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Measles cases have surged pre-COVID-19 and the pandemic has aggravated the problem. Most measles-associated morbidity and mortality arises from destruction of pre-existing immune memory by measles virus (MeV), a paramyxovirus of the morbillivirus genus. Therapeutic measles vaccination lacks efficacy, but little is known about preserving immune memory through antivirals and the effect of respiratory disease history on measles severity. We use a canine distemper virus (CDV)-ferret model as surrogate for measles and employ an orally efficacious paramyxovirus polymerase inhibitor to address these questions. A receptor tropism-intact recombinant CDV with low lethality reveals an 8-day advantage of antiviral treatment versus therapeutic vaccination in maintaining immune memory. Infection of female ferrets with influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks pre-CDV causes fatal hemorrhagic pneumonia with lung onslaught by commensal bacteria. RNAseq identifies CDV-induced overexpression of trefoil factor (TFF) peptides in the respiratory tract, which is absent in animals pre-infected with IAV. Severe outcomes of consecutive IAV/CDV infections are mitigated by oral antivirals even when initiated late. These findings validate the morbillivirus immune amnesia hypothesis, define measles treatment paradigms, and identify priming of the TFF axis through prior respiratory infections as risk factor for exacerbated morbillivirus disease. Measles virus infection causes immunosuppression and it's unclear whether this can be prevented by antivirals. Here, using a canine distemper virus ferret model, the authors show that measles-like immune amnesia and lethal exacerbated respiratory disease after consecutive respiratory virus infections can be mitigated by oral antiviral therapy initiated at the onset of primary clinical signs. [ABSTRACT FROM AUTHOR]

Research has been conducted on Newcastle disease virus F protein which lies in a cylindrical cavity. Results demonstrate that the features of the cavity are conserved within the Paramyxovirinae, and that cavity's specific structural features are important for paramyxovirus fusion initiation.

The morbillivirus genus comprises major human and animal pathogens, including the highly contagious measles virus. Morbilliviruses feature single stranded negative sense RNA genomes that are wrapped by a plasma membrane-derived lipid envelope. Genomes are encapsidated by the viral nucleocapsid protein forming ribonucleoprotein complexes, and only the encapsidated RNA is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp). In this review, we discuss recent breakthroughs towards the structural and functional understanding of the morbillivirus polymerase complex. Considering the clinical burden imposed by members of the morbillivirus genus, the development of novel antiviral therapeutics is urgently needed. The viral polymerase complex presents unique structural and enzymatic properties that can serve as attractive candidates for druggable targets. We evaluate distinct strategies for therapeutic intervention and examine how high-resolution insight into the organization of the polymerase complex may pave the path towards the structure-based design and optimization of next-generation RdRp inhibitors. [ABSTRACT FROM AUTHOR]

Infection of cells by enveloped viruses requires merger of the viral envelope membrane with target cell membranes, resulting in the formation of fusion pores through which the viral genome is released. Since lipid membranes do not mix spontaneously, the fusion process is energy-dependent and mediated by viral envelope glycoprotein complexes. On the basis of their structural and mechanistic properties, three distinct classes of viral fusion proteins have been identified to date. Despite their diversity, basic principles of viral membrane fusion, simultaneous engagement of both donor and target membrane and refolding into hairpin-like structures, have emerged as universally conserved. This article provides an overview of the basic principles of viral membrane fusion common to all enveloped viruses and discusses the specific structural and functional features of the different fusion protein classes by example of the paramyxovirus, flavivirus and rhabdovirus families. [ABSTRACT FROM AUTHOR]

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family. [ABSTRACT FROM AUTHOR]

Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen-directed inhibitor of RdRp activity. [ABSTRACT FROM AUTHOR]

Members of the Paramyxovirinae subfamily rely on the concerted action of two envelope glycoprotein complexes, attachment protein H and the fusion (F) protein oligomer, to achieve membrane fusion for viral entry. Despite advances in X-ray information, the organization of the physiological attachment (H) oligomer in functional fusion complexes and the molecular mechanism linking H receptor binding with F triggering remain unknown. Here, we have applied an integrated approach based on biochemical and functional assays to the problem. Blue native PAGE analysis indicates that native H complexes extract predominantly in the form of loosely assembled tetramers from purified measles virus (MeV) particles and cells transiently expressing the viral envelope glycoproteins. To gain functional insight, we have established a bimolecular complementation (BiC) assay for MeV H, on the basis of the hypothesis that physical interaction of H with F complexes, F triggering, and receptor binding constitute distinct events. Having experimentally confirmed three distinct H complementation groups, implementation of H BiC (H-BiC) reveals that a high-affinity receptor-to-paramyxovirus H monomer stoichiometry below parity is sufficient for fusion initiation, that F binding and fusion initiation are separable in H oligomers, and that a higher relative amount of F binding-competent than F fusion initiation- or receptor binding-competent H monomers per oligomer is required for optimal fusion. By capitalizing on these findings, H-BiC activity profiles confirm the organization of H into tetramers or higher-order multimers in functional fusion complexes. Results are interpreted in light of a model in which receptor binding may affect the oligomeric organization of the attachment protein complex. [ABSTRACT FROM AUTHOR]

Several members of the paramyxovirus family constitute major human pathogens that, collectively, are responsible for major morbidity and mortality worldwide. In an effort to develop novel therapeutics against measles virus (MV), a prominent member of the paramyxovirus family, the authors report a high-throughput screening protocol that uses a nonrecombinant primary MV strain as targets. Implementation of the assay has yielded 60 hit candidates from a 137,500-entry library. Counterscreening and generation of dose-response curves narrows this pool to 35 compounds with active concentrations ≤15.3 μM against the MV-Alaska strain and specificity indices ranging from 36 to >500. Library mining for structural analogs of several confirmed hits combined with retesting of identified candidates reveals a high accuracy of primary hit identification. Eleven of the confirmed hits interfere with viral entry, whereas the remaining 24 compounds target postentry steps of the viral life cycle. Activity testing against selected members of the paramyxovirus family reveals 3 patterns of activity: 1) exclusively MV-specific blockers, 2) inhibitors of MV and related viruses of the same genus, and 3) broader range inhibitors with activity against a different Paramyxovirinae genus. Representatives of the last class may open avenues for the development of broad-range paramyxovirus inhibitors through hit-tolead chemistry. [ABSTRACT FROM AUTHOR]

Measles virus (MeV), a morbillivirus within the paramyxovirus family, expresses two envelope glycoproteins. The attachment (H) protein mediates receptor binding, followed by triggering of the fusion (F) protein, which leads to merger of the viral envelope with target cell membranes. Receptor binding by members of related paramyxovirus genera rearranges the head domains of the attachment proteins, liberating an F-contact domain within the attachment protein helical stalk. However, morbillivirus glycoproteins first assemble intracellularly prior to receptor binding, raising the question of whether alternative protein-protein interfaces are involved or whether an entirely distinct triggering principle is employed. To test these possibilities, we generated headless H stem mutants of progressively shorter length. Conformationally restricted H stems remained capable of intracellular assembly with a standard F protein and a soluble MeV F mutant. Proteolytic maturation of F, but not the altered biochemical conditions at the cell surface, reduces the strength of glycoprotein interaction, readying the complexes for triggering. F mutants stabilized in the prefusion conformation interact with H intracellularly and at the cell surface, while destabilized F mutants interact only intracellularly, prior to F maturation. These results showcase an MeV entry machinery that functionally varies conserved motifs of the proposed paramyxovirus infection pathway. Intracellular and plasma membrane-resident MeV glycoprotein complexes employ the same protein-protein interface. F maturation prepares for complex separation after triggering, and the H head domains in prereceptor-bound conformation prevent premature stalk rearrangements and F activation. Intracellular preassembly affects MeV fusion profiles and may contribute to the high cell-to-cell fusion activity characteristic of the morbillivirus genus. [ABSTRACT FROM AUTHOR]

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design. IMPORTANCE: Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family. [ABSTRACT FROM AUTHOR]

Paramyxovirus attachment and fusion (F) envelope glycoprotein complexes mediate membrane fusion required for viral entry. The measles virus (MeV) attachment (H) protein stalk domain is thought to directly engage F for fusion promotion. However, past attempts to generate truncated, fusion-triggering-competent H-stem constructs remained fruitless. In this study, we addressed the problem by testing the hypothesis that truncated MeV H stalks may require stabilizing oligomerization tags to maintain intracellular transport competence and F-triggering activity. We engineered H-stems of different lengths with added 4-helix bundle tetramerization domains and demonstrate restored cell surface expression, efficient interaction with F, and fusion promotion activity of these constructs. The stability of the 4-helix bundle tags and the relative orientations of the helical wheels of H-stems and oligomerization tags govern the kinetics of fusion promotion, revealing a balance between H stalk conformational stability and F-triggering activity. Recombinant MeV particles expressing a bioactive H-stem construct in the place of full-length H are viable, albeit severely growth impaired. Overall, we demonstrate that the MeV H stalk represents the effector domain for MeV F triggering. Fusion promotion appears linked to the conformational flexibility of the stalk, which must be tightly regulated in viral particles to ensure efficient virus entry. While the pathways toward assembly of functional fusion complexes may differ among diverse members of the paramyxovirus family, central elements of the triggering machinery emerge as highly conserved. [ABSTRACT FROM AUTHOR]

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism. [ABSTRACT FROM AUTHOR]

The measles virus (MeV), a member of the paramyxovirusfamily, is an important cause of pediatric morbidity and mortalityworldwide. In an effort to provide therapeutic treatments for improvedmeasles management, we previously identified a small, non-nucleosideorganic inhibitor of the viral RNA-dependent RNA polymerase by meansof high-throughput screening. Subsequent structure–activityrelationship (SAR) studies around the corresponding pyrazole carboxamidescaffold led to the discovery of 2(AS-136a), a firstgeneration lead with low nanomolar potency against life MeV and attractivephysical properties suitable for development. However, its poor watersolubility and low oral bioavailability (F) in rat suggested that the lead could benefit from further SAR studiesto improve the biophysical characteristics of the compound. Optimizationof in vitro potency and aqueous solubility led to the discovery of 2o(ERDRP-00519), a potent inhibitor of MeV (EC50= 60 nM) with an aqueous solubility of approximately 60 μg/mL.The agent shows a 10-fold exposure (AUC/Cmax) increase in the rat model relative to 2, displaysnear dose proportionality in the range of 10–50 mg/kg, andexhibits good oral bioavailability (F= 39%). The significant solubility increase appears linked to the improvedoral bioavailability. [ABSTRACT FROM AUTHOR]

Paramyxovirinae envelope glycoproteins constitute a premier model to dissect how specific and dynamic interactions in multisubunit membrane protein complexes can control deep-seated conformational rearrangements. However, individual residues that determine reciprocal specificity of the viral attachment and fusion (F) proteins have not been identified. We have developed an assay based on a pair of canine distemper virus (CDV) F proteins (strains Onderstepoort (ODP) and Lederle) that share ∼95% identity but differ in their ability to form functional complexes with the measles virus (MV) attachment protein (H). Characterization of CDV F chimeras and mutagenesis reveals four residues in CDV F-ODP (positions 164, 219, 233, and 317) required for productive interaction with MV H. Mutating these residues to the Lederle type disrupts triggering of F-ODP by MV H without affecting functionality when co-expressed with CDV H. Co-immunoprecipitation shows a stronger physical interaction of F-ODP than F-Lederle with MV H. Mutagenesis of MV F highlights the MV residues homologous to CDV F residues 233 and 317 as determinants for physical glycoprotein interaction and fusion activity under homotypic conditions. In assay reversal, the introduction of sections of the CDV H stalk into MV H shows a five-residue fragment (residues 110-114) to mediate specificity for CDV F-Lederle. All of the MV H stalk chimeras are surface-expressed, show hemadsorption activity, and trigger MV F. Combining the five-residue H chimera with the CDV F-ODP quadruple mutant partially restores activity, indicating that the residues identified in either glycoprotein contribute interdependently to the formation of functional complexes. Their localization in structural models of F and H suggests that placement in particular of F residue 233 in close proximity to the 110-114 region of H is structurally conceivable. [ABSTRACT FROM AUTHOR]

Abstract: High-throughput screening has identified 1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)phenyl]-1H-pyrazole-5-carboxamide 16677 as a novel and potent (IC50 =35–145nM) inhibitor against multiple primary isolates of diverse measles virus (MV) genotypes currently circulating worldwide. The synthesis of 16677 and several analogs together with effects on MV replication is described. The most potent analog displays nanomolar inhibition against the MV and a selectivity ratio (CC50/IC50) of ca. 16,500. [Copyright &y& Elsevier]

Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability. [ABSTRACT FROM AUTHOR]

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid α-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F. [ABSTRACT FROM AUTHOR]

In search of target sites for the development of paramyxovirus inhibitors, we have engineered disulfide bridges to introduce covalent links into the prefusion F protein trimer of measles virus. F-Edm-452C/460C, predicted to bridge head and stalk domains of different F monomers, shows a high degree of proteolytic maturation and surface expression, predominantly as stable, dithiothreitol-sensitive trimers, but no fusion activity. Reduction of disulfide bridges partially restores activity. These findings underscore the importance of reversible intersubunit interactions between the stalk and head domains for F activity. Noncovalent small molecules mimicking this behavior may constitute a potent strategy for preventing paramyxovirus entry. [ABSTRACT FROM AUTHOR]