Antivirals targeting paramyxovirus membrane fusion.

The Paramyxoviridae family includes enveloped single-stranded negative-sense RNA viruses such as measles, mumps, human parainfluenza, canine distemper, Hendra, and Nipah viruses, which cause a tremendous global health burden. The ability of paramyxoviral glycoproteins to merge viral and host membranes allows entry of the viral genome into host cells, as well as cell-cell fusion, an important contributor to disease progression. Recent molecular and structural advances in our understanding of the paramyxovirus membrane fusion machinery gave rise to various therapeutic approaches aiming at inhibiting viral infection, spread, and cytopathic effects. These therapeutic approaches include peptide mimics, antibodies, and small molecule inhibitors with various levels of success at inhibiting viral entry, increasing the potential of effective antiviral therapeutic development.

Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Paramyxovirus-Like Particles as Protein Delivery Vehicles.

We have developed a flexible platform for delivery of proteins to target cell interiors using paramyxovirus-like particles. The key enabling feature is an appendage, 15 to 30 amino acid residues in length, that is added to cargo proteins and that induces them to bind to the viral matrix (M) protein during virus-like particle (VLP) assembly. The cargo is then incorporated within the VLPs as they bud, using the same interactions that normally direct viral genome packaging. The appendage can also serve as an epitope tag for cargo detection using a nucleocapsid (NP) protein-specific monoclonal antibody. Using this approach, we generated Renilla luciferase-loaded VLPs, green fluorescent protein-loaded VLPs, superoxide dismutase-loaded VLPs, and Cre recombinase-loaded VLPs. In each case, the VLPs could efficiently deliver their functional cargos to target cells and, in the case of Cre recombinase, to target cell nuclei. The strategy was employed using two different VLP production platforms, one based on parainfluenza virus 5 (PIV5) and the other based on Nipah virus, and in both cases efficient cargo packaging and delivery could be achieved. These findings provide a foundation for development of paramyxovirus-like particles as tools for safe and efficient delivery of therapeutic proteins to cells and tissues. IMPORTANCE Therapeutic proteins including transcription factors and genome editors have enormous clinical potential but are currently limited in part due to the challenges of safely and efficiently delivering these proteins to the interiors of target cells. Here, we have developed a new strategy for protein delivery based on manipulation of paramyxovirus genome packaging interactions.

Phosphatidylinositol-3-kinase-Akt pathway in negative-stranded RNA virus infection: a minireview.

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.

Three genetically distinct ferlaviruses have varying effects on infected corn snakes (Pantherophis guttatus).

Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of ferlaviruses into four genogroups, and criteria for definition of ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups.

Regulation of measles virus gene expression by P protein coiled-coil properties.

The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the "a" or "d" hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness.

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.

Experimental Characterization of Fuzzy Protein Assemblies: Interactions of Paramyxoviral NTAIL Domains With Their Functional Partners.

In this chapter we detail various experimental approaches to characterize the fuzziness of complexes made of the C-terminal domain of the nucleoprotein (NTAIL) from three representative paramyxoviruses and of the C-terminal X domain (XD) of the homologous phosphoprotein. We discuss the advantages, the limitations, as well as the caveats of the various methods. We describe experimental data showing that paramyxoviral NTAIL-XD complexes are characterized by a considerable amount of conformational heterogeneity. We also detail recent data that revealed that NTAIL is highly malleable, i.e., it displays a partner-mediated polymorphism. All the results suggest that NTAIL plasticity and fuzziness play a role in the coordination and regulation of the NTAIL interaction network so as to ensure efficient transcription and replication.

How order and disorder within paramyxoviral nucleoproteins and phosphoproteins orchestrate the molecular interplay of transcription and replication.

In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and phosphoproteins (P). In particular, we focus on measles, Nipah, and Hendra viruses and highlight both commonalities and differences with respect to the closely related Sendai virus. The molecular mechanisms that control the disorder-to-order transition undergone by the intrinsically disordered C-terminal domain (NTAIL) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins are described in detail. By having a significant residual disorder, NTAIL-XD complexes are illustrative examples of "fuzziness", whose possible functional significance is discussed. Finally, the relevance of N-P interactions as promising targets for innovative antiviral approaches is underscored, and the functional advantages of structural disorder for paramyxoviruses are pinpointed.

Spectrum of Newcastle disease virus stability in gradients of temperature and pH.

Newcastle disease (ND) is one of the highly pathogenic viral diseases of avian species. The disease is endemic in many developing countries where agriculture serves as the primary source of national income. Newcastle disease virus (NDV) belongs to the family Paramyxoviridae and is well characterized member among the avian paramyxovirus serotypes. The failure of vaccination is one of the major causes of NDV outbreaks in field condition. The present study gives a brief picture about the biology of NDV genome and its proteins under different conditions of temperature and pH. Our results indicate that the NDV is non-infective above 42 °C and unstable above 72 °C. The study will be useful in defining an optimum storage condition for NDV without causing any deterioration in its viability.

Activation of paramyxovirus membrane fusion and virus entry.

The paramyxoviruses represent a diverse virus family responsible for a wide range of human and animal diseases. In contrast to other viruses, such as HIV and influenza virus, which use a single glycoprotein to mediate host receptor binding and virus entry, the paramyxoviruses require two distinct proteins. One of these is an attachment glycoprotein that binds receptor, while the second is a fusion glycoprotein, which undergoes conformational changes that drive virus-cell membrane fusion and virus entry. The details of how receptor binding by one protein activates the second to undergo conformational changes have been poorly understood until recently. Over the past couple of years, structural and functional data have accumulated on representative members of this family, including parainfluenza virus 5, Newcastle disease virus, measles virus, Nipah virus and others, which suggest a mechanistic convergence of activation models. Here we review the data indicating that paramyxovirus attachment glycoproteins shield activating residues within their N-terminal stalk domains, which are then exposed upon receptor binding, leading to the activation of the fusion protein by a 'provocateur' mechanism.

An anti-interferon activity shared by paramyxovirus C proteins: inhibition of Toll-like receptor 7/9-dependent alpha interferon induction.

Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN-α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll-like receptor (TLR) 7- and TLR9-dependent IFN-α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion.

Amino acid requirements for MDA5 and LGP2 recognition by paramyxovirus V proteins: a single arginine distinguishes MDA5 from RIG-I.

Paramyxovirus V proteins bind to MDA5 (melanoma differentiation-associated gene 5) and LGP2 (laboratory of genetics and physiology gene 2) but not RIG-I (retinoic acid-inducible gene I). The results demonstrate MDA5 R806 is essential for inhibition by diverse V proteins. Complementary substitution for the analogous RIG-I L714 confers V protein recognition. The analogous LGP2 R455 is required for recognition by measles V protein, but not other V proteins. These findings indicate that paramyxoviruses use a single amino acid to distinguish MDA5 from RIG-I and have evolved distinct contact sites for LGP2 interference.

Role of sequence and structure of the Hendra fusion protein fusion peptide in membrane fusion.

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion.

Paget's disease of bone.

OBJECTIVES: To review recent advance in understanding the causation and management of Paget's disease. DESIGN AND METHODS: We review recent publications concerning the aetiology of the disease and the use of biochemical markers of bone turnover in diagnosis and treatment. RESULTS: Epidemiologic studies suggest that Paget's disease is decreasing in prevalence and severity (implying that environmental factors are important) but there is also strong evidence of a genetic predisposition particularly through the SQSTM1 gene. Genome-wide association studies have identified polymorphisms at several other loci that are associated with the disease. Plasma alkaline phosphatase activity (ALP) is widely used, but less helpful in patients with disease of limited extent. Of the newer markers, plasma procollagen-1 N-peptide (PINP) performs best. Treatment with potent bisphosphonates usually produces long-term remission. CONCLUSIONS: Both genetic and environmental factors appear to be important in the aetiology of Paget's disease. Effective long-term disease suppression can be achieved with bisphosphonate treatment.

Capture and imaging of a prehairpin fusion intermediate of the paramyxovirus PIV5.

During cell entry, enveloped viruses fuse their viral membrane with a cellular membrane in a process driven by energetically favorable, large-scale conformational rearrangements of their fusion proteins. Structures of the pre- and postfusion states of the fusion proteins including paramyxovirus PIV5 F and influenza virus hemagglutinin suggest that this occurs via two intermediates. Following formation of an initial complex, the proteins structurally elongate, driving a hydrophobic N-terminal "fusion peptide" away from the protein surface into the target membrane. Paradoxically, this first conformation change moves the viral and cellular bilayers further apart. Next, the fusion proteins form a hairpin that drives the two membranes into close opposition. While the pre- and postfusion hairpin forms have been characterized crystallographically, the transiently extended prehairpin intermediate has not been visualized. To provide evidence for this extended intermediate we measured the interbilayer spacing of a paramyxovirus trapped in the process of fusing with solid-supported bilayers. A gold-labeled peptide that binds the prehairpin intermediate was used to stabilize and specifically image F-proteins in the prehairpin intermediate. The interbilayer spacing is precisely that predicted from a computational model of the prehairpin, providing strong evidence for its structure and functional role. Moreover, the F-proteins in the prehairpin conformation preferentially localize to a patch between the target and viral membranes, consistent with the fact that the formation of the prehairpin is triggered by local contacts between F- and neighboring viral receptor-binding proteins (HN) only when HN binds lipids in its target membrane.

The structure of the nucleoprotein binding domain of lyssavirus phosphoprotein reveals a structural relationship between the N-RNA binding domains of Rhabdoviridae and Paramyxoviridae.

The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.

Binding of rabies virus polymerase cofactor to recombinant circular nucleoprotein-RNA complexes.

In rabies virus, the attachment of the L polymerase (L) to the viral nucleocapsids (NCs)-a nucleoprotein (N)-RNA complex that serves as template for RNA transcription and replication-is mediated by the polymerase cofactor, the phosphoprotein (P). P forms dimers (P(2)) that bind through their C-terminal domains (P(CTD)) to the C-terminal region of the N. Recombinant circular N(m)-RNA complexes containing 9 to 12 protomers of N (hereafter, the subscript m denotes the number of N protomers) served here as model systems for studying the binding of P to NC-like N(m)-RNA complexes. Titration experiments show that there are only two equivalent and independent binding sites for P dimers on the N(m)-RNA rings and that each P dimer binds through a single P(CTD). A dissociation constant in the nanomolar range (160+/-20 nM) was measured by surface plasmon resonance, indicating a strong interaction between the two partners. Small-angle X-ray scattering (SAXS) data and small-angle neutron scattering data showed that binding of two P(CTD) had almost no effect on the size and shape of the N(m)-RNA rings, whereas binding of two P(2) significantly increased the size of the complexes. SAXS data and molecular modeling were used to add flexible loops (N(NTD) loop, amino acids 105-118; N(CTD) loop, amino acids 376-397) missing in the recently solved crystal structure of the circular N(11)-RNA complex and to build a model for the N(10)-RNA complex. Structural models for the N(m)-RNA-(P(CTD))(2) complexes were then built by docking the known P(CTD) structure onto the completed structures of the circular N(10)-RNA and N(11)-RNA complexes. A multiple-stage flexible docking procedure was used to generate decoys, and SAXS and biochemical data were used for filtering the models. In the refined model, the P(CTD) is bound to the C-terminal top of one N protomer (N(i)), with the C-terminal helix (alpha(6)) of P(CTD) lying on helix alpha(14) of N(i). By an induced-fit mechanism, the N(CTD) loop of the same protomer (N(i)) and that of the adjacent one (N(i)(-1)) mold around the P(CTD), making extensive protein-protein contacts that could explain the strong affinity of P for its template. The structural model is in agreement with available biochemical data and provides new insights on the mechanism of attachment of the polymerase complex to the NC template.

Specificity switching in virus-receptor complexes.

Several structures of complexes between viral attachment proteins and their cellular receptors have been determined recently, enhancing our understanding of the molecular recognition processes that guide formation of virus-receptor complexes. Moreover, these structures also highlight strategies by which highly similar viral proteins within a single virus family can adapt to engage different receptors. Consequences of such differences are altered tropism and pathogenicity. An improved understanding of the molecular details of this specificity switching in receptor binding will help to establish links between receptor tropism, spread, and disease. Moreover, it also has relevance for the design and use of viruses as gene delivery vehicles with altered properties as well as for the identification of target viral epitopes of new vaccines.

Functional interaction between paramyxovirus fusion and attachment proteins.

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 approximately 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.