The SI Strain of Measles Virus Derived from a Patient with Subacute Sclerosing Panencephalitis Possesses Typical Genome Alterations and Unique Amino Acid Changes That Modulate Receptor Specificity and Reduce Membrane Fusion Activity

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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A single amino acid substitution in the measles virus F2 protein reciprocally modulates membrane fusion activity in pathogenic and oncolytic strains

Virus Research ◽

10.1016/j.virusres.2013.12.016 ◽

2014 ◽

Vol 180 ◽

pp. 43-48 ◽

Cited By ~ 7

Author(s):

Sabine Heidmeier ◽

Jan R.H. Hanauer ◽

Katrin Friedrich ◽

Steffen Prüfer ◽

Irene C. Schneider ◽

...

Keyword(s):

Amino Acid ◽

Membrane Fusion ◽

Amino Acid Substitution ◽

Measles Virus ◽

Single Amino Acid Substitution ◽

Single Amino Acid ◽

Fusion Activity

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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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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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Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients

mBio ◽

10.1128/mbio.00081-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 34

Author(s):

Yohei Watanabe ◽

Yasuha Arai ◽

Tomo Daidoji ◽

Norihito Kawashita ◽

Madiha S. Ibrahim ◽

...

Keyword(s):

Viral Replication ◽

Membrane Fusion ◽

Compensatory Mechanism ◽

Fusion Activity ◽

Receptor Specificity ◽

Airway Epithelia ◽

Viral Receptor ◽

Human Airway ◽

H5n1 Influenza ◽

Human Airway Epithelia

ABSTRACTA change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses.IMPORTANCECirculation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism.

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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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Measles Virus Mutants Possessing the Fusion Protein with Enhanced Fusion Activity Spread Effectively in Neuronal Cells, but Not in Other Cells, without Causing Strong Cytopathology

Journal of Virology ◽

10.1128/jvi.03346-14 ◽

2014 ◽

Vol 89 (5) ◽

pp. 2710-2717 ◽

Cited By ~ 22

Author(s):

Shumpei Watanabe ◽

Shinji Ohno ◽

Yuta Shirogane ◽

Satoshi O. Suzuki ◽

Ritsuko Koga ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Fusion Protein ◽

Measles Virus ◽

Subacute Sclerosing Panencephalitis ◽

Neuronal Cells ◽

Extracellular Domain ◽

Fusion Activity ◽

F Protein ◽

The Central Nervous System

ABSTRACTSubacute sclerosing panencephalitis (SSPE) is caused by persistent measles virus (MV) infection in the central nervous system (CNS). Since human neurons, its main target cells, do not express known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), it remains to be understood how MV infects and spreads in them. We have recently reported that fusion-enhancing substitutions in the extracellular domain of the MV fusion (F) protein (T461I and S103I/N462S/N465S), which are found in multiple SSPE virus isolates, promote MV spread in human neuroblastoma cell lines and brains of suckling hamsters. In this study, we show that hyperfusogenic viruses with these substitutions also spread efficiently in human primary neuron cultures without inducing syncytia. These substitutions were found to destabilize the prefusion conformation of the F protein trimer, thereby enhancing fusion activity. However, these hyperfusogenic viruses exhibited stronger cytopathology and produced lower titers at later time points in SLAM- or nectin 4-expressing cells compared to the wild-type MV. Although these viruses spread efficiently in the brains of SLAM knock-in mice, they did not in the spleens. Taken together, the results suggest that enhanced fusion activity is beneficial for MV to spread in neuronal cells where no cytopathology occurs, but detrimental to other types of cells due to strong cytopathology. Acquisition of enhanced fusion activity through substitutions in the extracellular domain of the F protein may be crucial for MV's extensive spread in the CNS and development of SSPE.IMPORTANCESubacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). Its cause is not well understood, and no effective therapy is currently available. Recently, we have reported that enhanced fusion activity of MV through the mutations in its fusion protein is a major determinant of efficient virus spread in human neuronal cells and brains of suckling hamsters. In this study, we show that those mutations render the conformation of the fusion protein less stable, thereby making it hyperfusogenic. Our results also show that enhanced fusion activity is beneficial for MV to spread in the CNS but detrimental to other types of cells in peripheral tissues, which are strongly damaged by the virus. Our findings provide important insight into the mechanism for the development of SSPE after MV infection.

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The Ig-Like V-Type Domain of Paired Ig-Like Type 2 Receptor Alpha Is Critical for Herpes Simplex Virus Type 1-Mediated Membrane Fusion

Journal of Virology ◽

10.1128/jvi.01039-10 ◽

2010 ◽

Vol 84 (17) ◽

pp. 8664-8672 ◽

Cited By ~ 21

Author(s):

Qing Fan ◽

Richard Longnecker

Keyword(s):

Amino Acid ◽

Herpes Simplex ◽

Membrane Fusion ◽

Fusion Activity ◽

Acid Protein ◽

Simplex Virus ◽

Amino Acid Protein ◽

Hsv 1

ABSTRACT Paired immunoglobulin (Ig)-like type 2 receptor alpha (PILRα) and PILRβ are paired receptors that are highly homologous to each other. When engaged by ligand, PILRα is inhibitory whereas PILRβ is activating. PILRα is a newly identified herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) receptor and is associated with membrane fusion and entry activity of HSV-1. PILRα is a 303-amino-acid protein with an Ig-like V (variable)-type domain from amino acid 31 to 150, whereas PILRβ is a 217-amino-acid protein with an Ig-like V-type domain from amino acid 21 to 143. We report that PILRβ is not a receptor for HSV-1 and HSV-2. Domain swaps between PILRα and PILRβ reveal that the Ig-like V-type domain of PILRα, but not PILRβ, plays a critical role in cell membrane fusion activity and the binding of PILRα to gB. Individual replacement of 13 amino acids in PILRα showed that most of these mutations had no effect on cell fusion activity. However, mutation of the tryptophan residue at amino acid 139 significantly impaired cell fusion activity for HSV-1 and eliminated binding to gB.

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The Pre-Transmembrane Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein Is Critical for Membrane Fusion and Virus Infectivity

Journal of Virology ◽

10.1128/jvi.01085-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 10993-11004 ◽

Cited By ~ 16

Author(s):

Zhaofei Li ◽

Gary W. Blissard

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Membrane Fusion ◽

Transmembrane Domain ◽

Aromatic Amino Acids ◽

Autographa Californica ◽

Fusion Pore ◽

Virus Infectivity ◽

Viral Fusion ◽

Fusion Activity

ABSTRACT The envelope glycoprotein, GP64, of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a gp64null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity.

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