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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.

mentioning

confidence: 95%

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

Navaratnarajah

Kumar

Generous

et al. 2014

“…Atomic structures of the attachment proteins (HN, H, or G) reveal a globular head, harboring a typical sialidase domain created by a six-bladed ␤-propeller fold (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). PIV1 to PIV5, MuV, and NDV have HN-type receptor-binding proteins possessing both hemagglutinating and neuraminidase activities, and HN binds sialic acid as a receptor through a central binding site within the ␤-propeller fold.…”

Section: Paramyxoviruses Are a Large Family Of Membrane-enveloped Negmentioning

confidence: 99%

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

Bose

Song

Jardetzky

et al. 2014

113

Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirusmediated membrane fusion. IMPORTANCE Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [

“…The ectodomains contain a membrane proximal stalk domain that supports the membrane-distal cuboidal head region (10,14). X-ray structures of many paramyxovirus attachment protein head domains invariably revealed a six-bladed beta-propeller structure, which serves as the receptor docking region (6,10,(15)(16)(17)(18)(19)(20). Partial crystal structures of the Newcastle disease virus (NDV) and parainfluenza virus type 5 (PIV5) attachment protein (HN) stalks highlighted a common four-helical-bundle (4HB) conformation (21,22).…”

mentioning

confidence: 99%

“…It is thought that H binding to a specific receptor on target cells triggers oligomeric conformational changes of the H stalk domain, which may, in turn, translate into F activation (5)(6)(7)(8)(9)(10)(11). As a consequence, prefusion F trimers undergo a series of conformational changes, which are associated with plasma membrane fusion activity, fusion pore formation, and cell entry (5,12,13).…”

mentioning

confidence: 99%

Molecular Determinants Defining the Triggering Range of Prefusion F Complexes of Canine Distemper Virus

Avila

Alves

Khosravi

et al. 2014

The morbillivirus cell entry machinery consists of a fusion (F) protein trimer that refolds to mediate membrane fusion following receptor-induced conformational changes in its binding partner, the tetrameric attachment (H) protein. To identify molecular determinants that control F refolding, we generated F chimeras between measles virus (MeV) and canine distemper virus (CDV). We located a central pocket in the globular head domain of CDV F that regulates the stability of the metastable, prefusion conformational state of the F trimer. Most mutations introduced into this "pocket'" appeared to mediate a destabilizing effect, a phenotype associated with enhanced membrane fusion activity. Strikingly, under specific triggering conditions (i.e., variation of receptor type and H protein origin), some F mutants also exhibited resistance to a potent morbillivirus entry inhibitor, which is known to block F triggering by enhancing the stability of prefusion F trimers. Our data reveal that the molecular nature of the F stimulus and the intrinsic stability of metastable prefusion F both regulate the efficiency of F refolding and escape from smallmolecule refolding blockers. IMPORTANCEWith the aim to better characterize the thermodynamic basis of morbillivirus membrane fusion for cell entry and spread, we report here that the activation energy barrier of prefusion F trimers together with the molecular nature of the triggering "stimulus" (attachment protein and receptor types) define a "triggering range," which governs the initiation of the membrane fusion process. A central "pocket" microdomain in the globular F head contributes substantially to the regulation of the conformational stability of the prefusion complexes. The triggering range also defines the mechanism of viral escape from entry inhibitors and describes how the cellular environment can affect membrane fusion efficiency.