Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering.

Department of Molecular Biosciences and Howard Hughes Medical Institute, Northwestern University, Evanston, IL 60208.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 09/2012; 109(39):E2625-34. DOI: 10.1073/pnas.1213813109
Source: PubMed

ABSTRACT The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion.

Download full-text


Available from: Sayantan Bose, Jun 23, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: Measles virus (MeV), a morbillivirus within the paramyxovirus family, expresses two envelope glycoproteins. The attachment (H) protein mediates receptor binding, followed by triggering of the fusion (F) protein, which leads to merger of the viral envelope with target cell membranes. Receptor binding by members of related paramyxovirus genera rearranges the head domains of the attachment proteins, liberating an F-contact domain within the attachment protein helical stalk. However, morbillivirus glycoproteins first assemble intracellularly prior to receptor binding, raising the question of whether alternative protein-protein interfaces are involved or an entirely distinct triggering principle is employed. To test these possibilities, we generated headless H-stem mutants of progressively shorter length. Conformationally restricted H-stems remained capable of intracellular assembly with standard F and a soluble MeV F mutant. Proteolytic maturation of F, but not the altered biochemical conditions at the cell surface, reduces the strength of glycoprotein interaction, readying the complexes for triggering. F mutants stabilized in the prefusion conformation interact with H intracellularly and at the cell surface, while destabilized F mutants interact only intracellularly, prior to F maturation. These results showcase a MeV entry machinery that functionally varies conserved motifs of the proposed paramyxovirus infection pathway. Intracellular and plasma membrane-resident MeV glycoprotein complexes employ the same protein-protein interface. F maturation prepares for complex separation after triggering, and the H head domains in pre-receptor bound conformation prevent premature stalk rearrangements and F activation. Intracellular preassembly affects MeV fusion profiles and may contribute to the high cell-to-cell fusion activity characteristic for the morbillivirus genus.
    Journal of Virology 11/2014; 89(2). DOI:10.1128/JVI.02754-14 · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    PLoS Pathogens 05/2015; 11(5):e1004880. DOI:10.1371/journal.ppat.1004880 · 8.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The crystal structure of the F protein (prefusion form) of paramyxovirus parainfluenza virus 5 (PIV5), WR isolate, was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Journal of Virology 01/2015; 89(6). DOI:10.1128/JVI.03221-14 · 4.65 Impact Factor