Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process

Abstract

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the “head-to-spacer” interaction, which subsequently “unlocks” the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk “spacer” microdomain as a possible drug target for antiviral therapy.

Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM

“…Cell entry of morbilliviruses relies on the concerted action of two surface glycoproteins (H and F) that undergo conformational changes upon the binding of H to a host cell receptor, resulting in membrane fusion (26,27,48,(56)(57)(58)(59). In this study, we aimed at improving our fundamental understanding of the first step that triggers the CDV membrane fusion machinery.…”

“…We thus hypothesize that, besides the essential proper positioning of the H head's lateral region with the front H-binding site of the V domain of SLAM, productive triggering of the morbillivirus membrane fusion machinery may rely not only on proper affinity and binding between the two partners but also on a local conformational change. With regard to the recently proposed safety catch model of morbillivirus F activation (27,39), this local structural modification may involve a repositioning of the side chains of residues SLAM E123 and H R533 (or the analogous R529 residue of CDV H) that will translate into the initiation of the large-scale movements of the heads (putatively reaching conformational intermediates, such as the X and/or V shapes), ultimately leading to structural rearrangements of the H stalk. Although structural information argues against large-scale conformational changes occurring within the paramyxovirus attachment protein monomeric head unit as a consequence of receptor binding (22,(29)(30)(31)(60)(61)(62)(63)(64), subtle local modifications cannot be excluded in all models proposed for F activation (26,27,36,40).…”

“…Rather, our recent mechanistic study was suggestive of a model where the H proteins of morbilliviruses may initially assume a conformation (putatively a "4 heads down-like" state) where the head units lock the inherent F-triggering capacity of the stalk domain (referred to as the "H-autorepressed state") (27). Although we cannot exclude that the V or X shape of MeV H defines the autorepressed conformation, our model rather indicates that the crystal structure(s) determined for MeV H are either not biologically relevant or do not represent the initial pre-F-triggering state.…”

“…Morbilliviruses have evolved finely tuned entry machineries composed of two tightly interacting surface glycoproteins, of which one is a tetrameric attachment protein (H), whose ectodomain is composed of a membrane-proximal helical F-contacting/activating stalk region supporting a membrane-distal receptor-binding head domain, and the other is a trimeric fusion protein (F) (23)(24)(25)(26). It is proposed that upon receptor engagement, H proteins undergo sequential conformational changes that activate F trimers (27). In turn, prefusion F structures undergo a series of irreversible structural rearrangements that lead to the merging of the viral envelope with the host cell plasma membrane at neutral pH (24,28).…”

“…Although we cannot exclude that the V or X shape of MeV H defines the autorepressed conformation, our model rather indicates that the crystal structure(s) determined for MeV H are either not biologically relevant or do not represent the initial pre-F-triggering state. Upon binding to its cognate receptor, the putative autorepressed H structure would experience a first conformational change characterized by movements of the heads (perhaps at this stage reaching the V and/or X shape configuration) that would result in unlocking the stalk domain, which in turn would be freed to undergo a second structural rearrangement strictly required for productive F activation (referred to as the "safety catch" model) (27,39).…”