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...

In the absence of ligand, some nuclear receptors, including retinoic acid receptor (RAR), act as transcriptional repressors by recruiting corepressor complexes to target genes. This constitutive repression is crucial in metazoan reproduction, development and homeostasis. However, its specific molecular determinants had remained obscure. Using structural, biochemical and cell-based assays, we show that the basal repressive activity of RAR is conferred by an extended beta-strand that forms an antiparallel beta-sheet with specific corepressor residues. Agonist binding induces a beta-strand-to-alpha-helix transition that allows for helix H11 formation, which in turn provokes corepressor release, repositioning of helix H12 and coactivator recruitment. Several lines of evidence suggest that this structural switch could be implicated in the intrinsic repressor function of other nuclear receptors. Finally, we report on the molecular mechanism by which inverse agonists strengthen corepressor interaction and enhance gene silencing by RAR.

Retinoic acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidylinositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAPK signaling pathways through a rapid, nongenomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor, retinoic acid receptor (RAR), on the basis of the pharmacological profile of the activation, loss, and gain of function experiments with mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells, and the physical association between liganded RAR and PI3K activity. The association of RAR with the two subunits of PI3K was differentially regulated by the ligand. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARalpha and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR proved to be relevant for PI3K activation. A chimerical RAR fusing c-Src myristylation domain to the N terminus of RARalpha (Myr-RARalpha) was targeted to plasma membrane. Transfection of Myr-RARalpha to mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells and COS-7 cells results in strong activation of the PI3K signaling pathway, although both in the absence as well in the presence of RA. Our results support a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signaling complex involving RAR and the subunits of PI3K.

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.

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The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes. Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling. Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays. 9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand.

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Influenza virus (IFV) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. Given its high capacity to change antigenically, acquired immunity is often not effective to limit IFV infection and therefore vaccination must be constantly redesigned to achieve effective protection. Improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of IFV disease. In the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of IFV infection. This review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on IFV clearance and inflammatory-mediated lung tissue damage. In particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation–coagulation interactions during IFV infection. Studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory IFV disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract.