The Measles Virus Hemagglutinin Stalk: Structures and Functions of the Central Fusion Activation and Membrane-Proximal Segments

Abstract

The measles virus (MeV) membrane fusion apparatus consists of a fusion protein trimer and an attachment protein tetramer. To trigger membrane fusion, the heads of the MeV attachment protein, hemagglutinin (H), bind cellular receptors while the 96-residue-long H stalk transmits the triggering signal. Structural and functional studies of the triggering mechanism of other paramyxoviruses suggest that receptor binding to their hemagglutinin-neuraminidase (HN) results in signal transmission through the central segments of their stalks. To gain insight into H-stalk structure and function, we individually replaced its residues with cysteine. We then assessed how stable the mutant proteins are, how efficiently they can be cross-linked by disulfide bonds, whether cross-linking results in loss of function, and, in this case, whether disulfide bond reduction restores function. While many residues in the central segment of the stalk and in the spacer segment above it can be efficiently cross-linked by engineered disulfide bonds, we report here that residues 59 to 79 cannot, suggesting that the 20 membrane-proximal residues are not engaged in a tetrameric structure. Rescue-of-function studies by disulfide bond reduction resulted in the redefinition and extension of the central fusion-activation segment as covering residues 84 to 117. In particular, we identified four residues located between positions 92 and 99, the function of which cannot be restored by disulfide bond reduction after cysteine mutagenesis. These mutant H proteins reached the cell surface as complex oligomers but could not trigger membrane fusion. We discuss these observations in the context of the stalk exposure model of membrane fusion triggering by paramyxoviruses. IMPORTANCEMeasles virus, while being targeted for eradication, still causes significant morbidity and mortality. Here, we seek to understand how it enters cells by membrane fusion. Two viral integral membrane glycoproteins (hemagglutinin tetramers and fusion protein trimers) mediate the concerted receptor recognition and membrane fusion processes. Since previous studies have suggested that the hemagglutinin stalk transmits the triggering signal to the fusion protein trimer, we completed an analysis of its structure and function by systematic Cys mutagenesis. We report that while certain residues of the central stalk segment confer specificity to the interaction with the fusion protein trimer, others are necessary to allow folding of the H-oligomer in a standard conformation conducive to fusion triggering, and still other residues sustain the conformational change that transmits the fusiontriggering signal.

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.…”

A Structurally Unresolved Head Segment of Defined Length Favors Proper Measles Virus Hemagglutinin Tetramerization and Efficient Membrane Fusion Triggering

“…Figure 3B plots the mean fluorescence intensities of different MeV H mutants as percentages of that of the standard H protein at the cell surface. Only deletion mutant Del_167-175 reached the cell surface with less than 5% of the efficiency of standard H. The remaining mutants were present at the cell surface at levels 45 to 95% of that of standard H, which is sufficient for full fusion support function (32,33). Mutant Ala_167-175 reached the cell surface at ϳ65% efficiency and exhibited full fusion support function.…”

“…Length compensation with Ser/Gly repeats fully restored the fusion-triggering function of a 9-residue deletion and partially restored the function of the entire 17-residue deletion. Thus, in combination with the dimeric top segments of the H stalk (32,33), the unresolved 167-183 H-head segment may act as a leash, which must be long enough for the heads to move about the stalk and adopt the metastable "heads-down" conformation.…”

“…The semiquantitative cell-to-cell fusion assay was performed as described previously (33). Briefly, 0.5 g each of three plasmids, encoding the H protein, the F protein, and green fluorescent protein (GFP), were transfected into 1.0 ϫ 10 5 Vero cells in 24-well plates using Lipofectamine 2000 (Life Technologies, Grand Island, NY) according to the manufacturer's instructions.…”

“…All these membranebound receptors may "pull" on head dimers, destabilizing them (11). The central segment of the H stalk then integrates and relays the triggering signals to the F trimer, which unfolds to fuse membranes (29)(30)(31)(32)(33).…”