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

Abstract

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.

Author Summary

Human T-cell leukaemia virus types-1 (HTLV-1) and bovine leukaemia virus (BLV) are divergent blood borne viruses that cause hematological malignancies in humans and cattle respectively. In common with other enveloped viruses, infection of cells by HTLV-1 and BLV is dependent on the membrane fusion properties of the viral envelope glycoproteins. Here we have solved the crystal structure of the BLV transmembrane glycoprotein, and, through a functional and comparative analysis with HTLV-1, we have identified features that are critical to fusion protein function. In particular, we demonstrate that electrostatic interactions with small ions dramatically stabilize the assembly and fusion-associated forms of the BLV TM, but are not required for the cell surface display of native pre-fusogenic envelope. Moreover, we show that charged residues that border a deep hydrophobic pocket contribute directly to appropriate folding of fusion-active envelope and are critical to membrane fusion. Importantly, the charged residues that border the pocket are key features that determine the specificity and activity of peptide inhibitors of envelope function. Our study demonstrates that charge-surrounded pockets and electrostatic interactions with small ions are significant leitmotifs of diverse class-1 fusion proteins and that these elements represent ideal targets for novel small-molecule inhibitors of viral entry.