HIV-1-infected cells can avoid cytotoxic T lymphocyte killing byclass II-associated invariant chain ͉ immune escape ͉ adaptor complexes

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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Nef alters the cell surface expression of several immunoreceptors, which may contribute to viral escape. We show that Nef modifies major histocompatibility complex class II (MHC II) intracellular trafficking and thereby its function. In the presence of Nef, mature, peptide-loaded MHC II were down-modulated at the cell surface and accumulated intracellularly, whereas immature (invariant [Ii] chain-associated) MHC II expression at the plasma membrane was increased. Antibody internalization experiments and subcellular fractionation analyses showed that immature MHC II were internalized from the plasma membrane but had limited access to lysosomes, explaining the reduced Ii chain degradation. Immunoelectron microscopy revealed that Nef expression induced a marked accumulation of multivesicular bodies (MVBs) containing Nef, MHC II, and high amounts of Ii chain. The Nef-induced up-regulation of surface Ii chain was inhibited by LY294002 exposure, indicating the involvement of a phosphatidylinositol 3-kinase, whose products play a key role in MVB biogenesis. Together, our results indicate that Nef induces an increase of the number of MVBs where MHC II complexes accumulate. Given that human immunodeficiency virus recruits the MVB machinery for its assembly process, our data raise the possibility that Nef is involved in viral assembly through its effect on MVBs. INTRODUCTIONThe nef gene is highly conserved in primate lentiviruses and expressed abundantly in the early stages and throughout the viral replication cycle. Despite its small size, the Nef protein has been credited with a remarkable number of distinct functions. In vivo studies showed Nef to be a key component in human immunodeficiency virus (HIV) pathogenesis (Kestler et al., 1991; Hanna et al., 1998a,b). However, the underlying cellular and molecular mechanisms of its contribution to disease progression and pathogenesis remain elusive. Nef also enhances virion infectivity and viral production (Miller et al., 1994;Spina et al., 1994;Schwartz et al., 1995;Dorfman et al., 2002). This phenomenon is possibly related to one of the best characterized functions of Nef, the downregulation of the CD4 cell surface receptor by a posttranslational mechanism (Garcia and Miller, 1991;Aiken et al., 1994;Lundquist et al., 2002). The reduction in cell surface CD4 facilitates viral release by preventing sequestration of the HIV gp120 protein through CD4 and increases the infectivity of viral particles (Lama et al., 1999;Ross et al., 1999).Nef interferes with the immune system by disrupting antigen recognition at different levels. In addition to the accelerated endocytosis of CD4, part of the T cell receptor machinery, Nef also enhances internalization of the costimulatory CD28 molecule, impeding effective T cell activation (Swigut et al., 2001). Furthermore, Nef induces apoptosis of virus-specific cytotoxic T-lymphocytes via expression of CD95L, while protecting infected cells by modifying the signal transduction pathway regulating apoptosis (Fackler andBaur,2002).Thismodificati...

Siah proteins are E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina), a protein required for the R7 photoreceptor development. We have previously found that the expression of human siah-1 and its mouse homologue siah-1b are induced by p53 during apoptosis and tumor reversion. So far, no evidence that the siah-1b gene is a direct transcriptional target of p53 has been provided. In the present study we investigate this issue. Northern blot analysis with a specific probe demonstrates an increase in siah-1b transcription on activation of endogenous and inducible exogenous p53. To explore whether this effect is directly mediated by p53 we analyzed 20 kb of chromosome X DNA, containing the siah-1b locus. A p53-binding site was identified in the siah-1b promoter, located at nucleotides ؊2155͞؊2103 relative to the translational start site. This site is composed of two halfsites, conforming to the p53-binding consensus sequence but separated by a nonclassical 33-bp spacer. In luciferase assays, p53 induces a substantial increase in siah-1b promoter activity. Gel shift and DNase-I-footprinting studies, combined with mutational analysis and chromatin immunoprecipitation, indicate that p53 effectively binds the siah-1b promoter in vitro and in vivo. Thus, the siah-1b gene is a direct transcriptional target of p53.

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