Probing the Fusion-Active Structures of Envelope of Human T-cell Leukaemia Virus Type-1 with Conformation-Specific Monoclonal Antibodies

Abstract

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.

Resistance to Neutralization by Antibodies Targeting the Coiled Coil of Fusion-active Envelope Is a Common Feature of Retroviruses

“…4). Moreover, these MAbs failed to bind to a set of overlapping but monomeric 27-35 amino acid synthetic peptides (23,24) that cover the entire core coiled coil of envelope (data not shown). Thus binding of each of the MAbs to the coiled coil is highly dependent on appropriate presentation of epitopes that are sensitive to the conformational status of the coiled coil.…”

“…1). This recombinant TM-derived fusion protein, referred to as MBP-fishhook, is trimeric as revealed by size exclusion chromatography and by native PAGE (12), and specifically binds to synthetic peptides that mimic the C-helical region of the HTLV-1 trimer-of-hairpins (12,24). Thus the recombinant TM fragment faithfully mimics the critical features of the exposed core coiled coil of fusion-active TM.…”

“…1B) that faithfully mimics the exposed coiled coil of fusion-active envelope (12,23,24). We therefore anticipated that some antibodies would exhibit a degree of conformational specificity to the epitopes recognized.…”

“…Plasmids-The plasmids pHTE-1 (22), pMAL-gp21hairpin (MBP-hairpin), pMAL-gp21fishhook (MBP-fishhook) (12), pMAL-N-helix (MBP-N-helix), and pMAL-STOP (MBP-stop) have been described previously (23,24).…”

“…Bio-P cr -400 (2.0 g/ml) was incubated with immobilized MBP-fishhook in the presence or absence of the test antibody at the indicated concentrations, at room temperature, for 1 h. Preliminary binding assays indicated that a peptide concentration of 2.0 g/ml is within the upper linear range of the concentration curve for binding of the peptide to the coiled coil (24). Subsequently, plates were washed (five times) to remove unbound peptide, and bound complexes were incubated for 1 h at room temperature with 100 l of streptavidin/horseradish peroxidase (Sigma) (1:10,000 dilution).…”