Interaction of the Hemagglutinin Stalk Region with Cell Adhesion Molecule (CADM) 1 and CADM2 Mediates the Spread between Neurons and Neuropathogenicity of Measles Virus with a Hyperfusogenic Fusion Protein.

Measles virus (MeV), the causative agent of measles, is an enveloped RNA virus of the family Paramyxoviridae, which remains an important cause of childhood morbidity and mortality. MeV has two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. During viral entry or virus-mediated fusion between infected cells and neighboring susceptible cells, the head domain of the H protein initially binds to its receptors, signaling lymphocytic activation molecule family member 1 (SLAM) and nectin-4, and then the stalk region of the H protein transmits the fusion-triggering signal to the F protein. MeV may persist in the human brain and cause a fatal neurodegenerative disease, subacute sclerosing panencephalitis (SSPE). Recently, we showed, using in vitro cell culture, that cell adhesion molecule (CADM) 1 and CADM2 are host factors that trigger hyperfusogenic mutant F proteins, causing cell-to-cell fusion and the transfer of the MeV genome between neurons. Unlike conventional receptors, CADM1 and CADM2 interact in cis (on the same membrane) with the H protein and then trigger membrane fusion. Here, we show that alanine substitutions in part of the stalk region (positions 171-175) abolish the ability of the H protein to mediate membrane fusion triggered by CADM1 and CADM2, but not by SLAM. The recombinant hyperfusogenic MeV carrying this mutant H protein loses its ability to spread in primary mouse neurons as well as its neurovirulence in experimentally infected suckling hamsters. These results indicate that CADM1 and CADM2 are key molecules for MeV propagation in the brain and its neurovirulence in vivo. IMPORTANCE Measles is an acute febrile illness with skin rash. Despite the availability of highly effective vaccines, measles is still an important cause of childhood morbidity and mortality in many countries. The World Health Organization estimates that more than 120,000 people died from measles worldwide in 2021. Measles virus (MeV), the causative agent of measles, can also cause a fatal progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. There is currently no effective treatment for this disease. In this study, using recombinant MeVs with altered receptor usage patterns, we show that cell adhesion molecule (CADM) 1 and CADM2 are host factors critical for MeV spread in neurons and its neurovirulence. These findings further our understanding of the molecular mechanism of MeV neuropathogenicity.

Collective fusion activity determines neurotropism of an en bloc transmitted enveloped virus.

Measles virus (MeV), which is usually non-neurotropic, sometimes persists in the brain and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection, serving as a model for persistent viral infections. The persisting MeVs have hyperfusogenic mutant fusion (F) proteins that likely enable cell-cell fusion at synapses and "en bloc transmission" between neurons. We here show that during persistence, F protein fusogenicity is generally enhanced by cumulative mutations, yet mutations paradoxically reducing the fusogenicity may be selected alongside the wild-type (non-neurotropic) MeV genome. A mutant F protein having SSPE-derived substitutions exhibits lower fusogenicity than the hyperfusogenic F protein containing some of those substitutions, but by the wild-type F protein coexpression, the fusogenicity of the former F protein is enhanced, while that of the latter is nearly abolished. These findings advance the understanding of the long-term process of MeV neuropathogenicity and provide critical insight into the genotype-phenotype relationships of en bloc transmitted viruses.

Short-Stalk Isoforms of CADM1 and CADM2 Trigger Neuropathogenic Measles Virus-Mediated Membrane Fusion by Interacting with the Viral Hemagglutinin.

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 recently showed that cell adhesion molecule 1 (CADM1; also known as IGSF4A, Necl-2, and SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, and 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 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.

A defective viral genome strategy elicits broad protective immunity against respiratory viruses.

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.

Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome.

During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.

CADM1 and CADM2 Trigger Neuropathogenic Measles Virus-Mediated Membrane Fusion by Acting in cis.

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae, is still an important cause of childhood morbidity and mortality worldwide. MeV 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). The disease is fatal, and no effective therapy is currently available. Although transsynaptic cell-to-cell transmission is thought to account for MeV propagation in the brain, neurons do not express the known receptors for MeV. Recent studies have shown that hyperfusogenic changes in the MeV fusion (F) protein play a key role in MeV propagation in the brain. However, how such mutant viruses spread in neurons remains unexplained. Here, we show that cell adhesion molecule 1 (CADM1; also known as IGSF4A, Necl-2, and SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors that enable MeV to cause membrane fusion in cells lacking the known receptors and to spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the envelope. However, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same cell membrane, causing the fusion protein triggering and membrane fusion. Knockdown of CADM1 and CADM2 inhibits syncytium formation and virus transmission between neurons that are both mediated by hyperfusogenic F proteins. Thus, our results unravel the molecular mechanism (receptor-mimicking cis-acting fusion triggering) by which MeV spreads transsynaptically between neurons, thereby causing SSPE. IMPORTANCE Measles virus (MeV), an enveloped RNA virus, is the causative agent of measles, which is still an important cause of childhood morbidity and mortality worldwide. Persistent MeV infection in the brain causes a fatal progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. However, how MeV spreads in neurons, which are mainly affected in SSPE, remains largely unknown. In this study, we demonstrate that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the viral membrane (envelope). Remarkably, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same membrane, triggering the fusion protein and causing membrane fusion, as viral receptors usually do in trans. Careful screening may lead to more examples of such "receptor-mimicking cis-acting fusion triggering" in other viruses.

Lysosome-Associated Membrane Proteins Support the Furin-Mediated Processing of the Mumps Virus Fusion Protein.

Mumps virus (MuV), an enveloped RNA virus of the Paramyxoviridae family and the causative agent of mumps, affects the salivary glands and other glandular tissues as well as the central nervous system. The virus enters the cell by inducing the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by MuV envelope proteins: the hemagglutinin-neuraminidase and fusion (F) protein. Cleavage of the MuV F protein (MuV-F) into two subunits by the cellular protease furin is a prerequisite for fusion and virus infectivity. Here, we show that 293T (a derivative of HEK293) cells do not produce syncytia upon expression of MuV envelope proteins or MuV infection. This failure is caused by the inefficient MuV-F cleavage despite the presence of functional furin in 293T cells. An expression cloning strategy revealed that overexpression of lysosome-associated membrane proteins (LAMPs) confers on 293T cells the ability to produce syncytia upon expression of MuV envelope proteins. The LAMP family comprises the ubiquitously expressed LAMP1 and LAMP2, the interferon-stimulated gene product LAMP3, and the cell type-specific proteins. The expression level of the LAMP3 gene, but not of LAMP1 and LAMP2 genes, differed markedly between 293T and HEK293 cells. Overexpression of LAMP1, LAMP2, or LAMP3 allowed 293T cells to process MuV-F efficiently. Furthermore, these LAMPs were found to interact with both MuV-F and furin. Our results indicate that LAMPs support the furin-mediated cleavage of MuV-F and that, among them, LAMP3 may be critical for the process, at least in certain cells.IMPORTANCE The cellular protease furin mediates proteolytic cleavage of many host and pathogen proteins and plays an important role in viral envelope glycoprotein maturation. MuV, an enveloped RNA virus of the Paramyxoviridae family and an important human pathogen, enters the cell through the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by the viral attachment protein and the F protein. Cleavage of MuV-F into two subunits by furin is a prerequisite for fusion and virus infectivity. Here, we show that LAMPs support the furin-mediated cleavage of MuV-F. Expression levels of LAMPs affect the processing of MuV-F and MuV-mediated membrane fusion. Among LAMPs, the interferon-stimulated gene product LAMP3 is most critical in certain cells. Our study provides potential targets for anti-MuV therapeutics.

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

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.

Disruption of the Dimer-Dimer Interaction of the Mumps Virus Attachment Protein Head Domain, Aided by an Anion Located at the Interface, Compromises Membrane Fusion Triggering.

Mumps virus (MuV), an enveloped negative-strand RNA virus belonging to the family Paramyxoviridae, enters the host cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin-neuraminidase (MuV-HN) and a fusion (F) protein. However, how the binding of MuV-HN to glycan receptors triggers membrane fusion is not well understood. The crystal structure of the MuV-HN head domain forms a tetramer (dimer of dimers) like other paramyxovirus attachment proteins. In the structure, a sulfate ion (SO42-) was found at the interface between two dimers, which may be replaced by a hydrogen phosphate ion (HPO42-) under physiological conditions. The anion is captured by the side chain of a positively charged arginine residue at position 139 of one monomer each from both dimers. Substitution of alanine or lysine for arginine at this position compromised the fusion support activity of MuV-HN without affecting its cell surface expression, glycan-receptor binding, and interaction with the F protein. Furthermore, the substitution appeared to affect the tetramer formation of the head domain as revealed by blue native-PAGE analysis. These results, together with our previous similar findings with the measles virus attachment protein head domain, suggest that the dimer-dimer interaction within the tetramer may play an important role in triggering membrane fusion during paramyxovirus entry.IMPORTANCE Despite the use of effective live vaccines, mumps outbreaks still occur worldwide. Mumps virus (MuV) infection typically causes flu-like symptoms and parotid gland swelling but sometimes leads to orchitis, oophoritis, and neurological complications, such as meningitis, encephalitis, and deafness. MuV enters the host cell through membrane fusion mediated by two viral proteins, a receptor-binding attachment protein, and a fusion protein, but its detailed mechanism is not fully understood. In this study, we show that the tetramer (dimer of dimers) formation of the MuV attachment protein head domain is supported by an anion located at the interface between two dimers and that the dimer-dimer interaction plays an important role in triggering the activation of the fusion protein and causing membrane fusion. These results not only further our understanding of MuV entry but provide useful information about a possible target for antiviral drugs.

Consensus and variations in cell line specificity among human metapneumovirus strains.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

Cooperation between different variants: A unique potential for virus evolution.

RNA viruses exist as quasispecies containing many variants within their populations because of the error prone nature of viral RNA-dependent RNA polymerases. Quasispecies are not a simple collection of individual variants. Instead, internal interactions among variants provide quasispecies with unique evolvability. An example is 'cooperation' between wild-type and defective measles viruses, in which co-existence of a wild-type and a mutant genome produces a new phenotype. Such internal interactions presuppose efficient co-transmission of multiple genomes to the same cell, which is achieved by polyploid virions of some virus families or by a high multiplicity of infection. Recent studies have revealed that multiple viral genomes can also be transmitted simultaneously ('bloc transmission') by other mechanisms, strengthening the concept of internal interactions among viral quasispecies. Elucidation of the mechanisms of virus evolution, including internal interactions and bloc transmission, may provide rational strategies to solve such important problems of virus infections as drug-resistance, immune evasion, and acquisition of the new tropism and host range.

New Insights into Measles Virus Brain Infections.

Measles virus (MeV) may persist in the brain, causing fatal neurodegenerative diseases, subacute sclerosing panencephalitis, and measles inclusion-body encephalitis. However, the mechanism of MeV propagation in the brain remains unexplained because human neurons affected by the diseases do not express the known receptors for MeV. Recent studies have revealed that certain changes in the ectodomain of the MeV fusion (F) protein play a key role in MeV spread in the brain. These changes destabilize the prefusion form of the F protein and render it hyperfusogenic, which in turn allows the virus to propagate in neurons. Based on crystal structures of the F protein, effective fusion inhibitors could be developed to treat these diseases.

Structures of the prefusion form of measles virus fusion protein in complex with inhibitors.

Measles virus (MeV), a major cause of childhood morbidity and mortality, is highly immunotropic and one of the most contagious pathogens. MeV may establish, albeit rarely, persistent infection in the central nervous system, causing fatal and intractable neurodegenerative diseases such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. Recent studies have suggested that particular substitutions in the MeV fusion (F) protein are involved in the pathogenesis by destabilizing the F protein and endowing it with hyperfusogenicity. Here we show the crystal structures of the prefusion MeV-F alone and in complex with the small compound AS-48 or a fusion inhibitor peptide. Notably, these independently developed inhibitors bind the same hydrophobic pocket located at the region connecting the head and stalk of MeV-F, where a number of substitutions in MeV isolates from neurodegenerative diseases are also localized. Since these inhibitors could suppress membrane fusion mediated by most of the hyperfusogenic MeV-F mutants, the development of more effective inhibitors based on the structures may be warranted to treat MeV-induced neurodegenerative diseases.

Cooperative Interaction Within RNA Virus Mutant Spectra.

RNA viruses usually consist of mutant spectra because of high error rates of viral RNA polymerases. Growth competition occurs among different viral variants, and the fittest clones predominate under given conditions. Individual variants, however, may not be entirely independent of each other, and internal interactions within mutant spectra can occur. Examples of cooperative and interfering interactions that exert enhancing and suppressing effects on replication of the wild-type virus, respectively, have been described, but their underlying mechanisms have not been well defined. It was recently found that the cooperation between wild-type and variant measles virus genomes produces a new phenotype through the heterooligomer formation of a viral protein. This observation provides a molecular mechanism underlying cooperative interactions within mutant spectra. Careful attention to individual sequences, in addition to consensus sequences, may disclose further examples of internal interactions within mutant spectra.

[Human Metapneumovirus Infection].

Human metapneumovirus (HMPV) was first isolated in 2001 from young children with symptoms of acute respiratory infections. Studies revealed that HMPV has circulated worldwide for more than 50 years in hu- man populations. HMPV is classified into two major, distinct groups, A and B, which show antigenic differ- ences, but clinical symptoms of infection with these groups are indistinguishable. The symptoms are often severe and similar to those of respiratory syncytial virus infections. HMPV is detected in more than 10% of children under five years old suffering from acute respiratory infections. Elderly people are also affected with HMPV and may develop severe respiratory diseases. Recently, point-of-care testing based on immu- nochromatography became available in Japan under the coverage of medical insurance, further revealing clini- cal pictures of HMPV infections and improving clinical treatment and control measures. Sensitive nucleo- tide amplification techniques are also available for the detection of HMPV. However, the virus titers and amounts of viral antigens decline significantly after 5 days of illness. Therefore, laboratory testing to detect HMPV antigens or genomes should be conducted using clinical specimens before 4 days of illness. Assays to detect immunoglobulin specific to HMPV (ELISA and neutralizing assay) have also been established, alt- hough they have not yet been approved as extracorporeal diagnostic medicines. [Review].

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.

UNLABELLED: Subacute 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. IMPORTANCE: Subacute 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.

Cooperation: another mechanism of viral evolution.

RNA viruses evolve rapidly under selection pressure as a result of the high error rates of viral RNA polymerase. 'Cooperation' between wild type and variant measles virus (MV) genomes through the heterooligomer formation of a viral protein has recently been shown to act as a mechanism of viral evolution. This type of cooperation between genomes producing a new phenotype may have implications for various aspects of evolution, including the expansion of viral tropism and host range, the emergence of segmented viral genomes, and the evolution of heteromultimeric molecules. It also lends support to the concept of the quasispecies acting as a unit of selection.

Mutations in the putative dimer-dimer interfaces of the measles virus hemagglutinin head domain affect membrane fusion triggering.

Measles virus (MV), an enveloped RNA virus belonging to the Paramyxoviridae family, enters the cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin (H) and a fusion (F) protein. The crystal structure of the receptor-binding head domain of MV-H bound to its cellular receptor revealed that the MV-H head domain forms a tetrameric assembly (dimer of dimers), which occurs in two forms (forms I and II). In this study, we show that mutations in the putative dimer-dimer interface of the head domain in either form inhibit the ability of MV-H to support membrane fusion, without greatly affecting its cell surface expression, receptor binding, and interaction with the F protein. Notably, some anti-MV-H neutralizing monoclonal antibodies are directed to the region around the dimer-dimer interface in form I rather than receptor-binding sites. These observations suggest that the dimer-dimer interactions of the MV-H head domain, especially that in form I, contribute to triggering membrane fusion, and that conformational shift of head domain tetramers plays a role in the process. Furthermore, our results indicate that although the stalk and transmembrane regions may be mainly responsible for the tetramer formation of MV-H, the head domain alone can form tetramers, albeit at a low efficiency.

Mutant fusion proteins with enhanced fusion activity promote measles virus spread in human neuronal cells and brains of suckling hamsters.

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein.

Cooperation between different RNA virus genomes produces a new phenotype.

An RNA virus population generally evolves rapidly under selection pressure, because of high error rates of the viral RNA polymerase. Measles virus, an enveloped RNA virus, has a fusion protein mediating fusion of the viral envelope with the cell membrane. Here we observe that a non-fusogenic recombinant measles virus evolves, after passages, into mutant viruses which regain the ability to induce membrane fusion. Unexpectedly, we identify a mutant virus possessing two types of genomes within a single virion: one genome encoding the wild-type fusion protein, the other a mutant version with a single amino-acid substitution. Neither the wild-type nor mutant protein by itself is able to mediate membrane fusion, but both together exhibit enhanced fusion activity through hetero-oligomer formation. Our results reveal a molecular mechanism for the 'cooperation' between different RNA virus genomes, which may have implications in viral evolution and in the evolution of other macromolecules.