4664536_monash_81682.pdf (6.89 MB)
Studies on the activation mechanism of the HIV-1 membrane fusion glycoprotein, gp41
thesisposted on 2017-05-18, 04:17 authored by Khasawneh, Ashraf Ibrahim
Retrovirus entry follows receptor binding by the surface-exposed glycoprotein, which triggers the transmembrane glycoprotein (TM) to refold into a membrane fusion-active state. The trimer of hairpins is the best-characterized TM protein conformer and is composed of a central trimeric coiled coil associated with an outer antiparallel layer of 3 C-terminal segments. The trimer of hairpins represents the end point of a refolding pathway that apposes and catalyses fusion of the virus and cell membranes. The structure of pretriggered or „prefusion‟ TM is unknown. Cysteine replacement mutagenesis was applied to the human T cell leukemia virus type 1 TM, gp21, to ask if it resembles the prefusion form of its structural homologue, Ebola virus GP2, which is maintained as a trimer via a short coiled coil. Structural homology predicted that Ala-375 of gp21 is at the N-terminus of a corresponding short coiled coil and is oriented towards the 3-fold symmetry axis. The A375C substitution enabled interprotomer disulfide bonding, indicating that gp21 protomers are in close proximity in the Ala-375 region. I propose that prefusion gp21 resembles GP2 with a short coiled coil maintaining trimerization and functioning as a structural scaffold for the transition to fusion-active conformations. The fusion activation of the human immunodeficiency virus type-1 TM, gp41, involves its transition to the trimer of hairpins via a prehairpin intermediate. Cysteine-replacement mutagenesis was used to examine the structures of prefusion gp41 and recombinant models of the prehairpin and trimer of hairpins. The T569C mutation, in the C-terminal segment of the predicted coiled coil enabled quantitative interprotomer disulfide bonding in prefusion gp41, whereas T538C in the N-terminal region did not. Thus the coiled coil appears to be also present in prefusion gp41. By contrast, S538C led to efficient interprotomer disulfide formation in the prehairpin and trimer of hairpins. Threonine-538 is within the N-terminal “polar segment” which mediates functionally important hydrophobic interactions with the membrane-proximal ectodomain region (MPER) in the trimer of hairpins. A model for gp41 conformational activation is proposed, whereby a trimeric coiled coil in prefusion gp41 becomes extended into the polar segment in the prehairpin, providing a packing surface for C-terminal sequences to form a hairpin structure that extends to the terminal membrane-interactive sequences. The disulfide-bonded region (DSR) of gp41 mediates association with the receptor-binding glycoprotein, gp120, and transmission of the activation signal from receptor-bound gp120. Forced evolution of the W596L.K601D DSR mutant that lacks gp120-gp41 association, was used to identify functional determinants that are linked to the DSR and therefore may be involved in the activation process. A D601H pseudoreversion in the DSR restored gp120-gp41 association but required an additional D674E mutation in the MPER for optimised replication competence. In an independent culture, D601H emerged together with D674N and deletion of Thr-389-Trp-390 in variable region 4 of gp120. Conservative substitutions at Asp-674 modulated virus entry in the context of W596L.K601H, indicating for the first time that the MPER is functionally linked to the association/activation synapse of gp120-gp41. My results reveal new information about the fusion-activation mechanism employed by retroviruses.