The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based ...technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.
We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)‐ATPases required ...for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide‐loaded, SDS‐stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B‐treated cells, a slow increase (> 20‐fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface‐disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor‐treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans‐Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway.
Sensing of incoming viruses is a pivotal task of dendritic cells (DCs). Human primary blood DCs encompass various subsets that are diverse in their susceptibility and response to HIV-1. The recent ...identification of the blood Axl+DC subset, endowed with unique capacities to bind, replicate, and transmit HIV-1 prompted us to evaluate its anti-viral response. We demonstrate that HIV-1 induced two main broad and intense transcriptional programs in different Axl+DCs potentially induced by different sensors; an NF-κB-mediated program that led to DC maturation and efficient CD4+ T cell activation, and a program mediated by STAT1/2 that activated type I IFN and ISG responses. These responses were absent from cDC2 exposed to HIV-1 except when viral replication was allowed. Finally, Axl+DCs actively replicating HIV-1 identified by quantification of viral transcripts exhibited a mixed NF-κB/ISG innate response. Our results suggest that the route of HIV-1 entry may dictate different innate sensing pathways by DCs.
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•Axl+DCs are unique among human blood DC subsets in their response to HIV-1•HIV-1 induces 2 transcriptional programs in Axl+DCs, likely through different sensors•The NF-ΚB-mediated program is RT-independent and activates T cell help functions•The STA1/2 program induces type I IFN and ISG responses through STING activation
Biological sciences; Clinical microbiology; Microbiology; Molecular biology; Transcriptomics
Macrophages are important targets for HIV‐1 infection and harbor the virions in an as yet unidentified organelle. To determine the location of HIV‐1 in these cells, an extensive analysis of primary ...human macrophages infected in vitro with HIV‐1 was carried out by immuno‐electron microscopy. Virus particles were found to accumulate in intracellular multivesicular compartments which were enriched in major histocompatibility complex class II molecules and CD63. These features are characteristics of major histocompatibility complex class II compartments where maturing class II molecules acquire their peptide cargo. The membrane‐delimited, electron‐dense virus particles of 100–110 nm diameter labeled strongly for HIV‐1 p24 antigen, major histocompatibility complex class II molecules, CD63 and, to a lesser extent for HIV‐1 gp120 envelope protein and Lamp 1. Our data suggest that virus particles may access the lumen of the major histocompatibility complex class II compartment by budding from the limiting membrane, thus acquiring proteins of this membrane such as class II and CD63. Viral assembly and budding would therefore occur in macrophages by a process similar to the formation of the internal vesicles in multivesicular bodies and at the same location. This could account for the particular content in lipids and proteins previously found in the membrane wrapping HIV particles. Our observations also suggest direct fusion of the virus containing major histocompatibility complex class II compartment with the plasma membrane, leading to massive release of viral particles into the extracellular medium.
Newly synthesized MHC II α- and β-chains associated with the invariant chain chaperone (Ii) enter the endocytic pathway for Ii degradation and loading with peptides before transport to the cell ...surface. It is unclear how αβIi complexes are sorted from the Golgi apparatus and directed to endosomes. However, indirect evidence tends to support direct transport involving the AP1 clathrin adaptor complex. Surprisingly, we show here that knocking down the production of AP1 by RNA interference did not affect the trafficking of αβIi complexes. In contrast, AP2 depletion led to a large increase in surface levels of αβIi complexes, inhibited their rapid internalization, and strongly delayed the appearance of mature MHC II in intracellular compartments. Thus, in the cell systems studied here, rapid internalization of αβIi complexes via an AP2-dependent pathway represents a key step for MHC II delivery to endosomes and lysosomes.
HIV-1-infected cells can avoid cytotoxic T lymphocyte killing by Nef-mediated down-regulation of surface MHC I. Here, we show that HIV-1 Nef inhibits MHC II restricted peptide presentation to ...specific T cells and thus may affect the induction of antiviral immune responses. Nef mediates this effect by reducing the surface level of mature (i.e., peptide-loaded) MHC II while increasing levels of immature MHC II, which are functionally incompetent because of their association with the invariant chain. Nef was the only HIV-1 gene product to possess this capacity, which was also observed in the context of the whole HIV-1 genome. Other proteins of the endocytic pathway were not affected by Nef expression, suggesting that Nef effects on MHC II did not result from a general alteration of the endocytic pathway. Response patterns to previously characterized mutations of Nef differed for Nef-induced modulation of mature and immature MHC II. Furthermore, the doses of Nef required to observe each of the two effects were clearly different, suggesting that Nef could affect MHC II peptide presentation through distinct mechanisms. Cooperation between those mechanisms may enable Nef to efficiently inhibit MHC II function.
Exosomes are small vesicles (60–100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen‐presenting cells (APC), such as B ...lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC‐derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II‐bearing exosomes in vitro. The rat mast cell line, RBL‐2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide HLA‐DR1–hemagglutinin (HA). These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross‐linking. Exosomes bearing DR1–HA(306–318) complexes activated HA/DR1‐specific T cells only weakly, whereas the cross‐linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.
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