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  • GDC-0449 To summarize our work highlights the facts that

    2018-10-23

    To summarize, our work highlights the facts that ECs maintain HIV-specific memory B cell responses associated to effective antiviral humoral activities and that Env-specific memory B cell responses are positively associated with the neutralization breadth in HLA-B*57+ ECs. We propose that promoting HIV-specific B cell polyfunctional responses by therapeutic vaccination might be highly beneficial in cART treated patients. The following are the supplementary data related to this article.
    Funding Sources
    Conflicts of Interest
    Author Contributions
    Acknowledgments
    Introduction The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is a small protein (101aa in most clinical isolates, or 86aa in the widely utilized HXB2 laboratory strain), acting as a transcriptional activator of viral gene expression. At the viral long terminal repeat (LTR) promoter, the protein binds a cis-acting RNA element (trans-activation-responsive region, TAR) present at the 5′-end of each viral transcript (Berkhout et al., 1989). Through this interaction, Tat activates HIV-1 transcription by promoting the assembly of transcriptionally active complexes at the LTR by multiple protein-protein interactions (Giacca, 2004; Ott et al., 2011). Besides regulating HIV-1 gene expression, >20years ago it was first demonstrated that Tat also possesses the unusual property of entering cells when present in the extracellular milieu (Frankel and Pabo, 1988; Green and Loewenstein, 1988). This property was later extensively characterized and shown to depend on a 9-aa long, arginine-rich sequence (aa 49–57), corresponding to the Tat basic domain, which also mediates nuclear transport and TAR binding. Work performed in different laboratories has shown that short peptides corresponding to this amino GDC-0449 stretch can be used as biotechnological tools for the intracellular delivery of heterologous proteins, drugs, viral vectors, siRNAs and nanoparticles (Fittipaldi and Giacca, 2005; Jones and Sayers, 2012; Schmidt et al., 2010; Zhang and Wang, 2012). We, and others have previously shown that extracellular Tat binds heparin through its basic domain (Mann and Frankel, 1991; Rusnati et al., 1997). We also showed that membrane bound-heparan sulfate proteoglycans (HSPG) are the cell surface receptor for Tat internalization, since cells that are genetically impaired in the synthesis of these molecules fail to internalize the extracellular protein (Tyagi et al., 2001). A few studies have also provided evidence in support of extracellular Tat release from the expressing cells (Becker-Hapak et al., 2001; Chang et al., 1997; Tasciotti and Giacca, 2005; Tyagi et al., 2001). The mechanism underlying this process, however, has remained largely elusive. The protein does not contain an N-terminal signal peptide driving its secretion from the ER-Golgi pathway and, accordingly, protein export is insensitive to drugs which disrupt the integrity of such organelles (Chang et al., 1997). Thus, Tat is a member of the small group of heterogeneous proteins that exit the cells by a process termed “unconventional” or “non-classical” protein secretion (Nickel and Rabouille, 2009). Recent data show that recruitment of Tat to the inner leaflet of the plasma membrane involves binding to membrane-associated phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) (Rayne et al., 2010), with the consequent formation of membrane pores (Zeitler et al., 2015); similar events occur for the unconventional secretion of FGF-2 (Temmerman et al., 2008). The mechanism for extracellular release of Tat and the molecular identity of the secretory machinery involved, however, remain elusive.
    Materials and Methods
    Results
    Discussion Our experiments indicate that the α subunit of the cellular Na+,K+-ATPase mediates unconventional Tat secretion in a ouabain-sensitive manner. Extracellular Tat release was affected neither by methylamine (a drug which blocks endosomal recycling and impairs IL1B and FGF-2 non canonical secretion, implying a vesicular intermediate in their release (Hamon et al., 1997; Rubartelli et al., 1990; Zhou et al., 2002)), nor glyburide (a sulfonylurea interfering with the ABC-1 transporter essential for the secretion of Galectin-1 (Flieger et al., 2003; Hamon et al., 1997)). In contrast, Tat secretion was markedly sensitive to ouabain, an inhibitor of the Na+,K+-ATPase. The observations that Tat secretion still occurred in the presence of curcumin, an inhibitor of all P-type ATPases (including the Na+,K+-ATPase) and that the rat D716N α1 mutant, which is impaired in catalytic function (Lane et al., 1993), still rescued Tat secretion in human cells treated with ouabain, are concordant in indicating that the effect of the Na+,K+-ATPase on extracellular Tat release is independent from its enzymatic activity while still demands physical binding of α1 to Tat.