HIV-1 Nef triggers Vav-mediated signaling pathway leading to functional and morphological differentiation of dendritic cells

MARIA GIOVANNA QUARANTA,* BENEDETTA MATTIOLI,* FRANCESCA SPADARO,* ELISABETTA STRAFACE,† LUCIANA GIORDANI,* CARLO RAMONI,* WALTER MALORNI,†AND MARINA VIORA*,1

*Department of Immunology, †Department of Ultrastructures, Istituto Superiore di Sanita`, 00161 Rome, Italy

Summary

Regulated migration of dendritic cells (DC), which is primed by different transcriptional factors leading to modulation of genes, plays a central role in induction of physiological immune responses and this process necessities plasticity of cytoskeleton. DCs are the first target of HIV and, by clustering and activating T-cells, may both activate antiviral immunity and facilitate virus dissemination. HIV-1 Nef protein is expressed early during infection and has been shown to be critical for viral pathogenesis in vivo, enhancing viral replication and infectivity. Depending on its intracellular localization, Nef interferes with cellular signal transduction pathway, also exogenous Nef inhibits the induction of a specific antibody response, participating in AIDS pathogenesis. In this paper, authors investigated Nef-induced DC differentiation, focusing on the interference of Nef in the signaling pathways that regulates DC maturation. They found that exogenous Nef enters immature DCs, targets Vav, activating its signaling cascade via Rac1, leading to cytoskeleton rearrangements, leading to cytoskeleton rearrangement and NF-kB activation.

Following information contains brief description of what was done during research experiment. Recombinant HIV-1 Nef protein was obtained from E.Coli. The Nef uptake by DC was elaluated by flow cytometry using FITC conjugate Nef. CLSM and IVM analysis were used to investigate the subcellular localization of Nef in the mature DC; kinetic analysis was carried out to analyze whether Nef signaling influences subcellular Vav distribution. NF-kB p65/NF-kB p50 transcription factor kits were used to analyze the rates of NF-kB activation from total lysates from untreated and Nef – or LPS-treated DCs. Nef induced morphological changes and cytoskeleton and vinculin rearrangements which were tested by SEM. Using DC/T cell conjugate analyses and fluorescent staining, it was testified that Nef improves immunological synapse formation between DCs and CD4+ T cells. Nef also enhanced nucleotide exchange rate of Rac1 and Cdc42 which was analyzed through particular protein fusion.

Summary of results is depicted in the figure:

Figure 8. Signaling events in Nef-induced DC maturation. Nef enters immature DCs leading to Vav phosphorylation. This is achieved by either direct interaction with Vav or an upstream activation of Src kinases, which in turn phosphorylate Vav. P-Vav is then able to activate Rac1 and Cdc42 proteins that trigger a series of downstream signaling events. Those include rearrangement in the actin and vinculin cytoskeleton, leading to morphological changes, and NF-kB translocation to the nucleus, which yields DC phenotypical and functional differentiation. In this scenario, Nef changes the local environment of the DCs resulting in their activation.

Significance:

Considering that mucosal DCs and blood DC-SIGN⁺ DCs represent the first HIV-1 targets upon sexual transmission and transmission via blood, in this study immature DCs are used as the closest model for in vivo primary HIV infection to investigate the effect of Nef on DC maturation. This work outlines Nef induced, Vav-dependent signaling pathway by which HIV-1 may take advantage of DCs favoring non-specific T cell activation, permitting immune system invasion that leads to AIDS. Understanding of the virus invasion mechanism is a necessary key to creation of effective treatment with knowledge of the steps that can be blocked in that pathway. The Nef-induced signaling pathway regulating DC functional and morphological differentiation could provide opportunities for therapeutic manipulation of immune system responses in vivo.

Article can be found on this link: http://www.fasebj.org/cgi/reprint/17/14/2025.pdf