Caspase inhibition activates HIV in latently infected cells. Role of tumor necrosis factor receptor 1 and CD95

Stimulation of tumor necrosis factor receptor 1 (TNF-R1) triggers both caspase-dependent and caspase-independent signaling activities. The caspase-dependent signaling pathway induces apoptotic cell death in susceptible cells, whereas the caspase-independent signaling cascade leads to activation of nuclear factor kappa B and induces antiapoptotic signaling activities. Stimulation of nuclear factor kappa B via TNF-R1 is known to activate human immunodeficiency virus (HIV) replication in infected cells. Here we show that the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD) activates HIV replication in the chronically infected T-cell line ACH-2. Virus activation was caused by a sensitization of TNF-R1 toward endogenously produced tumor necrosis factor alpha (TNF-alpha). Neutralizing anti-TNF-alpha antibodies completely abolished the virus-inducing activity of ZVAD. Treatment of cells with TNF-alpha in the presence of ZVAD caused increased expression of TNF-alpha and induced enhanced virus replication. Activation of CD95, another member of the TNF receptor family, similarly triggered HIV replication, which was further enhanced in the presence of ZVAD. Our data show that caspase inhibitors sensitize both CD95 and TNF-R1 to mediate activation of HIV in latently infected cells. Activation of HIV replication in latent virus reservoirs is currently discussed as a therapeutic strategy to achieve eradication of HIV in patients treated with antiretroviral therapy. Our results point to a novel role for caspase inhibitors as activators of virus replication in vivo.     

Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization

Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schr?der, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., H?llt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.     

Cnidarians as a model system for understanding evolution and regeneration

Hydra and Podocolyne are two cnidarian animals which provide complementary advantages for analysing developmental mechanisms possibly reflecting the basic developmental processes shared by most bilaterians. Interestingly, these mechanisms remain accessible all along the life of these animals, which bud and regenerate, whatever their age. The Hydra polyp permits a direct study of the molecular cascades linking amputation to regeneration. Podocoryne displays a complete life cycle, polyp and medusa stages with a fast and inducible sexual cycle and an unparalleled In vitro transdifferentiation potential. In both cases, a large number of evolutionarily conserved molecular markers are available, and analysis of their regulation highlights the molecular mechanisms which underly pattern formation in these two species.     

Evaluation in rhesus macaques of Tat and rev-targeted immunization as a preventive vaccine against mucosal challenge with SHIV-BX08

Recent evidence suggests that a CD8-mediated cytotoxic T-cell response against the regulatory proteins of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) may control infection after pathogenic virus challenge. Here, we evaluated whether vaccination with Tat or Tat and Rev could significantly reduce viral load in nonhuman primates. Rhesus macaques were primed with Semliki forest Virus (SFV) expressing HIV-1 tat (SFV-tat) and HIV-1 rev (SFV-rev) and boosted with modified vaccinia virus Ankara (MVA) expressing tat and rev. A second group of monkey was primed with SFV-tat only and boosted with MVA-tat. A third group received a tat and rev DNA/MVA prime-boost vaccine regimen. Monitoring of anti-Tat and anti-Rev antibody responses or antigen-specific IFN-gamma production, as measured by enzyme-linked immunospot assays revealed no clear differences between the three groups. These results suggest that priming with either DNA or SFV seemed to be equivalent, but the additive or synergistic effect of a rev vaccine could not be clearly established. The animals were challenged by the rectal route 9 weeks after the last booster immunization, using 10 MID(50) of a SHIV-BX08 stock. Postchallenge follow-up of the monkeys included testing seroconversion to Gag and Env antigens, measuring virus infectivity in PBMC by cocultivation with noninfected human cells, and monitoring of plasma viral load. None of the animals was protected from infection as assessed by PCR, but peak viremia was reduced more than 200-fold compared to sham controls in one third (6/18) of vaccinated macaques, whatever the vaccine regimen they received. Interestingly, among these six protected animals four did not seroconvert. Altogether, these results clearly indicated that the addition of early HIV proteins like Tat and Rev in a multicomponent preventive vaccine including structural proteins like Env or Gag may be beneficial in preventive vaccinal strategies.     

Embryonic development in the primitive bilaterian Neochildia fusca: normal morphogenesis and isolation of POU genes Brn-1 and Brn-3

Neochildia fusca is a member of the taxon Acoela, a group of flatworms that, according to some recent molecular phylogenetic analyses, are distinct from other flatworms and constitute a basal branch with a sister taxon relationship to the rest of the Bilateria. In this paper, we analyze early neural development in this species and report the sequence and expression of two Pit-Oct-Unc (POU) genes, NeocBrn-1 and NeocBrn-3. Homologs of these highly conserved genes play a role in neural fate determination in vertebrates, Drosophila and Caenorhabditis elegans. Acoels, including Neochildia, have a unique invariant pattern of early cleavage called duet spiral cleavage. In subsequent cell divisions descendants of the first three micromere duets form an outer layer of epidermal and neural progenitors surrounding the meso/endoderm progenitors, which are themselves descended from the macromere duet 4A, B and the micromere duet 4a, b. Organ formation begins at mid-embryonic stages with the epidermal primordium adopting a ciliated epithelial shape. Sub-epidermally, a bilaterally symmetric brain primordium can be seen at the anterior pole. Laterally and posteriorly, myoblasts form a thin layer underneath the epidermis. In late embryos and juveniles of Neochildia, the brain is formed by a 3-4 cell-diameter-thick layer of neurons forming a cortex surrounding a neuropile that is relatively free of cell bodies. A highly regular "orthogonal" array of muscle fibers penetrates the brain. We have isolated and partially sequenced homologs of the vertebrate Brn-1 and Brn-3 genes, which we call NeocBrn-1 and NeocBrn-3, respectively. These sequences contain and span portions of the POU-specific domain and a homeodomain, and are sequence similar to their respective homologs in vertebrates and Drosophila. RT-PCR reveals that NeocBrn-1 and NeocBrn-3 are expressed from mid-embryonic to adult stages. Whole-mount in situ hybridization shows expression of both genes in distinct subsets of nerve cells in juvenile and adult worms. NeocBrn-1 also appears in a subset of intra-epidermal gland cells. These observations are an initial step towards reconstructing the neural development of a key group of bilaterians, the Acoela. These flatworms, by virtue of their distinct morphology, development and phylogenetically basal placement, are likely to provide key insights into the interpretation of the evolution of metazoan neural architecture.     

Interaction between EGFR signaling and DE-cadherin during nervous system morphogenesis

Dynamically regulated cell adhesion plays an important role during animal morphogenesis. Here we use the formation of the visual system in Drosophila embryos as a model system to investigate the function of the Drosophila classic cadherin, DE-cadherin, which is encoded by the shotgun (shg) gene. The visual system is derived from the optic placode which normally invaginates from the surface ectoderm of the embryo and gives rise to two separate structures, the larval eye (Bolwig's organ) and the optic lobe. The optic placode dissociates and undergoes apoptotic cell death in the absence of DE-cadherin, whereas overexpression of DE-cadherin results in the failure of optic placode cells to invaginate and of Bolwig's organ precursors to separate from the placode. These findings indicate that dynamically regulated levels of DE-cadherin are essential for normal optic placode development. It was shown previously that overexpression of DE-cadherin can disrupt Wingless signaling through titration of Armadillo out of the cytoplasm to the membrane. However, the observed defects are likely the consequence of altered DE-cadherin mediated adhesion rather than a result of compromising Wingless signaling, as overexpression of a DE-cadherin-alpha-catenin fusion protein, which lacks Armadillo binding sites, causes similar defects as DE-cadherin overexpression. We further studied the genetic interaction between DE-cadherin and the Drosophila EGF receptor homolog, EGFR. If EGFR function is eliminated, optic placode defects resemble those following DE-cadherin overexpression, which suggests that loss of EGFR results in an increased adhesion of optic placode cells. An interaction between EGFR and DE-cadherin is further supported by the finding that expression of a constitutively active EGFR enhances the phenotype of a weak shg mutation, whereas a mutation in rhomboid (rho) (an activator of the EGFR ligand Spitz) partially suppresses the shg mutant phenotype. Finally, EGFR can be co-immunoprecipitated with anti-DE-cadherin and anti-Armadillo antibodies from embryonic protein extracts. We propose that EGFR signaling plays a role in morphogenesis by modulating cell adhesion.     

Decreased Rubisco activity leads to dramatic changes of nitrate metabolism,amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants

Tobacco transformants that express an antisense RBCS construct were used to investigate the consequences of a lesion in photosynthetic carbon metabolism for nitrogen metabolism and secondary metabolism. The results show that an inhibition of photosynthesis and decrease in sugar levels leads to a general inhibition of nitrogen metabolism, and dramatic changes in the levels of secondary metabolites. The response was particularly clear in plants that received excess nitrogen. In these conditions, a decrease of Rubisco activity led to an inhibition of nitrate reductase activity, accumulation of nitrate, a decrease of amino acid levels that was larger than the decrease of sugars, and a large decrease of chlorogenic acid and of nicotine, which are the major carbon- and nitrogen-rich secondary metabolites in tobacco leaves, respectively. Similar changes were seen when nitrogen-replete wild-type tobacco was grown in low light. The inhibition of nitrogen metabolism was partly masked when wild-type plants and antisense RBCS transformants were compared in marginal or in limiting nitrogen, because the lower growth rate of the transformants alleviated the nitrogen deficiency, leading to an increase of amino acids. In these conditions, chlorogenic acid always decreased but the decrease of nicotine was ameliorated or reversed. When the changes in internal pools are compared across all the genotypes and growth conditions, two conclusions emerge. First, decreased levels of primary metabolites lead to a dramatic decrease in the levels of secondary metabolites. Second, changes of the amino acid : sugar ratio are accompanied by changes of the nicotine:chlorogenic acid ratio.     

Rickettsia monacensis sp. nov., a spotted fever group Rickettsia,from ticks (Ixodes ricinus) collected in a European city park

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.     

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.     

The IFN-inducible Golgi- and endoplasmic reticulum- associated 47-kDa GTPase IIGP is transiently expressed during listeriosis

Members of the 47-kDa GTPase family are implicated in an IFN-gamma-induced, as yet unclear, mechanism that confers innate resistance against infection with intracellular pathogens. Overt immunological parameters are apparently uncompromised in mice deficient for individual members and the prototype of this family, IGTP, localizes to the endoplasmic reticulum. This suggests that these GTPases are involved in intracellular defense. We analyzed the expression of the 47-kDa GTPase cognate, IIGP, in splenic sections from mice infected with the intracellular pathogen Listeria monocytogenes by immunohistochemistry. An early transient IIGP induction was observed revealing the IFN-gamma responsiveness of cellular subcompartments within the spleen in early listeriosis. Marginal metallophilic macrophages and endothelial cells within the red and white pulp strongly expressed IIGP, while other splenocytes remained negative. In vitro analyses show that both type I and type II IFNs are prime stimuli for IIGP induction in various cells, including L. monocytogenes-infected or LPS-stimulated macrophages, endothelial cells, and activated T cells. Contrary to the subcellular localization of IGTP, IIGP was predominantly associated with the Golgi apparatus and also localizes to the endoplasmic reticulum. We conclude that IIGP exerts a distinct role in IFN-induced intracellular membrane trafficking or processing.