analyzed protein expression and activation of key players of the Akt/ mTOR pathway namely Akt, mTOR, and P70S6K in eleven A, B and AB thymomas as well as in normal thymuses. While only Akt and phospho-Akt were expressed in normal thymuses, both Akt and mTOR were activated in thymomas. Phospho-P70S6K was expressed in all thymic tumors whatever their subtypes, and absent in normal thymus. Interestingly, we report the activation of Akt, mTOR and P70S6 proteins in primary thymic epithelial cells maintained for short period of time after their derivation from seven AB and B thymomas. Finally, we showed that rapamycin (100 nM) significantly reduced proliferation of thymoma- derived epithelial cells without inducing cell death. Our results suggest that the activation of the Akt/ mTOR pathway might participate to the cell proliferation associated with tumor growth. Ultimately, our data enhance the potential role of thymic epithelial cells derived from tissue specimens for in vitro exploration of molecular abnormalities in rare thymic tumors.

Detection of chikungunya virus-specific IgM on laser-cut paper-based device using pseudo-particles as capture antigen.

The incidence of arbovirus infections has increased dramatically in recent decades, affecting hundreds of millions of people each year. The Togaviridae family includes the chikungunya virus (CHIKV), which is typically transmitted by Aedes mosquitoes and causes a wide range of symptoms from flu-like fever to severe arthralgia. Although conventional diagnostic tests can provide early diagnosis of CHIKV infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop efficient, affordable, simple, rapid, and robust diagnostic tools that can be used in point-of-care settings. Early diagnosis is crucial to improve patient management and to reduce the risk of complications. A glass-fiber laser-cut microfluidic device (paper-based analytical device [PAD]) was designed and evaluated in a proof of principle context, for the analysis of 30 µL of patient serum. Biological raw materials used for the functionalization of the PAD were first screened by MAC-ELISA (IgM capture enzyme-linked immunosorbent assay) for CHIKV Immunoglobulin M (IgM) capture and then evaluated on the PAD using various human samples. Compared with viral lysate traditionally used for chikungunya (CHIK) serology, CHIKV pseudo-particles (PPs) have proven to be powerful antigens for specific IgM capture. The PAD was able to detect CHIKV IgM in human sera in less than 10 minutes. Results obtained in patient sera showed a sensitivity of 70.6% and a specificity of around 98%. The PAD showed few cross-reactions with other tropical viral diseases. The PAD could help health workers in the early diagnosis of tropical diseases such as CHIK, which require specific management protocols in at-risk populations.

A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism.

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.

Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients.

UNLABELLED: Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. IMPORTANCE: Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B lymphocytes that conferred to a nonlymphotropic HCV the ability to enter B cells, thus providing a platform for characterization of HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell-derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and nonhepatotropic HCV populations may be important for effective treatment of chronically infected patients.

Chemoresistance of human monocyte-derived dendritic cells is regulated by IL-17A.

Dendritic cells initiate adaptive immune responses, leading either to control cancer by effector T cells or to exacerbate cancer by regulatory T cells that inhibit IFN-γ-mediated Th1-type response. Dendritic cells can also induce Th17-type immunity, mediated by IL-17A. However, the controversial role of this cytokine in cancer requires further investigations. We generated dendritic cells from peripheral blood monocytes to investigate lifespan, phenotype and chemoresistance of dendritic cells, treated with IL-17A with or without IFN-γ. Studying the expression of Bcl-2 family members, we demonstrated that dendritic cells constitutively express one pro-survival Bcl-2 member: MCL1. Immature dendritic cells were CD40(low)HLADR(low) CD1a(+) MCL1(+), did not express CD14, CD68 or BCL2A1, and displayed a short 2-day lifespan. IL-17A-treated DC exhibited a semi-mature (CD40(high) HLADR(low)) pre-M2 (CCL22(+) CD206(+) CD163(+) IL1RN(+) IL-10(-) CXCL10(-) IL-12(-)) mixed (CD1a(+) CD14+ CD68(+)) macrophage-dendritic cell phenotype. They efficiently exerted mannose receptor-mediated endocytosis and did not produce superoxide anions, in the absence of TLR engagement. Interestingly, IL-17A promoted a long-term survival of dendritic cells, beyond 12 days, that correlated to BCL2A1 induction, a pro-survival Bcl-2 family member. BCL2A1 transcription was activated by NF-κB, downstream of IL-17A transduction. Thus, immature dendritic cells only express MCL1, whereas IL-17A-treated dendritic cells concomitantly expressed two pro-survival Bcl-2 family members: MCL1 and BCL2A1. These latter developed chemoresistance to 11 of the 17 chemotherapy agents tested. However, high doses of either vinblastine or cytarabine decreased MCL1 expression and induced dendritic cell death. When IL-17A is produced in vivo, administration of anti-IL-17A biotherapy may impair dendritic cell survival by targeting BCL2A1 expression. Consequently, depending on the effector or regulatory role of dendritic cells, blocking IL-17A, may be either dangerous or beneficial for cancer outcomes, thus contributing to the apparent controversy around the role of IL-17A in cancer.

Netrin-1 up-regulation in inflammatory bowel diseases is required for colorectal cancer progression.

Chronic inflammation and cancer are intimately associated. This is particularly true for inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease, which show a major increased risk for colorectal cancer. While the understanding of the molecular pathogenesis of IBD has recently improved, the mechanisms that link these chronic inflammatory states to colorectal cancer development are in large part unknown. One of these mechanisms is NF-kappaB pathway activation which in turn may contribute to tumor formation by providing anti-apoptotic survival signals to the epithelial cells. Based on the observation that netrin-1, the anti-apoptotic ligand for the dependence receptors DCC and UNC5H is up-regulated in colonic crypts in response to NF-kappaB, we show here that colorectal cancers from inflammatory bowel diseases patients have selected up-regulation of netrin-1. Moreover, we demonstrate that this inflammation-driven netrin-1 up-regulation is causal for colorectal cancer development as interference with netrin-1 autocrine loop in a mouse model for ulcerative colitis-associated colorectal cancer, while showing no effect on inflammation, inhibits colorectal cancer progression.

NF-kappaB regulates netrin-1 expression and affects the conditional tumor suppressive activity of the netrin-1 receptors.

BACKGROUND & AIMS: Netrin-1 was recently proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. Because netrin-1 receptors belong to the family of dependence receptors, a selective advantage for a tumor is either to lose netrin-1 receptors or to gain autocrine expression of netrin-1. We have investigated whether netrin-1 is up-regulated in colorectal cancer and have searched for a link between NF-kappaB activation and netrin-1 up-regulation. METHODS: The level of netrin-1, netrin-1 receptors, ie, DCC, UNC5H1, UNC5H2, UNC5H3, and the proinflammatory markers cyclooxygenase-2 and inhibitor of nuclear factor-kappaB (IkappaB) alpha were analyzed in a panel of 59 primary sporadic colorectal carcinomas. Netrin-1 expression was investigated in tumor cells and in mouse colonic crypts in response to NF-kappaB activation but also in a mouse model of inflammation-induced colorectal cancer. Binding of NF-kappaB to netrin-1 promoter and effect of NF-kappaB activation to the proapoptotic activity of UNC5H2 were also analyzed. RESULTS: We show that colorectal tumors with a gain of netrin-1 are tumors that display increased activation of the NF-kappaB pathway. Moreover, netrin-1 up-regulation, which is associated with tumor formation in mice, is observed in mouse colonic crypts in response to NF-kappaB activation but also in a mouse model of inflammation-induced colorectal cancer. We demonstrate that the netrin-1 gene is a direct transcriptional target of NF-kappaB. We show that NF-kappaB-induced netrin-1 expression inhibits proapoptotic activity of the netrin-1 receptors. CONCLUSIONS: We propose that NF-kappaB activation that occurs in response to inflammation confers a selective advantage for tumor development through NF-kappaB-mediated netrin-1 up-regulation.

Lipid raft localization and palmitoylation: identification of two requirements for cell death induction by the tumor suppressors UNC5H.

UNC5H receptors (UNC5H1, UNC5H2, UNC5H3) are putative tumor suppressors whose expression is lost in numerous cancers. These receptors have been shown to belong to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand netrin-1 is absent, thus conferring a state of cellular dependence towards ligand presence. Along this line, these receptors may limit tumor progression because they induce the death of tumor cells that grow in settings of ligand unavailability. We show here that UNC5H receptors are localized to cholesterol-and sphingolipid-enriched membrane domains called lipid rafts. We then demonstrate that the lipid raft localization of UNC5H2 is required for the pro-apoptotic activity of unbound UNC5H2. We also propose that this lipid raft localization is probably mediated via the recruitment of adaptor protein(s) within the death domain of UNC5H2 but is not dependent on the post-translational modification by palmitoylation of UNC5H2 even though this palmitoylation is required for UNC5H2 pro-apoptotic activity. Moreover we show that the interaction of UNC5H2 with the downstream pro-apoptotic serine threonine kinase DAPk is dependent on both UNC5H2 lipid raft localization and palmitoylation. Thus, we propose that the UNC5H dependence receptors require lipid raft localization and palmitoylation to trigger apoptosis.

PIAS-1 is a checkpoint regulator which affects exit from G1 and G2 by sumoylation of p73.

p73 is a recently described member of the p53 family, and, like p53, it undergoes a number of posttranslational modifications. Here we show, by yeast two-hybrid screening, pull-down assays, and coimmunoprecipitation, that p73alpha, -beta, and -gamma bind to the protein inhibitor of activated STAT-1 (PIAS-1) and that this binding stabilizes p73. PIAS-1 also sumoylates p73alpha, although not the C-terminally truncated isoforms p73beta and -gamma, and this requires the RING finger domain of PIAS-1. The DeltaNp73alpha isoform can also bind, and be sumoylated by, PIAS-1. PIAS-1-mediated sumoylation decreases p73 transcriptional activity on several target promoters, such as Bax. p73 is colocalized in the nucleus with PIAS-1, and sumoylated p73 is located exclusively in the nuclear matrix. PIAS-1 is expressed predominantly during S phase, and PIAS-1 overexpression reduces p73-mediated transcription of p21, with a reduction of cells in G(1) and cell cycle reentry. Inhibition of endogenous PIAS-1 by RNA interference reduces the proportion of cells in S phase and induces G(2) arrest. These data suggest that PIAS-1, acting partly through binding and sumoylation of p73, is an important component of the cell cycle machinery.

p73 and p63 protein stability: the way to regulate function?

While the p53 homologue p73 has been found to be involved in tumorigenesis, the molecular mechanisms involved in this function are still not fully evident. The presence of two distinct promoters allows the formation of two proteins with opposite effects: while TA-p73 shows pro-apoptotic effects, DeltaN-p73 has an evident anti-apoptotic function. The relative expression of the two proteins is in fact related to the prognosis of several cancers. Since both p73 and p63, the other member of the same family, share the ability to interact with each other, it is important to understand the mechanisms that control the degradation and stability of both proteins, and their relative isoforms. p73 and p63 stability is regulated not only by protein modifications (phosphorylation, acetylation) but also by its degradation in the proteasome. To this end, the interaction with Mdm2, p300/CBP, and SUMO-1 are discussed in details.