Nonhelical leash and alpha-helical structures determine the potency of a peptide antagonist of human T-cell leukemia virus entry

Viral fusion proteins mediate the entry of enveloped viral particles into cells by inducing fusion of the viral and target cell membranes. Activated fusion proteins undergo a cascade of conformational transitions and ultimately resolve into a compact trimer of hairpins or six-helix bundle structure, which pulls the interacting membranes together to promote lipid mixing. Significantly, synthetic peptides based on a C-terminal region of the trimer of hairpins are potent inhibitors of membrane fusion and viral entry, and such peptides are typically extensively α-helical. In contrast, an atypical peptide inhibitor of human T-cell leukemia virus (HTLV) includes α-helical and nonhelical leash segments. We demonstrate that both the C helix and C-terminal leash are critical to the inhibitory activities of these peptides. Amino acid side chains in the leash and C helix extend into deep hydrophobic pockets at the membrane-proximal end of the HTLV type 1 (HTLV-1) coiled coil, and these contacts are necessary for potent antagonism of membrane fusion. In addition, a single amino acid substitution within the inhibitory peptide improves peptide interaction with the core coiled coil and yields a peptide with enhanced potency. We suggest that the deep pockets on the coiled coil are ideal targets for small-molecule inhibitors of HTLV-1 entry into cells. Moreover, the extended nature of the HTLV-1-inhibitory peptide suggests that such peptides may be intrinsically amenable to modifications designed to improve inhibitory activity. Finally, we propose that leash-like mimetic peptides may be of value as entry inhibitors for other clinically important viral infections

Regulation of nucleolar signalling to p53 through NEDDylation of L11

Several studies have shown that ribosomal proteins (RPs) are important mediators of p53 activation in response to nucleolar disruption; however, the pathways that control this signalling function of RPs are currently unknown. We have recently shown that RPs are targets for the ubiquitin-like molecule NEDD8, and that NEDDylation protects RPs from destabilization. Here, we identify NEDD8 as a crucial regulator of L11 RP signalling to p53. A decrease in L11 NEDDylation during nucleolar stress causes relocalization of L11 from the nucleolus to the nucleoplasm. This not only provides the signal for p53 activation, but also makes L11 susceptible to degradation. Mouse double minute 2 (MDM2) -mediated NEDDylation protects L11 from degradation and this is required for p53 stabilization during nucleolar stress. By controlling the correct localization and stability of L11, NEDD8 acts as a crucial, new regulator of nucleolar signalling to p53

Antibodies to the Envelope Glycoprotein of Human T Cell Leukemia Virus Type 1 Robustly Activate Cell-Mediated Cytotoxic Responses and Directly Neutralize Viral Infectivity at Multiple Steps of the Entry Process

Abstract 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.</jats:p

Conformation-specific antibodies targeting the trimer-of-hairpins motif of the human T-cell leukemia virus type 1 transmembrane glycoprotein recognize the viral envelope but fail to neutralize viral entry

Human T-cell leukemia virus type 1 (HTLV-1) entry into cells is dependent upon the viral envelope glycoprotein-catalyzed fusion of the viral and cellular membranes. Following receptor activation of the envelope, the transmembrane glycoprotein (TM) is thought to undergo a series of fusogenic conformational transitions through a rod-like prehairpin intermediate to a compact 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 TM is a valid target for antiviral therapy. To assess the utility of TM as a vaccine target and to explore further the function of TM in HTLV-1 pathogenesis, we have begun to examine the immunological properties of TM. Here we demonstrate that a recombinant trimer-of-hairpins form of the TM ectodomain is strongly immunogenic. Monoclonal antibodies raised against the TM immunogen specifically bind to trimeric forms of TM, including structures thought to be important for membrane fusion. Importantly, these antibodies recognize the envelope on virally infected cells but, surprisingly, fail to neutralize envelope-mediated membrane fusion or infection by pseudotyped viral particles. Our data imply that, even in the absence of overt membrane fusion, there are multiple forms of TM on virally infected cells and that some of these display fusion-associated structures. Finally, we demonstrate that many of the antibodies possess the ability to recruit complement to TM, suggesting that envelope-derived immunogens capable of eliciting a combination of neutralizing and complement-fixing antibodies would be of value as subunit vaccines for intervention in HTLV infections