Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses.

We investigated the interactions between human monocyte-derived dendritic cells (DCs) and Ag-activated circulating TCR-γδ-expressing lymphocytes (Vδ2). Coculture of immature DCs (iDCs) with peripheral blood Vδ2 T cells activated with either pyrophosphomonoesters (isopentenyl pyrophosphate; IPP) or aminobiphosphonates (pamidronate; PAM) led to a significant up-modulation of CD86 and MHC class I molecules and to the acquisition of functional features typical of activated DCs. DC activation induced by both IPP- and PAM-stimulated γδ T cells was mostly mediated by TNF-α and IFN-γ secreted by activated lymphocytes. However, the effect of PAM-activated γδ T cells, but not that of IPP-activated cells, required cell-to-cell contact. Reciprocally, activation of Vδ2 T cells by PAM, but not by IPP, was dependent on cell contact with iDCs. In fact, when PAM-stimulated DC-γδ T cell cocultures were separated by a semipermeable membrane or treated with blocking anti-CD86 Abs, induction of CD25 and CD69 as well as IFN-γ and TNF-α secretion by Vδ2 cells were strongly reduced. These results demonstrate for the first time a bidirectional activating interaction between iDCs and PAM-stimulated γδ T lymphocytes, thus suggesting a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drugs.

Human immunodeficiency virus type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry. It was recently demonstrated that HIV-1 glycoprotein 120 (gp120) elevated calcium and activated several ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. This study shows that chemokine receptor engagement by both CCR5-dependent (R5) and CXCR4-dependent (X4) gp120 led to rapid phosphorylation of the focal adhesion-related tyrosine kinase Pyk2 in macrophages. Pyk2 phosphorylation was also induced by macrophage inflammatory protein-1␤ (MIP-1␤) and stromal cell-derived factor-1␣, chemokine ligands for CCR5 and CXCR4. Activation was blocked by EGTA and by a potent blocker of calcium release-activated Ca ؉؉ (CRAC) channels, but was insensitive to pertussis toxin (PTX), implicating CRAC-mediated extracellular Ca ؉؉ influx but not G␣ i protein-dependent mechanisms. Coreceptor engagement by gp120 and chemokines also activated 2 members of the mitogen-activated protein kinase (MAPK) superfamily, c-Jun amino-terminal kinase/stress-activated protein kinase and p38 MAPK. Furthermore, gp120-stimulated macrophages secreted the chemokines monocyte chemotactic protein-1 and MIP-1␤ in a manner that was dependent on MAPK activation. Thus, the gp120 signaling cascade in macrophages includes coreceptor binding, PTX-insensitive signal transduction, ionic signaling including Ca ؉؉ influx, and activation of Pyk2 and MAPK pathways, and leads to secretion of inflammatory mediators. HIV-1 Env signaling through these pathways may contribute to dysregulation of uninfected macrophage functions, new target cell recruitment, or modulation of macrophage infection. IntroductionHuman immunodeficiency virus type 1 (HIV-1) infection is initiated by the formation of a trimolecular complex on the cell surface, consisting of the HIV-1 envelope (Env) glycoprotein 120 (gp120), the principal cellular receptor CD4, and a chemokine receptor that functions as a coreceptor (reviewed by Berger 1 ). Conformational changes induced in gp120 on binding to CD4 increase its affinity for the coreceptor, leading to fusion between the viral and cellular membranes. The chemokine receptors that serve as HIV coreceptors are members of the 7 transmembrane G protein-coupled receptor (GPCR) family. CCR5 and CXCR4 play a dominant role in HIV-1 entry into primary cells, and are the principal coreceptors used by macrophage (M)-tropic (R5) and T-cell line (T)-tropic (X4) isolates, respectively.Given their normal cell signaling function, the interaction of gp120 with the chemokine receptors may, in addition to facilitating viral entry, also result in activation of cellular responses that could modulate the activation status of the cells and affect postentry stages of HIV replication (reviewed by Popik and Pitha 2 ). Although chemokine receptors and their ligands play central roles in both HIV infection and in immune regulation, how signaling pathways mediated by CCR5 and CXCR4 contribute to the immunopathogenesis...