No abstract

Efficient human immunodeficiency virus (HIV)-1 infection depends on multiple interactions between the viral gp41/gp120envelope (Env) proteins and cell surface receptors. However, cytoskeleton-associated proteins that modify membrane dynamics may also regulate the formation of the HIV-mediated fusion pore and hence viral infection. Because the effects of HDAC6-tubulin deacetylase on cortical ␣-tubulin regulate cell migration and immune synapse organization, we explored the possible role of HDAC6 in HIV-1-envelope-mediated cell fusion and infection. The binding of the gp120 protein to CD4؉ -permissive cells increased the level of acetylated ␣-tubulin in a CD4-dependent manner. Furthermore, overexpression of active HDAC6 inhibited the acetylation of ␣-tubulin, and remarkably, prevented HIV-1 envelopedependent cell fusion and infection without affecting the expression and codistribution of HIV-1 receptors. In contrast, knockdown of HDAC6 expression or inhibition of its tubulin deacetylase activity strongly enhanced HIV-1 infection and syncytia formation. These results demonstrate that HDAC6 plays a significant role in regulating HIV-1 infection and Env-mediated syncytia formation. INTRODUCTIONHuman immunodeficiency virus (HIV) infection is initiated by the virus binding to the cell surface after CD4 engagement and HIV-1 fusion with the plasma membrane (Stein et al., 1987;Maddon et al., 1988;McClure et al., 1988;Moore et al., 1997). The main coreceptors for HIV-1 infection have been shown to be CXCR4 and CCR5, which represented a major advance in understanding the mechanism of the HIV-1 infection (Cocchi et al., 1995;Alkhatib et al., 1996;Bleul et al., 1996;Choe et al., 1996;Deng et al., 1996;Doranz et al., 1996;Dragic et al., 1996;Feng et al., 1996). As a result, it has been proposed that the sequential binding of gp120 viral protein to CD4 and to one coreceptor induces specific conformational changes in gp41, facilitating viral fusion with cell membrane (Clapham and McKnight, 2002).The cell cytoskeleton is involved in the early events of viral infection, regulating viral penetration and genome uncoating, the movement of viral capsids, and integration of the viral genome. Accordingly, actin and microtubules are required for the efficient entry of herpes simplex type 1 and simian virus 40, respectively (Pelkmans et al., 2002;Marozin et al., 2004). It has been shown that disrupting the actin network can inhibit infection of HIV-1 and fusion with the host cell (Iyengar et al., 1998;Jernigan et al., 2000), presumably by disrupting the colocalization of CD4 and CXCR4 (Iyengar et al., 1998). In addition, the actin cytoskeleton seems to be necessary for activation of the reverse transcription complex (Bukrinskaya et al., 1998). However, little is known about the role of cytoskeleton-related enzymes in the control of HIV fusion and infection.Histone deacetylase 6 (HDAC6) is exclusively located in the cytoplasm, and it regulates the acetylation of ␣-tubulin (Hubbert et al., 2002;Matsuyama et al., 2002;Haggarty et al., 2003;Z...

EWI-2 and EWI-F, two members of a novel subfamily of Ig proteins, are direct partners of tetraspanins CD9 (Tspan29) and CD81 (Tspan28). These EWI proteins contain a stretch of basic charged amino acids in their cytoplasmic domains that may act as binding sites for actin-linking ezrin-radixin-moesin (ERM) proteins. Confocal microscopy analysis revealed that EWI-2 and EWI-F colocalized with ERM proteins at microspikes and microvilli of adherent cells and at the cellular uropod in polarized migrating leukocytes. Immunoprecipitation studies showed the association of EWI-2 and EWI-F with ERM proteins in vivo. Moreover, pulldown experiments and protein-protein binding assays with glutathione S-transferase fusion proteins containing the cytoplasmic domains of EWI proteins corroborated the strong and direct interaction between ERMs and these proteins. The active role of ERMs was further confirmed by double transfections with the N-terminal domain of moesin, which acts as a dominant negative form of ERMs, and was able to delocalize EWIs from the uropod of polarized leukocytes. In addition, direct association of EWI partner CD81 C-terminal domain with ERMs was also demonstrated. Functionally, silencing of endogenous EWI-2 expression by short interfering RNA in lymphoid CEM cells augmented cell migration, cellular polarity, and increased phosphorylation of ERMs. Hence, EWI proteins, through their direct interaction with ERM proteins, act as linkers to connect tetraspanin-associated microdomains to actin cytoskeleton regulating cell motility and polarity.

The integrin ␣4␤1 (VLA-4) not only mediates the adhesion and transendothelial migration of leukocytes, but also provides costimulatory signals that contribute to the activation of T lymphocytes. However, the behavior of ␣4␤1 during the formation of the immune synapse is currently unknown. Here, we show that ␣4␤1 is recruited to both human and murine antigen-dependent immune synapses, when the antigen-presenting cell is a B lymphocyte or a dendritic cell, colocalizing with LFA-1 at the peripheral supramolecular activation complex. However, when conjugates are formed in the presence of anti-␣4 antibodies, VLA-4 colocalizes with the CD3-chain at the center of the synapse. In addition, antibody engagement of ␣4 integrin promotes polarization toward a T helper 1 (Th1) response in human in vitro models of CD4 ؉ T cell differentiation and naïve T cell priming by dendritic cells. The in vivo administration of anti-␣4 integrin antibodies also induces an immune deviation to Th1 response that dampens a Th2-driven autoimmune nephritis in Brown Norway rats. These data reveal a regulatory role of ␣4 integrins on T lymphocyte-antigen presenting cell cognate immune interactions.A fter the recognition of antigens (Ag) presented by dendritic cells (DCs), naïve T lymphocytes proliferate and differentiate into T helper (Th) 1 or 2 effector cells. These effector lymphocytes are characterized by distinct patterns of cytokine production and homing behavior. Th1 cells mainly produce IFN-␥ and IL-2 and have a key role in the cellular immune responses. Conversely, Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and promote the humoral immune responses (1). DCs are the only Ag-presenting cells (APCs) involved in the priming of naïve Th cells and their polarization toward Th1 or Th2 differentiation. To acquire this capacity, DCs must undergo a maturation process characterized by the loss of their Ag-capturing capacity and the increase of their expression of costimulatory and adhesion molecules, including ␣4␤1 integrin (2). However, other APCs (e.g., B lymphocytes) are also involved in regulating the cytokine profiles of Th cell responses, indicating the importance of postpriming events (3).The interaction between T cells and APCs plays an important role in directing Th cell polarization. The strength of antigenic stimulation, the duration of T cell receptor engagement, the presence of different cytokines, and the participation of distinct costimulatory molecules are critical in determining the phenotype of differentiated T cells. The cytokine IL-12, high doses of Ag, and CD28͞B7-1 interaction promote Th1 differentiation, whereas an environment enriched in IL-4, low doses of Ag, and CD28͞B7-2 or inducible costimulator (ICOS)͞ICOS ligand participation promote Th2 responses (4).Integrins are a large family of ␣␤ heterodimeric transmembrane proteins that mediate cell-cell and cell-extracellular matrix adhesion. Several integrins, lymphocyte functionassociated (LFA-1; ␣ L ␤ 2 ), very late activation antigen-4 (VLA-4; ␣4␤1), and VLA-1 (␣1␤1) have ...