The electrogenic sodium/calcium exchanger (NCX) mediates bidirectional calcium transport controlled by the transmembrane sodium gradient. NCX inactivation occurs in the absence of phosphatidylinositol 4,5-bisphosphate and is facilitated by palmitoylation of a single cysteine at position 739 within the large intracellular loop of NCX. The aim of this investigation was to identify the structural determinants of NCX1 palmitoylation. Full-length NCX1 (FL-NCX1) and a YFP fusion protein of the NCX1 large intracellular loop (YFP-NCX1) were expressed in HEK cells. Single amino acid changes around Cys-739 in FL-NCX1 and deletions on the N-terminal side of Cys-739 in YFP-NCX1 did not affect NCX1 palmitoylation, with the exception of the rare human polymorphism S738F, which enhanced FL-NCX1 palmitoylation, and D741A, which modestly reduced it. In contrast, deletion of a 21-amino acid segment enriched in aromatic amino acids on the C-terminal side of Cys-739 abolished YFP-NCX1 palmitoylation. We hypothesized that this segment forms an amphipathic α-helix whose properties facilitate Cys-739 palmitoylation. Introduction of negatively charged amino acids to the hydrophobic face or of helix-breaking prolines impaired palmitoylation of both YFP-NCX1 and FL-NCX1. Alanine mutations on the hydrophilic face of the helix significantly reduced FL-NCX1 palmitoylation. Of note, when the helix-containing segment was introduced adjacent to cysteines that are not normally palmitoylated, they became palmitoylation sites. In conclusion, we have identified an amphipathic α-helix in the NCX1 large intracellular loop that controls NCX1 palmitoylation. NCX1 palmitoylation is governed by a distal secondary structure element rather than by local primary sequence.

Infection of cells by human T cell leukaemia virus (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to the cell surface receptor Glut-1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. In the absence of membrane fusion viral entry into the host cell cannot occur. Thus, envelope is a prime target for the development of anti-viral vaccines and small-molecule antagonists of viral infection. Indeed, we have shown that HTLV-1 infection can be blocked at all stages of the entry process including, viral attachment, primary receptor binding and the post-binding steps of viral entry. To extend our studies, we have expressed recombinant protein fragments that mimic the core-coiled-coil region and six-helix bundle of fusion-active HTLV-1 envelope. Using these recombinant proteins as immunogens we have generated monoclonal antibodies (mAbs) against the fusionactive and post-fusion conformations of HTLV-1 envelope. Most importantly, we have now used these conformation-specific mAbs to probe the events that culminate in membrane fusion. We demonstrate that these monoclonal antibodies can be used to detect viral envelope on infected cells and to monitor the process of cell-to-cell viral transfer. Our recent results will be presented, and the implications of our results for HTLV-1 pathogenesis will be discussed.

Fusion of the viral and cellular membranes is a critical step in the infection of cells by the human T-cell leukemia virus type 1 (HTLV-1) and this process is catalysed by the viral envelope glycoproteins. During fusion, the transmembrane glycoprotein (TM) is thought to undergo a transition from a rod-like pre-hairpin conformation that is stabilized by a trimeric coiled coil to a more compact six-helix-bundle or trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that the pre-hairpin motif is a valid target for antiviral therapy. Here, a stable, trimeric TM derivative that mimics the coiled-coil structure of fusion-active TM has been used to develop a plate-based assay to identify reagents that interfere with the formation of the six-helix bundle. The assay discriminates effectively between strong, weak and inactive peptide inhibitors of membrane fusion and has been used to identify a monoclonal antibody (mAb) that disrupts six-helix-bundle formation efficiently in vitro. The mAb is reactive with the C-helical region of TM, indicating that this region of TM is immunogenic. However, the mAb failed to neutralize HTLV-1 envelope-mediated membrane fusion, suggesting that, on native viral envelope, the epitope recognized by the mAb is obscured during fusion. This novel mAb will be of value in the immunological characterization of fusion-active structures of HTLV-1 TM. Moreover, the assay developed here will aid the search for therapeutic antibodies, peptides and small-molecule inhibitors targeting envelope and the HTLV-1 entry process. INTRODUCTIONEntry of retrovirus capsids into the host-cell cytoplasm requires fusion of the lipid membranes that surround both the virus and the target cell. Membrane fusion is catalysed by the viral envelope glycoproteins (Env), which are presented on the surface of the virus or infected cell as a trimer of surface glycoprotein (SU) subunits anchored to a trimer of transmembrane glycoproteins (TM). Binding of SU to a cellsurface receptor is thought to induce dramatic changes in Env conformation that convert Env from a metastable, nonfusogenic state to a fusion-active form (reviewed by Eckert & Kim, 2001;Sodroski, 1999). The receptor-stimulated changes in Env conformation promote insertion of the fusion peptide of TM into the target-cell membrane and, through a cascade of molecular rearrangements, promote membrane fusion (Eckert & Kim, 2001;Sodroski, 1999). For both human immunodeficiency virus (HIV) and human Tcell leukemia virus (HTLV), this model of virus entry is supported by the observations that mutations within Env (Delamarre et al., 1994(Delamarre et al., , 1997(Delamarre et al., , 1999Pique et al., 1990Pique et al., , 1992Pique et al., , 1993Poon & Chen, 1998;Rosenberg et al., 1997), anti-Env antibodies (Baba et al., 1993;Blanchard et al., 1999;Burton et al., 2004Burton et al., , 2005 Desgranges et al., 1994;Kuroki et al., 1992;Palker et al., 199...

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1–infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1–infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.