No abstract

The HIV Nef protein mediates endocytosis of surface receptors that correlates with disease progression, but the link between this Nef function and HIV pathogenesis is not clear. Here, we report that Nef-mediated activation of membrane trafficking is bidirectional, connecting endocytosis with exocytosis as occurs in activated T cells. Nef expression induced an extensive secretory activity in infected and, surprisingly, also in noninfected T cells, leading to the massive release of microvesicle clusters, a phenotype observed in vitro and in 36%-87% of primary CD4 T cells from HIV-infected individuals. Consistent with exocytosis in noninfected cells, Nef is transferred to bystander cells upon cell-to-cell contact and subsequently induces secretion in an Erk1/2-dependent manner. Thus, HIV Nef alters membrane dynamics, mimicking those of activated T cells and causing a transfer of infected cell signaling (TOS) to bystander cells. This mechanism may help explain the detrimental effect on bystander cells seen in HIV infection.

IntroductionThe pathology of acquired immunodeficiency syndrome (AIDS) should be considered as the sum of effects generated by human immunodeficiency virus (HIV) replication, with direct T-cell destruction, and by a deep alteration in the pattern of soluble factors. The latter largely depends on the extent of viral replication, because it is hindered by effective antiretroviral therapy. HIV/ simian immunodeficiency virus (SIV) Nef, a multifunctional 27-to 34-kd protein expressed early in viral replication, was demonstrated to be a strong candidate for many of the pathogenic effects of HIV/SIV. This was first highlighted by the evidence that monkeys failed to develop the disease on infection with nef-deleted SIV, 1 and later confirmed by the observation that nef transgenic mice developed a syndrome strictly related to AIDS. 2 Furthermore, Nef perturbs the pattern of secreted factors in different cell types. As an example, the engagement of Nef with the chain of T-cell receptor leads to release of Fas-ligand from T cells in an antigenindependent manner. 3 In addition, the expression of Nef in monocytes/macrophages leads to a massive release of macrophage inflammatory protein 1␣/␤ (MIP-1␣/␤) chemokines and of still unknown factor-activating lymphocytes. 4 These soluble factors possibly promote recruitment and activation of T lymphocytes, which become susceptible to HIV replication. Nef is also able to alter some cellular functions, that is, CD4 internalization/ recycling, 5-7 major histocompatibility complex class I surface expression, 8 and src tyrosine kinases signaling (reviewed in Herna Remkema 9 ) through intracellular mechanisms.We were interested in investigating whether the expression of Nef influences the activation of signal tranducers and activators of transcription (STAT) molecules (reviewed in Schindler and Darnell, 10 Leaman et al, 11 Darnell,12 Stark et al, 13 and Bromberg and Darnell 14 ). STAT-governed pathways were first described by Darnell and colleagues by studying the interferon (IFN)-induced intracellular signal transduction. 15 Seven different STATs have been characterized so far. Activation of STATs is involved in the response of a wide number of cytokines, growth factors, and hormones. Typically, binding of cytokines with specific receptors lacking intrinsic kinase activity in its cytoplasmic tail induces receptor aggregation and recruitment of members of Janus kinases. These become activated by phosphorylating themselves and tyrosine residues of the receptor cytoplasmic tails. The receptor phosphotyrosines serve as docking sites for the binding of inactive STAT through the Src-homology 2 (SH2) domains. STAT monomers become phosphorylated at a constant tyrosine residue and dimerize. The activated dimers translocate to the nucleus thereby binding to specific DNA response elements, ultimately influencing gene expression programs. Hence, alterations in the STAT pathways could substantially influence cellular homeostasis. Few reports regarding the effects of HIV-1 infection on STAT activation/...

CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cell-substrate adhesion and cell migration through physical and functional interactions with integrin receptors. In contrast, little is known about the potential role of CD151 in controlling cell proliferation and survival. We have previously shown that ␤4 integrin, a major CD151 partner, not only acts as an adhesive receptor for laminins but also as an intracellular signaling platform promoting cell proliferation and invasive growth upon interaction with Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF). Here we show that RNAi-mediated silencing of CD151 expression in cancer cells impairs HGF-driven proliferation, anchorage-independent growth, protection from anoikis, and tumor progression in xenograft models in vivo. Mechanistically, we found that CD151 is crucially implicated in the formation of signaling complexes between Met and ␤4 integrin, a known amplifier of HGF-induced tumor cell growth and survival. CD151 depletion hampered HGF-induced phosphorylation of ␤4 integrin and the ensuing Grb2-Gab1 association, a signaling pathway leading to MAPK stimulation and cell growth. Accordingly, CD151 knockdown reduced HGF-triggered activation of MAPK but not AKT signaling cascade. These results indicate that CD151 controls Met-dependent neoplastic growth by enhancing receptor signaling through ␤4 integrin-mediated pathways, independent of cell-substrate adhesion.CD151 is a cell surface protein of the tetraspanin family. These proteins contain four transmembrane domains, two extracellular loops, and two short cytoplasmic tails (1, 2). Tetraspanins are major partners of integrins that regulate cell adhesion to extracellular matrix components as well as cellcell contacts, thereby modulating cell migration. In particular, CD151 is typically associated with ␣3␤1, ␣6␤1, and ␣6␤4 integrin receptor complexes for laminins (3, 4), and its expression is increased during epithelial-mesenchymal transition (5). Not surprisingly, the activity of tetraspanins as pro-migratory molecules has been implicated in tumor invasion and metastasis (6, 7). For example, elevated CD151 expression has been correlated with poor prognosis and increased metastasis in colon, lung, and prostate cancers (8 -10). In line with this, the association between integrin ␣6␤4 and CD151 has been linked to tumor cell motility, increased metastasis, and poor prognosis in colon cancer (11). More recently, it has been shown that a CD151-specific antibody can prevent tumor cell invasion and metastasis by modulating cell-substrate adhesion (12). Notably, although the function of CD151 in the regulation of integrin-dependent adhesion and migration is well established, its role in cell growth and survival signaling is poorly understood.Tetraspanins can interact with several partners in addition to integrins and form intricate protein networks in tetraspanin-enriched microdomains at the cell surface, which may function as platforms regulating receptor signal...