Applying Reverse Genetics to Study Measles Virus Interactions with the Host.

The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.

Brain tropism acquisition: The spatial dynamics and evolution of a measles virus collective infectious unit that drove lethal subacute sclerosing panencephalitis.

It is increasingly appreciated that pathogens can spread as infectious units constituted by multiple, genetically diverse genomes, also called collective infectious units or genome collectives. However, genetic characterization of the spatial dynamics of collective infectious units in animal hosts is demanding, and it is rarely feasible in humans. Measles virus (MeV), whose spread in lymphatic tissues and airway epithelia relies on collective infectious units, can, in rare cases, cause subacute sclerosing panencephalitis (SSPE), a lethal human brain disease. In different SSPE cases, MeV acquisition of brain tropism has been attributed to mutations affecting either the fusion or the matrix protein, or both, but the overarching mechanism driving brain adaptation is not understood. Here we analyzed MeV RNA from several spatially distinct brain regions of an individual who succumbed to SSPE. Surprisingly, we identified two major MeV genome subpopulations present at variable frequencies in all 15 brain specimens examined. Both genome types accumulated mutations like those shown to favor receptor-independent cell-cell spread in other SSPE cases. Most infected cells carried both genome types, suggesting the possibility of genetic complementation. We cannot definitively chart the history of the spread of this virus in the brain, but several observations suggest that mutant genomes generated in the frontal cortex moved outwards as a collective and diversified. During diversification, mutations affecting the cytoplasmic tails of both viral envelope proteins emerged and fluctuated in frequency across genetic backgrounds, suggesting convergent and potentially frequency-dependent evolution for modulation of fusogenicity. We propose that a collective infectious unit drove MeV pathogenesis in this brain. Re-examination of published data suggests that similar processes may have occurred in other SSPE cases. Our studies provide a primer for analyses of the evolution of collective infectious units of other pathogens that cause lethal disease in humans.

Oncolytic attenuated measles virus encoding NY-ESO-1 induces HLA I and II presentation of this tumor antigen by melanoma and dendritic cells.

Antitumor virotherapy stimulates the antitumor immune response during tumor cell lysis induced by oncolytic viruses (OVs). OV can be modified to express additional transgenes that enhance their therapeutic potential. In this study, we armed the spontaneously oncolytic Schwarz strain of measles viruses (MVs) with the gene encoding the cancer/testis antigen NY-ESO-1 to obtain MVny. We compared MV and MVny oncolytic activity and ability to induce NY-ESO-1 expression in six human melanoma cell lines. After MVny infection, we measured the capacity of melanoma cells to present NY-ESO-1 peptides to CD4 + and CD8 + T cell clones specific for this antigen. We assessed the ability of MVny to induce NY-ESO-1 expression and presentation in monocyte-derived dendritic cells (DCs). Our results show that MVny and MV oncolytic activity are similar with a faster cell lysis induced by MVny. We also observed that melanoma cell lines and DC expressed the NY-ESO-1 protein after MVny infection. In addition, MVny-infected melanoma cells and DCs were able to stimulate NY-ESO-1-specific CD4 + and CD8 + T cells. Finally, MVny was able to induce DC maturation. Altogether, these results show that MVny could be an interesting candidate to stimulate NY-ESO-1-specific T cells in melanoma patients with NY-ESO-1-expressing tumor cells.

Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA polymerase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppression of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe-1 did not contribute to progression of SSPE. Three mutations in the L protein strongly suppressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection.

A vector-encoded bispecific killer engager to harness virus-activated NK cells as anti-tumor effectors.

Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors.

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.

Downregulation of mitochondrial biogenesis by virus infection triggers antiviral responses by cyclic GMP-AMP synthase.

In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.

Measles virus exits human airway epithelia within dislodged metabolically active infectious centers.

Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2'-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV's strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.

Therapeutic targeting of measles virus polymerase with ERDRP-0519 suppresses all RNA synthesis activity.

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.

Type II interferon signaling in the brain during a viral infection with age-dependent pathogenesis.

Viral infections of the central nervous system (CNS) often cause disease in an age-dependent manner, with greater neuropathology during the fetal and neonatal periods. Transgenic CD46+ mice model these age-dependent outcomes through a measles virus infection of CNS neurons. Adult CD46+ mice control viral spread and survive the infection in an interferon gamma (IFNγ)-dependent manner, whereas neonatal CD46+ mice succumb despite similar IFNγ expression in the brain. Thus, we hypothesized that IFNγ signaling in the adult brain may be more robust, potentially due to greater basal expression of IFNγ signaling proteins. To test this hypothesis, we evaluated the expression of canonical IFNγ signaling proteins in the neonatal and adult brain, including the IFNγ receptor, Janus kinase (JAK) 1/2, and signal transducer and activator of transcription-1 (STAT1) in the absence of infection. We also analyzed the expression and activation of STAT1 and IFNγ-stimulated genes during MV infection. We found that neonatal brains have equivalent or greater JAK/STAT1 expression in the hippocampus and the cerebellum than adults. IFNγ receptor expression varied by cell type in the brain but was widely expressed on neuronal and glial cells. During MV infection, increased STAT1 expression and activation correlated with viral load in the hippocampus regardless of age, but not in the cerebellum where viral load was consistently undetectable in adults. These results suggest the neonatal brain is capable of initiating IFNγ signaling during a viral infection, but that downstream STAT1 activation is insufficient to limit viral spread.

Structural characteristics of measles virus entry.

Measles virus, a member of the genus Morbillivirus, is highly contagious and still shows considerable mortality with over 100000 deaths annually, although efficient attenuated vaccines exist. Recent studies of measles virus haemagglutinin (MeV-H) and its receptor, including crystallographic and electron microscopic structural analyses combined with functional assays, have revealed how the MeV-H protein recognizes its cognate receptors, SLAM and Nectin-4, and how the glycan shield ensures effective vaccination. In addition, the crystal structure of the MeV-F protein indicated its similarity to those of other paramyxoviruses. Taking into account these data, several models of viral entry/membrane fusion of measles viruses and related paramyxoviruses have been proposed. Furthermore, anti-MeV-F inhibitors targeted to specific regions to inhibit MeV-F protein activation were reported, with potency for preventing MeV infection. The inhibitors targeted for entry events may potentially be applied to treatment of MeV-derived diseases, although escape mutations and drug profiles should be considered.

Neurological Complications of Measles (Rubeola).

PURPOSE OF REVIEW: Owing to vaccine hesitancy, there has been a resurgence of measles infections in developed countries. Practitioners can expect to see an increase in patients with neurologic complications of measles. These devastating disorders include primary measles encephalitis, acute post measles encephalitis, subacute sclerosing panencephalitis (SSPE), and measles inclusion body encephalitis (MIBE). RECENT FINDINGS: Although there are many unanswered questions regarding the neurologic complications of measles, recent advances have led to better understanding of the mechanism of the spread of measles within the nervous system, particularly the disruption of F protein function, which raises the possibility of treatment with fusion-inhibiting molecules. Measles and its neurological complications are preventable and must be prevented. Neurologists must educate other clinicians and the public regarding the consequences of inadequate herd immunity to measles. More effective treatments for SSPE and MIBE may be available in the near future, but currently these remain lethal diseases.

Measles Virus Ribonucleoprotein Complexes Rapidly Spread across Well-Differentiated Primary Human Airway Epithelial Cells along F-Actin Rings.

Measles virus (MeV) is a highly contagious human pathogen that continues to be a worldwide health burden. One of the challenges for the study of MeV spread is the identification of model systems that accurately reflect how MeV behaves in humans. For our studies, we use unpassaged, well-differentiated primary cultures of airway epithelial cells from human donor lungs to examine MeV infection and spread. Here, we show that the main components of the MeV ribonucleoprotein complex (RNP), the nucleocapsid and phosphoprotein, colocalize with the apical and circumapical F-actin networks. To better understand how MeV infections spread across the airway epithelium, we generated a recombinant virus incorporating chimeric fluorescent proteins in its RNP complex. By live cell imaging, we observed rapid movement of RNPs along the circumapical F-actin rings of newly infected cells. This strikingly rapid mechanism of horizontal trafficking across epithelia is consistent with the opening of pores between columnar cells by the viral membrane fusion apparatus. Our work provides mechanistic insights into how MeV rapidly spreads through airway epithelial cells, contributing to its extremely contagious nature.IMPORTANCE The ability of viral particles to directly spread cell to cell within the airways without particle release is considered to be highly advantageous to many respiratory viruses. Our previous studies in well-differentiated, primary human airway epithelial cells suggest that measles virus (MeV) spreads cell to cell by eliciting the formation of intercellular membrane pores. Based on a newly generated ribonucleoprotein complex (RNP) "tracker" virus, we document by live-cell microscopy that MeV RNPs move along F-actin rings before entering a new cell. Thus, rather than diffusing through the cytoplasm of a newly infected columnar cell, RNPs take advantage of the cytoskeletal infrastructure to rapidly spread laterally across the human airway epithelium. This results in rapid horizontal spread through the epithelium that does not require particle release.

Specificity of Morbillivirus Hemagglutinins to Recognize SLAM of Different Species.

Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H-SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion.

Type I Interferon Receptor Signaling Drives Selective Permissiveness of Astrocytes and Microglia to Measles Virus during Brain Infection.

Fatal neurological syndromes can occur after measles virus (MeV) infection of the brain. The mechanisms controlling MeV spread within the central nervous system (CNS) remain poorly understood. We analyzed the role of type I interferon (IFN-I) receptor (IFNAR) signaling in the control of MeV infection in a murine model of brain infection. Using organotypic brain cultures (OBC) from wild-type and IFNAR-knockout (IFNARKO) transgenic mice ubiquitously expressing the human SLAM (CD150) receptor, the heterogeneity of the permissiveness of different CNS cell types to MeV infection was characterized. In the absence of IFNAR signaling, MeV propagated significantly better in explant slices. In OBC from IFNAR-competent mice, while astrocytes and microglia were infected on the day of explant preparation, they became refractory to infection with time, in contrast to neurons and oligodendrocytes, which remained permissive to infection. This selective loss of permissiveness to MeV infection was not observed in IFNARKO mouse OBC. Accordingly, the development of astrogliosis related to the OBC procedure was exacerbated in the presence of IFNAR signaling. In the hippocampus, this astrogliosis was characterized by a change in the astrocyte phenotype and by an increase of IFN-I transcripts. A proteome analysis showed the upregulation of 84 out of 111 secreted proteins. In the absence of IFNAR, only 27 secreted proteins were upregulated, and none of these were associated with antiviral activities. Our results highlight the essential role of the IFN-I response in astrogliosis and in the permissiveness of astrocytes and microglia that could control MeV propagation throughout the CNS.IMPORTANCE Measles virus (MeV) can infect the central nervous system (CNS), with dramatic consequences. The mechanisms controlling MeV invasion of the CNS remain ill-defined since most previous data were obtained from postmortem analysis. Here, we highlight for the first time the crucial role of the type I interferon (IFN-I) response not only in the control of CNS invasion but also in the early permissiveness of glial cells to measles virus infection.

Histopathological and Immunohistochemical Characteristics of Measles Exanthema: A Study of a Series of 13 Adult Cases and Review of the Literature.

Despite available vaccination, measles is one of the leading causes of death among young children in developing countries. In clinical practice, the spectrum of differential diagnoses of morbilliform exanthemas associated with fever is wide, and it can be hard to differentiate from other infectious eruptions, especially in adults or in atypical courses in immunocompromised patients. The goal of our study was to identify characteristic histomorphological and immunohistochemical patterns of measles exanthema through the study of 13 skin biopsy specimens obtained from 13 patients with this disease and a review of cases in the literature. Histopathological features of measles exanthema are quite distinctive and characterized by a combination of multinucleated keratinocytes, and individual and clustered necrotic keratinocytes in the epidermis with pronounced folliculosebaceous as well as acrosyringeal involvement. Immunohistochemical staining of skin biopsies with anti-measles virus (MeV) nucleoprotein and anti-MeV phosphoprotein can be of great value in confirming the diagnosis of measles. Both methods can serve as quick additional diagnostic tools for prompt implementation of quarantine measures and for providing medical assistance, even in patients in whom the clinician did not consider measles as a differential diagnosis of the rash due to the rarity of the disease in a putatively vaccinated community.

Measles Virus Bearing Measles Inclusion Body Encephalitis-Derived Fusion Protein Is Pathogenic after Infection via the Respiratory Route.

A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intrahost evolution of the wild-type (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of small-molecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for interhost spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37°C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro, neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV.IMPORTANCE Measles virus (MeV) infection can be severe in immunocompromised individuals and lead to complications, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE) occur even in the face of an intact immune response. While they are relatively rare complications of MeV infection, MIBE and SSPE are lethal. This work addresses the hypothesis that despite a dysregulated viral fusion complex, central nervous system (CNS)-adapted measles virus can spread outside the CNS within an infected host.

Analysis of a Subacute Sclerosing Panencephalitis Genotype B3 Virus from the 2009-2010 South African Measles Epidemic Shows That Hyperfusogenic F Proteins Contribute to Measles Virus Infection in the Brain.

During a measles virus (MeV) epidemic in 2009 in South Africa, measles inclusion body encephalitis (MIBE) was identified in several HIV-infected patients. Years later, children are presenting with subacute sclerosing panencephalitis (SSPE). To investigate the features of established MeV neuronal infections, viral sequences were analyzed from brain tissue samples of a single SSPE case and compared with MIBE sequences previously obtained from patients infected during the same epidemic. Both the SSPE and the MIBE viruses had amino acid substitutions in the ectodomain of the F protein that confer enhanced fusion properties. Functional analysis of the fusion complexes confirmed that both MIBE and SSPE F protein mutations promoted fusion with less dependence on interaction by the viral receptor-binding protein with known MeV receptors. While the SSPE F required the presence of a homotypic attachment protein, MeV H, in order to fuse, MIBE F did not. Both F proteins had decreased thermal stability compared to that of the corresponding wild-type F protein. Finally, recombinant viruses expressing MIBE or SSPE fusion complexes spread in the absence of known MeV receptors, with MIBE F-bearing viruses causing large syncytia in these cells. Our results suggest that alterations to the MeV fusion complex that promote fusion and cell-to-cell spread in the absence of known MeV receptors is a key property for infection of the brain.IMPORTANCE Measles virus can invade the central nervous system (CNS) and cause severe neurological complications, such as MIBE and SSPE. However, mechanisms by which MeV enters the CNS and triggers the disease remain unclear. We analyzed viruses from brain tissue of individuals with MIBE or SSPE, infected during the same epidemic, after the onset of neurological disease. Our findings indicate that the emergence of hyperfusogenic MeV F proteins is associated with infection of the brain. We also demonstrate that hyperfusogenic F proteins permit MeV to enter cells and spread without the need to engage nectin-4 or CD150, known receptors for MeV that are not present on neural cells.

An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function.

Measles virus genome encapsidation is essential for viral replication and is controlled by the intrinsically disordered phosphoprotein (P) maintaining the nucleoprotein in a monomeric form (N) before nucleocapsid assembly. All paramyxoviruses harbor highly disordered amino-terminal domains (PNTD) that are hundreds of amino acids in length and whose function remains unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we describe the structure and dynamics of the 90-kDa N0PNTD complex, comprising 450 disordered amino acids, at atomic resolution. NMR relaxation dispersion reveals the existence of an ultraweak N-interaction motif, hidden within the highly disordered PNTD, that allows PNTD to rapidly associate and dissociate from a specific site on N while tightly bound at the amino terminus, thereby hindering access to the surface of N. Mutation of this linear motif quenches the long-range dynamic coupling between the two interaction sites and completely abolishes viral transcription/replication in cell-based minigenome assays comprising integral viral replication machinery. This description transforms our understanding of intrinsic conformational disorder in paramyxoviral replication. The essential mechanism appears to be conserved across Paramyxoviridae, opening unique new perspectives for drug development against this family of pathogens.