Canine Distemper Virus Envelope Protein Interactions Modulated by Hydrophobic Residues in the Fusion Protein Globular Head

Avila, Mislay; Khosravi, Mojtaba; Alves, Lisa; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Plemper, Richard K.; Plattet, Philippe

doi:10.1128/jvi.01828-14

Cited by 21 publications

(18 citation statements)

References 40 publications

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“…In fact, recent functional data obtained with henipaviruses are consistent with this idea (41). However, although the latest mechanistic data obtained with morbilliviruses support the safety catch model for F activation (39,65,66), we cannot formally exclude at this stage that the proposed productive SLAM-H interactions translate into membrane fusion triggering by one of the alternative mechanisms (stalk exposure/induced fit [36], attachment protein oligomerization [40], and bidentate attachment protein-F interaction [41] …”

Section: Discussionmentioning

confidence: 56%

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

Khosravi

Bringolf

Röthlisberger

et al. 2016

J Virol

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Measles virus (MeV) and canine distemper virus (CDV) possess tetrameric attachment proteins (H) and trimeric fusion proteins, which cooperate with either SLAM or nectin 4 receptors to trigger membrane fusion for cell entry. While the MeV H-SLAM cocrystal structure revealed the binding interface, two distinct oligomeric H assemblies were also determined. In one of the conformations, two SLAM units were sandwiched between two discrete H head domains, thus spotlighting two binding interfaces ("front" and "back"). Here, we investigated the functional relevance of both interfaces in activating the CDV membrane fusion machinery. While alanine-scanning mutagenesis identified five critical regulatory residues in the front H-binding site of SLAM, the replacement of a conserved glutamate residue (E at position 123, replaced with A [E123A]) led to the most pronounced impact on fusion promotion. Intriguingly, while determination of the interaction of H with the receptor using soluble constructs revealed reduced binding for the identified SLAM mutants, no effect was recorded when physical interaction was investigated with the full-length counterparts of both molecules. Conversely, although mutagenesis of three strategically selected residues within the back H-binding site of SLAM did not substantially affect fusion triggering, nevertheless, the mutants weakened the H-SLAM interaction recorded with the membrane-anchored protein constructs. Collectively, our findings support a mode of binding between the attachment protein and the V domain of SLAM that is common to all morbilliviruses and suggest a major role of the SLAM residue E123, located at the front H-binding site, in triggering the fusion machinery. However, our data additionally support the hypothesis that other microdomain(s) of both glycoproteins (including the back H-binding site) might be required to achieve fully productive H-SLAM interactions. IMPORTANCEA complete understanding of the measles virus and canine distemper virus (CDV) cell entry molecular framework is still lacking, thus impeding the rational design of antivirals. Both viruses share many biological features that partially rely on the use of analogous Ig-like host cell receptors, namely, SLAM and nectin 4, for entering immune and epithelial cells, respectively. Here, we provide evidence that the mode of binding between the membrane-distal V domain of SLAM and the attachment protein (H) of morbilliviruses is very likely conserved. Moreover, although structural information revealed two discrete conformational states of H, one of the structures displayed two H-SLAM binding interfaces ("front" and "back"). Our data not only spotlight the front H-binding site of SLAM as the main determinant of membrane fusion promotion but suggest that the triggering efficiency of the viral entry machinery may rely on a local conformational change within the front H-SLAM interactive site rather than the binding affinity. Measles virus (MeV) and canine distemper virus (CDV) belong to the Morbillivirus genus of the P...

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Section: Discussionmentioning

confidence: 56%

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

Khosravi

Bringolf

Röthlisberger

et al. 2016

J Virol

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“…An insertion of a FLAG tag in the F ectodomain of the related morbilliviruses canine distemper virus (CDV) and measles virus (MeV) does not significantly modulate their bioactivity (40,41). In those studies, the FLAG tag was inserted in the F 2 subunit close to the furin cleavage site (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Although extracellular tags (HA) have been used for henipavirus Gs, they have not been described for the F glycoproteins (8). Here, we created well-expressed and fully functional extracellularly FLAG-tagged NiV F and HeV F at locations similar to those recently reported for members of the closely related Morbillivirus genus (35,40,41). Our results revealed equal homologous but unequal heterologous glycoprotein F-triggering capabilities.…”

mentioning

confidence: 92%

Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes

Bradel-Tretheway

Zamora

Stone

et al. 2019

J Virol

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Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus. The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs. IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.

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“…While intracellular assembly of morbillivirus H//F complexes [ 48 , 67 ] directly challenged the “stalk exposure/induced fit” hypotheses of paramyxovirus cell entry [ 40 , 46 ], the recently proposed model of membrane fusion activation mediated by CDV [ 68 ] and MeV [ 48 ] (referred to as the “safety catch” model in the latter study) reconciled the different datasets. In the present study, we not only deliver tangible mechanistic insight in support of the safety catch hypothesis, but also substantially extend the model by unravelling the molecular nature governing the dynamics of morbillivirus membrane fusion triggering; prior to receptor-binding, H-tetramers initially fold into an auto-repressed conformational state, where the inherent F-triggering activity of the stalk is silenced by a specific positioning of the head domains.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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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.

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Canine Distemper Virus Envelope Protein Interactions Modulated by Hydrophobic Residues in the Fusion Protein Globular Head

Cited by 21 publications

References 40 publications

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

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

Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes

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

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