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

ABSTRACTParamyxoviruses include several insidious and ubiquitous pathogens of humans and animals, with measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin [H]) tetramer and a fusion (F) protein trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or cocrystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high-molecular-weight H oligomers. Some oligomers had reduced fusion trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers.IMPORTANCEUnderstanding the molecular mechanism of membrane fusion triggering may allow development of new antiviral strategies. The fusion apparatus of paramyxoviruses consists of a receptor binding tetramer and a fusion protein trimer. Structural analyses of the receptor binding hemagglutinin-neuraminidases of certain paramyxoviruses suggest that fusion triggering is preceded by relocation of its head domains, facilitated by flexible linkers. Having noted a structurally unresolved 17-residue segment linking the globular heads to the tetrameric stalk of the measles virus hemagglutinin (H), we asked whether and how it may facilitate membrane fusion triggering. We conclude that, together with the top of the stalk, the flexible linker keeps H heads on a leash long enough to adopt a triggering-permissive conformation but short enough to limit roaming and improper contacts with heads of neighboring tetramers. All morbillivirus H-protein heads appear to be connected to their stalks through a “leash,” suggesting a conserved triggering mechanism.

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The Measles Virus Fusion Protein Transmembrane Region Modulates Availability of an Active Glycoprotein Complex and Fusion Efficiency

Journal of Virology ◽

10.1128/jvi.00779-08 ◽

2008 ◽

Vol 82 (22) ◽

pp. 11437-11445 ◽

Cited By ~ 24

Author(s):

Michael D. Mühlebach ◽

Vincent H. J. Leonard ◽

Roberto Cattaneo

Keyword(s):

Membrane Fusion ◽

Receptor Binding ◽

Measles Virus ◽

Protein Structures ◽

Proteolytic Processing ◽

Single Mutation ◽

Fusion Activity ◽

F Protein ◽

Glycoprotein Complex ◽

H Protein

ABSTRACT The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H) protein. Structures and single amino acids influencing fusion function have been identified in the F-protein ectodomain and cytoplasmic tail, but not in its transmembrane (TM) region. Since this region influences function of the envelope proteins of other viruses, we examined its role in the MV F protein. Alanine-scanning mutagenesis revealed that an F protein with a single mutation of a central TM region leucine (L507A) was more fusogenic than the unmodified F protein while retaining similar kinetics of proteolytic processing. In contrast, substitution of residues located near the edges of the lipid bilayer reduced fusion activity. This was true not only when the mutated F proteins were coexpressed with H but also in the context of infections with recombinant viruses. Analysis of the H-F complexes with reduced fusion activities revealed that more precursor (F0) than activated (F1+2) protein coprecipitated with H. In contrast, in complexes with enhanced fusion activity, including H-FL507A, the F0/F1+2 ratio shifted toward F1+2. Thus, fusion activity correlated with an active F-H protein complex, and the MV F protein TM region modulated availability of this complex.

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Short-stalk isoforms of CADM1 and CADM2 trigger neuropathogenic measles virus-mediated membrane fusion by interacting with the viral hemagglutinin

Journal of Virology ◽

10.1128/jvi.01949-21 ◽

2021 ◽

Author(s):

Ryuichi Takemoto ◽

Tateki Suzuki ◽

Takao Hashiguchi ◽

Yusuke Yanagi ◽

Yuta Shirogane

Keyword(s):

Cell Membrane ◽

Membrane Fusion ◽

Measles Virus ◽

Subacute Sclerosing Panencephalitis ◽

Febrile Illness ◽

Host Factors ◽

F Protein ◽

Short Stalk ◽

Head Domain ◽

H Protein

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae , usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). MeV bears two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. The H protein possesses a head domain that initially mediates receptor binding and a stalk domain that subsequently transmits the fusion-triggering signal to the F protein. We have recently shown that cell adhesion molecule 1 (CADM1, also known as IGSF4A, Necl-2, SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors enabling cell-cell membrane fusion mediated by hyperfusogenic F proteins of neuropathogenic MeVs as well as MeV spread between neurons lacking the known receptors. CADM1 and CADM2 interact in cis with the H protein on the same cell membrane, triggering hyperfusogenic F protein-mediated membrane fusion. Multiple isoforms of CADM1 and CADM2 containing various lengths of their stalk regions are generated by alternative splicing. Here we show that only short-stalk isoforms of CADM1 and CADM2 predominantly expressed in the brain induce hyperfusogenic F protein-mediated membrane fusion. While the known receptors interact in trans with the H protein through its head domain, these isoforms can interact in cis even with the H protein lacking the head domain and trigger membrane fusion, presumably through its stalk domain. Thus, our results unveil a new mechanism of viral fusion triggering by host factors. Importance Measles, an acute febrile illness with skin rash, is still an important cause of childhood morbidity and mortality worldwide. Measles virus (MeV), the causative agent of measles, may also cause a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. The disease is fatal, and no effective therapy is available. Recently, we have reported that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV cell-to-cell spread in neurons. These molecules interact in cis with the MeV attachment protein on the same cell membrane, triggering the fusion protein and causing membrane fusion. CADM1 and CADM2 are known to exist in multiple splice isoforms. In this study, we report that their short-stalk isoforms can induce membrane fusion by interacting in cis with the viral attachment protein independently of its receptor-binding head domain. This finding may have important implications for cis -acting fusion triggering by host factors.

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Weak cis and trans Interactions of the Hemagglutinin with Receptors Trigger Fusion Proteins of Neuropathogenic Measles Virus Isolates

Journal of Virology ◽

10.1128/jvi.01727-19 ◽

2019 ◽

Vol 94 (2) ◽

Cited By ~ 1

Author(s):

Yuta Shirogane ◽

Takao Hashiguchi ◽

Yusuke Yanagi

Keyword(s):

Membrane Fusion ◽

Measles Virus ◽

Subacute Sclerosing Panencephalitis ◽

Target Cells ◽

Wild Type ◽

F Protein ◽

The Brain ◽

H Protein ◽

In Cells ◽

Cell Cell

ABSTRACT Measles virus (MeV) is an enveloped RNA virus bearing two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. Upon receptor binding, the H protein triggers conformational changes of the F protein, causing membrane fusion and subsequent virus entry. MeV may persist in the brain, infecting neurons and causing fatal subacute sclerosing panencephalitis (SSPE). Since neurons do not express either of the MeV receptors, signaling lymphocytic activation molecule (SLAM; also called CD150) and nectin-4, how MeV propagates in neurons is unknown. Recent studies have shown that specific substitutions in the F protein found in MeV isolates from SSPE patients are critical for MeV neuropathogenicity by rendering the protein unstable and hyperfusogenic. Recombinant MeVs possessing the F proteins with such substitutions can spread in primary human neurons and in the brains of mice and hamsters and induce cell-cell fusion in cells lacking SLAM and nectin-4. Here, we show that receptor-blind mutant H proteins that have decreased binding affinities to receptors can support membrane fusion mediated by hyperfusogenic mutant F proteins, but not the wild-type F protein, in cells expressing the corresponding receptors. The results suggest that weak interactions of the H protein with certain molecules (putative neuron receptors) trigger hyperfusogenic F proteins in SSPE patients. Notably, where cell-cell contacts are ensured, the weak cis interaction of the H protein with SLAM on the same cell surface also could trigger hyperfusogenic F proteins. Some enveloped viruses may exploit such cis interactions with receptors to infect target cells, especially in cell-to-cell transmission. IMPORTANCE Measles virus (MeV) may persist in the brain, causing incurable subacute sclerosing panencephalitis (SSPE). Because neurons, the main target in SSPE, do not express receptors for wild-type (WT) MeV, how MeV propagates in the brain is a key question for the disease. Recent studies have demonstrated that specific substitutions in the MeV fusion (F) protein are critical for neuropathogenicity. Here, we show that weak cis and trans interactions of the MeV attachment protein with receptors that are not sufficient to trigger the WT MeV F protein can trigger the mutant F proteins from neuropathogenic MeV isolates. Our study not only provides an important clue to understand MeV neuropathogenicity but also reveals a novel viral strategy to expand cell tropism.

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Fully Retargeted Oncolytic Measles Virus Specific for the WUE-Ag Results in Specific Lysis of Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v106.11.3053.3053 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3053-3053 ◽

Cited By ~ 1

Author(s):

Horst-Dieter Hummel ◽

Takafumi Nakamura ◽

Gabriele Kuntz ◽

Hermann Einsele ◽

Steve J. Russell ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

Membrane Fusion ◽

Cell Lines ◽

Measles Virus ◽

Cell Types ◽

Myeloma Cells ◽

Syncytia Formation ◽

Rpmi 8226 ◽

H Protein

Abstract Effective cancer therapy with a minimum of undesired side effects in multiple myeloma (MM) requires specifity of the therapeutic agent against the neoplastic cells. One approach to reach this aim is virotherapy using oncolytic measles virus. Attenuated replication-competent Edmonston lineage strains of measles virus (MV-Edm) have proven anti-tumor activity against xenograft models of human multiple myeloma, ovarian cancer, lymphoma and glioma. The virus is selectively oncolytic, causing extensive lethal cell to cell fusion via CD46, which is more highly expressed on tumor cells than on normal cells. However, MV-Edm retains the capacity to infect a variety of nontransformed cell types via its native receptors, CD46 and SLAM which are present on many different cell types. One approach to avoid this risk is to engineer the viral attachment protein to ablate its natural tropisms and at the same time, redirect its specificity to interact with alternative tumor specific receptors. The native measles Hemagglutinin (H) protein recognizes CD46 or SLAM resulting in membrane fusion and syncytia formation of cells. In this work the H protein was engineered to restrict and retarget membrane fusion through the display of a single-chain antibody (scFv) recognizing the Wue-1-antigen known to be highly specific for MM cells and abrogation of native measles binding domains for CD46 and SLAM by mutation. This modified H protein (chimeric H) was cloned in a full-length viral backbone including EGFP enabling to detect infected cells and syncytia formation under the UV light emitting green fluorescence (EGFP). On the basis of the parental measles virus expressing EGFP (MV-GFP) two different viruses were generated: non-ablated virus expressing the chimeric H protein including the scFv Wue-1 still competent to infect cells expressing CD46 and SLAM (MV-W1) with the chimeric H protein but ablated for the interactions with CD46 and SLAM (MV-W2). The genetically modified viruses propagated as recently described (Nat. Biotech., Vol.23, Nr.2, Feb.2005, pp.209–214.). To determine if the fully retargeted MV-Edm would be able to infect MM cell lines selectively a first array of infection assays was performed using the MM cell lines RPMI 8226 and ARH-77 expressing the Wue-1 antigen as expected targets and K562 and healthy CD40L activated CD19 positive B cells as controls. 24 to 96 hours after infection with MV-GFP, MV-W1 and MV-W2 we observed syncytia formation and expression of EGFP with MV-GFP and MV-W1 in all cells indicating that the modification of the virus with the scFv-Wue-1 doesn’t alter the potential to infect and kill cells compared to the parental virus. In contrast MV-W2 was able to form only EGFP positive plaques with the Wue-1 antigen positive cells RPMI 8226 and ARH-77 but did not infect K562 or CD19 positve B cells both negative for Wue-1 antigen. These results indicate that the measles virus vaccine strain Edmonston B can be modified to express a scFv recognizing the Wue-1 antigen. In addition the native H protein can be mutated resulting in ablation of the natural tropism towards CD46 and SLAM positive cells. Viruses with these modifications can be rescued and propagated in vitro and selectively infected Multiple Myeloma cells without causing damage to normal B-cell progenitors.

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Importance of the Cytoplasmic Tails of the Measles Virus Glycoproteins for Fusogenic Activity and the Generation of Recombinant Measles Viruses

Journal of Virology ◽

10.1128/jvi.76.14.7174-7186.2002 ◽

2002 ◽

Vol 76 (14) ◽

pp. 7174-7186 ◽

Cited By ~ 53

Author(s):

Markus Moll ◽

Hans-Dieter Klenk ◽

Andrea Maisner

Keyword(s):

Amino Acids ◽

Cell Fusion ◽

Receptor Binding ◽

Measles Virus ◽

Local Density ◽

Biologically Active ◽

Host Cells ◽

Susceptible Host ◽

Cytoplasmic Tails ◽

H Protein

ABSTRACT The generation of replication-competent measles virus (MV) depends on the incorporation of biologically active, fusogenic glycoprotein complexes, which are required for attachment and penetration into susceptible host cells and for direct virus spread by cell-to-cell fusion. Whereas multiple studies have analyzed the importance of the ectodomains of the MV glycoproteins hemagglutinin (H) and fusion protein (F), we have investigated the role of the cytoplasmic tails of the F and H proteins for the formation of fusogenic complexes. Deletions in the cytoplasmic tails of transiently expressed MV glycoproteins were found to have varying effects on receptor binding, fusion, or fusion promotion activity. F tail truncation to only three amino acids did not affect fusion capacity. In contrast, truncation of the H cytoplasmic tail was limited. H protein mutants with cytoplasmic tails of <14 residues no longer supported F-mediated cell fusion, predominantly due to a decrease in surface expression and receptor binding. This indicates that a minimal length of the H protein tail of 14 amino acids is required to ensure a threshold local density to have sufficient accumulation of fusogenic H-F complexes. By using reverse genetics, a recombinant MV with an F tail of three amino acids (rMV-FcΔ30), as well as an MV with an H tail of 14 residues (rMV-HcΔ20), could be rescued, whereas generation of viruses with shorter H tails failed. Thus, glycoprotein truncation does not interfere with the successful generation of recombinant MV if fusion competence is maintained.

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Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity

Viruses ◽

10.3390/v13010128 ◽

2021 ◽

Vol 13 (1) ◽

pp. 128

Author(s):

Neeta Shrestha ◽

Flavio M. Gall ◽

Jonathan Vesin ◽

Marc Chambon ◽

Gerardo Turcatti ◽

...

Keyword(s):

Membrane Fusion ◽

Small Molecule ◽

High Throughput Screening ◽

Measles Virus ◽

Canine Distemper Virus ◽

Rna Virus ◽

Preventive Measure ◽

Close Relative ◽

Fusion Activity ◽

Canine Distemper

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

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A Quantitative and Kinetic Fusion Protein-Triggering Assay Can Discern Distinct Steps in the Nipah Virus Membrane Fusion Cascade

Journal of Virology ◽

10.1128/jvi.00469-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8033-8041 ◽

Cited By ~ 28

Author(s):

Hector C. Aguilar ◽

Vanessa Aspericueta ◽

Lindsey R. Robinson ◽

Karen E. Aanensen ◽

Benhur Lee

Keyword(s):

Membrane Fusion ◽

Receptor Binding ◽

Nipah Virus ◽

Heptad Repeat ◽

Critical Parameters ◽

Second Phase ◽

Two Phase ◽

Glycoprotein G ◽

Fusion Glycoprotein ◽

Functional Features

ABSTRACT The deadly paramyxovirus Nipah virus (NiV) contains a fusion glycoprotein (F) with canonical structural and functional features common to its class. Receptor binding to the NiV attachment glycoprotein (G) triggers F to undergo a two-phase conformational cascade: the first phase progresses from a metastable prefusion state to a prehairpin intermediate (PHI), while the second phase is marked by transition from the PHI to the six-helix-bundle hairpin. The PHI can be captured with peptides that mimic F's heptad repeat regions, and here we utilized a NiV heptad repeat peptide to quantify PHI formation and the half-lives (t 1/2) of the first and second fusion cascade phases. We found that ephrinB2 receptor binding to G triggered ∼2-fold more F than that triggered by ephrinB3, consistent with the increased rate and extent of fusion observed with ephrinB2- versus ephrinB3-expressing cells. In addition, for a series of hyper- and hypofusogenic F mutants, we quantified F-triggering capacities and measured the kinetics of their fusion cascade phases. Hyper- and hypofusogenicity can each be manifested through distinct stages of the fusion cascade, giving rise to vastly different half-lives for the first (t 1/2, 1.9 to 7.5 min) or second (t 1/2, 1.5 to 15.6 min) phase. While three mutants had a shorter first phase and a longer second phase than the wild-type protein, one mutant had the opposite phenotype. Thus, our results reveal multiple critical parameters that govern the paramyxovirus fusion cascade, and our assays should help efforts to elucidate other class I membrane fusion processes.

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Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering

Journal of Virology ◽

10.1128/jvi.00679-18 ◽

2018 ◽

Vol 92 (18) ◽

Cited By ~ 7

Author(s):

Michael Herren ◽

Neeta Shrestha ◽

Marianne Wyss ◽

Andreas Zurbriggen ◽

Philippe Plattet

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Surface ◽

Membrane Fusion ◽

Measles Virus ◽

Cell Entry ◽

Regulatory Role ◽

Fusion Activity ◽

Animal Pathogens

ABSTRACTMorbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerizationper sewas not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCEMeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

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