David W. Brighty

Human T-Cell Leukemia Virus Type 1 Envelope Glycoprotein gp46 Interacts with Cell Surface Heparan Sulfate Proteoglycans

ABSTRACT The major receptors required for attachment and entry of the human T-cell leukemia virus type 1 (HTLV-1) remain to be identified. Here we demonstrate that a functional, soluble form of the HTLV-1 surface envelope glycoprotein, gp46, fused to an immunoglobulin Fc region (gp46-Fc) binds to heparan sulfate proteoglycans (HSPGs) on mammalian cells. Substantial binding of gp46-Fc to HeLa and Chinese hamster ovary (CHO) K1 cells that express HSPGs was detected, whereas binding to the sister CHO lines 2244, which expresses no HSPGs, and 2241, which expresses no glycosaminoglycans (GAGs), was much reduced. Enzymatic removal of HSPGs from HeLa and CHO K1 cells also reduced gp46-Fc binding. Dextran sulfate inhibited gp46-Fc binding to HSPG-expressing cells in a dose-dependent manner, whereas chondroitin sulfate was less effective. By contrast, dextran sulfate inhibited gp46-Fc binding to GAG-negative cells such as CHO 2244, CHO 2241, and Jurkat T cells weakly or not at all. Dextran sulfate inhibited HTLV-1 envelope glycoprotein (Env)-pseudotyped virus infection of permissive, HSPG-expressing target cells and blocked syncytium formation between HTLV-1 Env-expressing cells and HSPG-expressing permissive target cells. Finally, HSPG-expressing cells were more permissive for HTLV-1 Env-pseudotyped virus infection than HSPG-negative cells. Thus, similar to other pathogenic viruses, HTLV-1 may have evolved to use HSPGs as cellular attachment receptors to facilitate its propagation.

Human T-Cell Leukemia Virus Type 1 Receptor Expression among Syncytium-Resistant Cell Lines Revealed by a Novel Surface Glycoprotein-Immunoadhesin

ABSTRACT The envelope glycoproteins of human T-cell leukemia virus type 1 (HTLV-1) perform functions that are crucial for virus entry into cells. The surface glycoprotein (SU) is responsible for viral recognition of, and binding to, target cells through its interaction with an unknown cell surface receptor. To facilitate molecular analysis of the receptor-binding properties of SU and to characterize the cellular receptor employed by HTLV-1, we have expressed a recombinant SU fused to the Fc domain of human immunoglobulin G. Here, we demonstrate that this novel SU-immunoadhesin retains both the biochemical properties of Fc and the receptor-binding specificity of the HTLV-1 SU. We use this SU-immunoadhesin to demonstrate, by direct cell surface binding assays, that the receptor used by HTLV-1 has been conserved through vertebrate evolution. Moreover, using murine-human somatic cell hybrids we provide data that do not support the previously assigned location for the HTLV-1 receptor on human chromosome 17. Most importantly, we show that many cell lines that are resistant to HTLV-1 envelope-mediated infection and syncytium formation express functional receptors that are recognized by the HTLV-1 SU. Based on our results, we suggest that for some HTLV-1-resistant cell lines the block to viral entry occurs at a late post-receptor-binding step of the entry process. Our findings will be of value in developing new strategies to identify the cellular receptor used by HTLV-1.

An Antiviral Peptide Targets a Coiled-Coil Domain of the Human T-Cell Leukemia Virus Envelope Glycoprotein

ABSTRACT Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.

Viral interference with host mRNA surveillance, the nonsense-mediated mRNA decay (NMD) pathway, through a new function of HTLV-1 Rex: implications for retroviral replication.

Nonsense-mediated mRNA decay (NMD) is an essential and conserved cellular mRNA quality control mechanism. RNA signals to express viral genes from overlapping open reading frames potentially initiate NMD, nevertheless it is not clear whether viral RNAs are sensitive to NMD or if viruses have evolved mechanisms to evade NMD. Here we demonstrate that the genomic and full-length mRNAs of Human-T-cell Leukemia Virus type-I (HTLV-1), a retrovirus responsible for Adult T-cell Leukemia (ATL), are sensitive to NMD. They exhibit accelerated turnover in NMD-activated cells, while siRNA-mediated knockdown of NMD-master-regulator, UPF1, promotes enhanced stability of them. These effects on RNA stability were recapitulated by a reporter construct encoding the HTLV-1 translational frameshift signal of gag-pol. In agreement with the RNA stability, viral protein expression from the integrated provirus was inversely correlated with cellular NMD activity. We further demonstrated that the viral RNA-binding protein, Rex, approves the stability of viral RNA by inhibiting NMD. Significantly, Rex establishes a general block to NMD, as both NMD-responsive reporter transcripts and natural host-encoded NMD substrates were stabilized in the presence of Rex. Thus, we suggest that Rex not only stabilizes viral transcripts, but also perturbs cellular mRNA metabolism and host cell homeostasis via inhibition of NMD.

Soluble recombinant HTLV-1 surface glycoprotein competitively inhibits syncytia formation and viral infection of cells

Efficient entry into, and infection of, human cells by human T-cell leukaemia virus type-1 (HTLV-1) is mediated by the viral envelope glycoproteins, gp46 and gp21. The gp46 surface glycoprotein binds to an as yet unidentified cell surface receptor, thereby, allowing the gp21 transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. In the absence of membrane fusion viral penetration and entry into the host cell cannot occur. The envelope glycoproteins are also a major target for neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and represent ideal constituents for a recombinant HTLV-1 vaccine. Given the importance of the envelope proteins in HTLV-1 pathogenesis there is increasing interest in obtaining sufficient quantities of these proteins for biochemical, biophysical and biological analyses. We have now developed a system for production of large amounts of a glycosylated and functional form of soluble recombinant gp46 (sRgp46), and have used this recombinant material for analysis of envelope function and receptor binding activity. We find that, the sRgp46 molecules expressed in our system are immunologically indistinguishable from the native virally expressed surface glycoproteins; that sRgp46 binds to T-cells in a dose dependent and saturable manner; and that cell surface binding by sRgp46 can be inhibited by neutralising antibodies. Importantly, we demonstrate that these sRgp46 molecules potently inhibit syncytia formation and viral infection of target cells, and that regions outwith the SU domain of envelope are not required for binding to target cells or for inhibiting membrane fusion. The sRgp46 produced in our study will provide new opportunities to investigate envelope-receptor interactions, and will be of utility in defining the conformationally sensitive antigenic determinants of the HTLV-1 surface glycoprotein.

A Leptomycin B-sensitive Homologue of Human CRM1 Promotes Nuclear Export of Nuclear Export Sequence-containing Proteins inDrosophila Cells

The Rev protein of human immunodeficiency virus is a nuclear shuttling protein that promotes nuclear export of mRNAs that encode the viral structural proteins Gag, Pol, and Env. Rev binds to a highly structured RNA motif, the Rev-responsive element (RRE), that is present in all Rev-responsive viral transcripts and facilitates their entry into a nuclear export pathway by recruiting cellular export factors. In mammalian and yeast cells, the principal export receptor engaged by Rev has been identified as the importin/transportin family member CRM1/exportin 1. CRM1 binds directly to a leucine-rich nuclear export sequence (NES) present in Rev, and similar motifs have been identified in a variety of cellular nuclear shuttling proteins. We and our colleagues previously demonstrated that, in transfected Drosophila cells, HIV-1 Rev is fully functional and promotes expression of the viral envelope glycoprotein. We now demonstrate that the fundamental mechanism of Rev action in insect cells is identical to that observed in the mammalian systems. In particular, we show that Drosophila cells express a leptomycin B-sensitive homologue of human CRM1 that supports Rev-dependent gene expression and is required for nuclear export of NES-containing proteins in insect cells.

The Synthetic Peptide P-197 Inhibits Human T-Cell Leukemia Virus Type 1 Envelope-Mediated Syncytium Formation by a Mechanism That Is Independent of Hsc70

ABSTRACT Entry of human T-cell leukemia virus type 1 (HTLV-1) into cells is mediated by the viral envelope glycoproteins gp46 and gp21. The gp46 surface glycoprotein binds to a poorly characterized cell surface receptor, thereby promoting the gp21-dependent fusion of the viral and cellular membranes. Interestingly, a synthetic peptide (P-197) simulating amino acids 197 to 216 of gp46 strongly inhibits envelope-dependent membrane fusion with Molt-4 target cells. It has been suggested that this peptide acts by competitively binding to Hsc70, a putative cellular receptor for HTLV-1. We now demonstrate that P-197 inhibits membrane fusion among diverse HTLV-1-permissive target cells. Importantly, most of these cells lack detectable levels of Hsc70, indicating that P-197 inhibits membrane fusion by a mechanism that is Hsc70 independent. We now suggest that competition for primary receptor binding is unlikely to account for the inhibitory activity of P-197. Understanding the mechanism by which P-197 functions may reveal concepts of general relevance to antiretroviral chemotherapy.

Charge-Surrounded Pockets and Electrostatic Interactions with Small Ions Modulate the Activity of Retroviral Fusion Proteins.

Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry.

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

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.

Highly specific inhibition of leukaemia virus membrane fusion by interaction of peptide antagonists with a conserved region of the coiled coil of envelope

BackgroundHuman T-cell leukaemia virus (HTLV-1) and bovine leukaemia virus (BLV) entry into cells is mediated by envelope glycoprotein catalyzed membrane fusion and is achieved by folding of the transmembrane glycoprotein (TM) from a rod-like pre-hairpin intermediate to a trimer-of-hairpins. For HTLV-1 and for several virus groups this process is sensitive to inhibition by peptides that mimic the C-terminal α-helical region of the trimer-of-hairpins.ResultsWe now show that amino acids that are conserved between BLV and HTLV-1 TM tend to map to the hydrophobic groove of the central triple-stranded coiled coil and to the leash and C-terminal α-helical region (LHR) of the trimer-of-hairpins. Remarkably, despite this conservation, BLV envelope was profoundly resistant to inhibition by HTLV-1-derived LHR-mimetics. Conversely, a BLV LHR-mimetic peptide antagonized BLV envelope-mediated membrane fusion but failed to inhibit HTLV-1-induced fusion. Notably, conserved leucine residues are critical to the inhibitory activity of the BLV LHR-based peptides. Homology modeling indicated that hydrophobic residues in the BLV LHR likely make direct contact with a pocket at the membrane-proximal end of the core coiled-coil and disruption of these interactions severely impaired the activity of the BLV inhibitor. Finally, the structural predictions assisted the design of a more potent antagonist of BLV membrane fusion.ConclusionA conserved region of the HTLV-1 and BLV coiled coil is a target for peptide inhibitors of envelope-mediated membrane fusion and HTLV-1 entry. Nevertheless, the LHR-based inhibitors are highly specific to the virus from which the peptide was derived. We provide a model structure for the BLV LHR and coiled coil, which will facilitate comparative analysis of leukaemia virus TM function and may provide information of value in the development of improved, therapeutically relevant, antagonists of HTLV-1 entry into cells.