Differential recognition of HIV-stimulated IL-1β and IL-18 secretion through NLR and NAIP signalling in monocyte-derived macrophages

Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1β and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the “priming” signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1β/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K⁺ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages.     

HIV-1 replication in dendritic cells occurs through a tetraspanin-containing compartment enriched in AP-3

Dendritic cells (DC) are crucial components of the early events of HIV infection. Dendritic cells capture and internalize HIV at mucosal surfaces and efficiently transfer the virus to CD4+ T cells in trans through infectious synapses (trans-infection pathway). Alternatively, HIV-1 replicates in DC (R5-HIV-1) (cis-infection pathway). Here, we analyzed HIV trafficking in DC during the trans-infection pathway as well as the cis-infection pathway. Confocal immunofluorescence microscopy demonstrated that after capture by DC, R5-HIV-1 and HIV-1 pseudotyped with vesicular stomatitis virus protein G colocalized in a viral compartment enriched in tetraspanins including CD81, CD82 and CD9, although at different levels, indicating a role of the viral envelope in targeting to the tetraspanin-rich compartment. Replication of R5-HIV-1 in DC (cis-infection pathway) also led to the accumulation, in an envelope-independent manner, of mature viral particles in a tetraspanin-rich compartment. A fraction of the HIV-1-containing compartments appeared directly accessible from the cell surface. In sharp contrast with the trans-infection pathway, the delta-subunit of the adaptor protein 3 (AP-3) complex was enriched on the HIV-1-containing compartment during R5-HIV-1 replication in DC (cis-infection pathway). Downregulation of AP-3 delta-adaptin reduced significantly viral particle release from HIV-1-infected DC. Together, these studies demonstrate a role for AP-3 in HIV replication in a tetraspanin-rich compartment in DC and contribute to the elucidation of the trafficking pathways required for DC-T cell transfer of HIV-1 infection, a critical step during the early events of HIV infection.     

Investigation of Ochratoxin A biosynthetic genes in<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> by DDRT-PCR experiments: differential expression of OTA genes

A defined minimal medium has been developed which is either restrictive or permissive for the production of ochratoxin A byPenicillium verrucosum simply by changing the nitrogen and carbon source. The combination of ammonium/glycerin promoted, whereas the combination nitrate/glucose repressed ochratoxin production.In parallel mutants ofP verrucosum have been constructed by restriction endonuclease mediated integration, which were not able to produce ochratoxin. Different types of transformants, which either produce no detectable amounts of ochratoxin A but ochratoxin α, strains which produce only ochratoxin B and strains which produces none of these secondary metabolites, have been observed.     

Thrombocytopenia in an animal model of malaria is associated with an increased caspase-mediated death of thrombocytes

Infection of mice with Plasmodium Berghei Anka (PbA) leads to a thrombocytopenia, due to a reduced platelet life span, eventually associated with a syndrome of severe or cerebral malaria (CM). Thrombocytopenia was associated with an increase in the number of microparticles (mcp) in plasma. More than >60% of these mcp were of platelet origin, as seen by staining with an anti-platelet antibody. The thrombocytopenia and the amount of mcp were decreased in mice treated with anti CD40L mAb, suggesting that CD40L is the main effector of the thrombocytopenia. Caspase-1, -3, -6, -8, -9 were activated in platelets from infected mice, as seen by the binding of labeled probes or the amount of pro-caspase-3. Treatment of infected mice with the caspases inhibitor ZVAD-fmk decreased the number of mcp and the thrombocytopenia, shoving that platelet caspases are responsible for platelet fragmentation. In addition, the caspase inhibitor also caused a decrease in the mortality associated with CM, indicating a critical role of caspases in the expression of CM.     

The HIV Nef protein alters Ca(2+) signaling in myelomonocytic cells through SH3-mediated protein-protein interactions

Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca(2+) signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef((PXXP)4(-))). In differentiated HL60 cells, Nef markedly altered cellular Ca(2+) signaling; the amount of intracellularly stored Ca(2+) was increased, and as a consequence, store-operated Ca(2+)-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca(2+) signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59(hck) co-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca(2+) stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca(2+) stores through SH3-mediated interactions in myelomonocytic cells.     

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.      

Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361). Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l). Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst. Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters. The two forms (cell- enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.      

The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection

Granuloma formation in the liver of mice infected with BCG coincides with local TNF synthesis. Injection of rabbit anti-TNF antibody, after 1 or 2 weeks of infection, dramatically interferes with the development of granulomas (both in number and size, large epithelioid cells failing to appear) and subsequent mycobacterial elimination. Furthermore, fully developed BCG granulomas, after 3 weeks of infection, rapidly regress after anti-TNF treatment. Antibody treatment also prevents or suppresses accumulation of TNF mRNA and protein, which resumes after disappearance of the antibody. Peritoneal macrophages exposed to TNF transiently accumulate TNF mRNA, and show an enhanced increase in TNF mRNA in response to gamma interferon. We propose that TNF released from macrophages in the microenvironment of developing granulomas is involved in a process of autoamplification: acting in an autocrine or paracrine way, it enhances its own synthesis and release, thus favoring further macrophage accumulation and differentiation leading to bacterial elimination.     

HIV-1 Vpu Neutralizes the Antiviral Factor Tetherin/BST-2 by Binding It and Directing Its Beta-TrCP2-Dependent Degradation

Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and β-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a β-TrCP2-dependent manner. Accordingly, in conditions where Vpu-β-TrCP2-tetherin interplay was not operative, including cells stably knocked down for β-TrCP2 expression or cells expressing a dominant negative form of β-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its β-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication.