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Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.

Current seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Several factors, including immunodominance of hemagglutinin over neuraminidase, instability of neuraminidase in vaccine formulations, and variable, nonstandardized amounts of neuraminidase in the vaccines, may contribute to this effect. However, vaccines that induce strong antineuraminidase immune responses would be beneficial, as they are highly protective. Furthermore, antigenic drift is slower for neuraminidase than for hemagglutinin, potentially providing broader coverage. Here, we designed stabilized recombinant versions of neuraminidase by replacing the N-terminal cytoplasmic domain, transmembrane, and extracellular stalk with tetramerization domains from the measles or Sendai virus phosphoprotein or from an Arabidopsis thaliana transcription factor. The measles virus tetramerization domain-based construct, termed N1-MPP, was chosen for further evaluation, as it retained antigenicity, neuraminidase activity, and structural integrity and provided robust protection in vivo against lethal virus challenge in the mouse model. We tested N1-MPP as a standalone vaccine, admixed with seasonal influenza virus vaccines, or given with seasonal influenza virus vaccines but in the other leg of the mouse. Admixture with different formulations of seasonal vaccines led to a weak neuraminidase response, suggesting a dominant effect of hemagglutinin over neuraminidase when administered in the same formulation. However, administration of neuraminidase alone or with seasonal vaccine administered in the alternate leg of the mouse induced robust antibody responses. Thus, this recombinant neuraminidase construct is a promising vaccine antigen that may enhance and broaden protection against seasonal influenza viruses. IMPORTANCE Influenza virus infections remain a high risk to human health, causing up to 650,000 deaths worldwide every year, with an enormous burden on the health care system. Since currently available seasonal vaccines are only partially effective and often mismatched to the circulating strains, a broader protective influenza virus vaccine is needed. Here, we generated a recombinant influenza virus vaccine candidate based on the more conserved neuraminidase surface glycoprotein in order to induce a robust and broader protective immune response against a variety of circulating influenza virus strains.

Intrinsically disordered proteins (IDPs), which in isolation do not adopt a well-defined tertiary structure but instead populate a structurally heterogeneous ensemble of interconverting states, play important roles in many biological pathways. IDPs often fold into ordered states upon binding to their physiological interaction partners (a so-called "folding-upon-binding" process), but it has proven difficult to obtain an atomic-level description of the structural mechanisms by which they do so. Here, we describe in atomic detail the folding-upon-binding mechanism of an IDP segment to its binding partner, as observed in unbiased molecular dynamics simulations. In our simulations, we observed over 70 binding and unbinding events between the α-helical molecular recognition element (α-MoRE) of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N TAIL ) and the X domain (XD) of the measles virus phosphoprotein complex. We found that folding-upon-binding primarily occurred through induced-folding pathways (in which intermolecular contacts form before or concurrently with the secondary structure of the disordered protein)-an observation supported by previous experiments-and that the transition state ensemble was characterized by formation of just a few key intermolecular contacts and was otherwise highly structurally heterogeneous. We found that when a large amount of helical content was present early in a transition path, N TAIL typically unfolded and then refolded after additional intermolecular contacts formed. We also found that, among conformations with similar numbers of intermolecular contacts, those with less helical content had a higher probability of ultimately forming the native complex than conformations with more helical content, which were more likely to unbind. These observations suggest that even after intermolecular contacts have formed, disordered regions can have a kinetic advantage over folded regions in the folding-upon-binding process.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The measles virus replication complex represents a potentially important, but as yet relatively unexplored target for viral inhibition. Little is known about the molecular mechanisms that underpin replication and transcription in paramyxoviruses. In recent years it has become clear that conformational dynamics play an important role in paramyxoviral replication, and that a complete understanding of the viral cycle requires a description of the structural plasticity of the different components. Here, we review recent progress in this direction, covering the dynamics of the nucleocapsid assembly process, high resolution structure and dynamics of protein:RNA interactions, and the investigation of the role of intrinsic conformational disorder in pre-assembly nucleoprotein/phosphoprotein complexes. Finally, we discuss the role of viral factories in the form of phase-separated membraneless organelles formed by measles virus phospho and nucleoproteins that promote the assembly of nucleocapsid structures., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Intrinsically Disordered Proteins (IDPs) are a class of protein that exert their function despite lacking a well-defined three-dimensional structure, which is sometimes achieved only upon binding to their natural ligands. This feature implies the folding of IDPs to be generally coupled with a binding event, representing an interesting challenge for kinetic studies. In this review, we recapitulate some of the most important findings of IDPs binding-induced folding mechanisms obtained by analyzing their binding kinetics. Furthermore, by focusing on the interaction between the Measles virus N TAIL protein, a prototypical IDP, and its physiological partner, the X domain, we recapitulate the major theoretical and experimental approaches that were used to describe binding induced folding., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The hemagglutinin (H) protein of measles viruses (MeV) mediates binding to the cellular receptors, CD46,human signaling lymphocyte activation molecule and nectin-4. Vaccine strains primarily contain H-proteins possessing MeV-H: Y481 and can utilize CD46. Reports suggest that a single amino acid change in MeV-H at position 481 in wild type strains renders them inefficient in utilizing CD46. The in-depth molecular mechanism by which substitutions at 481 and another reported critical residue position 546 affects CD46 binding affinity however remains elusive. We used molecular docking studies of CD46 with MeV-H possessing Y481 N/D to understand the in-depth molecular mechanism involved. It was found that loss in either of the hydrogen bond (H-bond) contacts (MeV-H:481-CD46:65, MeV-H:546-CD46:63) in the central contact region prevented efficient CD46 binding. Y481 N could form the specific H-bond, while G546S H-bond could be formed only in conjunction with Y481, revealing the significance of these residues in determining CD46 receptor binding potential. Elucidating the underlying molecular mechanism of receptor usage by the MeV has implications to understanding cellular tropism, viral pathogenesis and therapy., (Copyright © 2019. Published by Elsevier Ltd.)

Nipah virus is a highly lethal zoonotic pathogen found in Southeast Asia that has caused human encephalitis outbreaks with 40%-70% mortality. NiV encodes its own RNA-dependent RNA polymerase within the large protein, L. Efficient polymerase activity requires the phosphoprotein, P, which tethers L to its template, the viral nucleocapsid. P is a multifunctional protein with modular domains. The central P multimerization domain is composed of a long, tetrameric coiled coil. We investigated the importance of structural features found in this domain for polymerase function using a newly constructed NiV bicistronic minigenome assay. We identified a conserved basic patch and central kink in the coiled coil that are important for polymerase function, with R555 being absolutely essential. This basic patch and central kink are conserved in the related human pathogens measles and mumps viruses, suggesting that this mechanism may be conserved., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The interaction between N TAIL and XD from the measles virus represents a paradigmatic example of molecular recognition between an intrinsically disordered protein and a folded partner. By binding to XD, a small portion of N TAIL (classically denoted as MoRE) undergoes a disorder-to-order transition, populating an α-helical structure, while the reminder of the protein remains disordered. Here, we demonstrate an unexpected crosstalk between such a disordered region and the adjacent molecular recognition element (MoRE). This result was obtained by producing a series of truncation and site-directed variants of N TAIL while measuring the effects on the kinetics of folding and binding. We show that the disordered region of N TAIL exerts its inhibitory role by slowing the folding step of the MoRE, thereby tuning the affinity of the interaction.

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly., Competing Interests: The authors declare no conflict of interest.

Protein thermal shift assay (TSA) has been extensively used in investigation of protein stabilization (for protein biopharmaceutics stabilization, protein crystallization studies or screening of recombinant proteins) and drug discovery (screening of ligands or inhibitors). This work aimed to analyze thermal shift assay results in comparison to protein polymerization (multimerization and aggregation) propensity and test the most stabilizing formulations for their stabilization effect on enveloped viruses. Influence of protein concentration, buffer pH and molarity was tested on three proteins (immunoglobulin G, ovalbumin, and albumin) and results showed that each of these factors has an impact on determined shift in protein melting point T m , and the impact was similar for all three proteins. In case of ovalbumin, molecular dynamics simulations were performed with the goal to understanding molecular basis of protein's thermal stability dependence on pH. Effect of three denaturing agents in a wide concentration range on T m showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on T m for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing potential using cell culture infectivity assay (CCID 50 ) and results showed lack of correlation with TSA results. Only weak correlation of T m shift and protein polymerization measured by SEC-HPLC was obtained, meaning that polymerization cannot be predicted from T m shifts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Measles (MEV) and mumps virus (MUV) are enveloped, non-segmented, negative single stranded RNA viruses of the family Paramyxoviridae, and are the cause of measles and mumps, respectively, both preventable by vaccination. Aside from proteins coded by the viral genome, viruses are considered to contain host cell proteins (HCPs). The presence of extracellular vesicles (ECVs), which are often co-purified with viruses due to their similarity in size, density and composition, also contributes to HCPs detected in virus preparations, and this has often been neglected. The aim was to identify which virus-coded proteins are present in MEV and MUV virions, and to try to detect which HCPs, if any, are incorporated inside the virions or adsorbed on their outer surface, and which are more likely to be a contamination from co-purified ECVs., Methods: MUV, MEV and ECVs were purified by ultracentrifugation, hydrophobic interaction chromatography and immunoaffinity chromatography, proteins in the samples were resolved by SDS-PAGE and subjected to identification by MALDI-TOF/TOF-MS. A comparative analysis of HCPs present in all samples was carried out., Results: By proteomics approach, it was verified that almost all virus-coded proteins are present in MEV and MUV particles. Protein C in MEV which was until now considered to be non-structural viral protein, was found to be present inside the MeV virions. Results on the presence of HCPs in differently purified virus preparations imply that actin, annexins, cyclophilin A, moesin and integrin β1 are part of the virions., Conclusions: All HCPs detected in the viruses are present in ECVs as well, indicating their possible function in vesicle formation, or that most of them are only present in ECVs. Only five HCPs were constantly present in purified virus preparations, regardless of the purification method used, implying they are likely the integral part of the virions. The approach described here is helpful for further investigation of HCPs in other virus preparations.

Intrinsically disordered proteins (IDPs) possess a peculiar amino acid composition that makes them very soluble. Nevertheless, they can encounter aggregation in physiological and pathological contexts. In this work, we addressed the issue of how electrostatic charges can influence aggregation propensity by using the N-terminus moiety of the measles virus phosphoprotein, PNT, as a model IDP. Taking advantage of the high sequence designability of IDPs, we have produced an array of PNT variants sharing the same hydrophobicity, but differing in net charges per residue and isoelectric points (pI). The solubility and conformational properties of these proteins were analysed through biochemical and biophysical techniques in a wide range of pH values and compared with those of the green fluorescence protein (GFP), a globular protein with lower net charge per residue, but similar hydrophobicity. Tested proteins showed a solubility minimum close to their pI, as expected, but the pH-dependent decrease of solubility was not uniform and driven by the net charge per residue of each variant. A parallel behaviour was observed also in fusion proteins between PNT variants and GFP, which minimally contributes to the solubility of chimeras. Our data suggest that the overall solubility of a protein can be dictated by protein regions endowed with higher net charge per residue and, hence, prompter to respond to pH changes. This finding could be exploited for biotechnical purposes, such as the design of solubility/aggregation tags, and in studies aimed to clarify the pathological and physiological behaviour of IDPs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Intrinsically disordered proteins (IDPs) are functionally active despite lacking a well-defined three-dimensional structure. Such proteins often undergo a disorder-to-order transition, or induced folding, when binding to their specific physiological partner. Because of cooperativity, the folding and binding steps typically appear as a single event, and therefore, induced folding is extremely difficult to characterize experimentally. In this perspective, the interaction between the disordered C-terminal domain of the measles virus nucleoprotein N TAIL and the folded X domain of the viral phosphoprotein (XD) is particularly interesting because the inherent complexity of the observed kinetics allows characterization of the binding and folding steps individually. Here we present a detailed structural description of the folding and binding events occurring in the recognition between N TAIL and XD. This result was achieved by measuring the effect of single-amino acid substitutions in N TAIL on the reaction mechanism. Analysis of the experimental data allowed us (i) to identify the key residues involved in the initial recognition between the two molecules and (ii) to depict the general features of the folding pathway of N TAIL . Furthermore, an analysis of the changes in stability obtained for the whole set of variants highlights how the sequence of this IDP has not been selected during evolution to fold efficiently. This feature might be a consequence of the weakly funneled nature of the energy landscape of IDPs in their unbound state and represents a plausible explanation of their highly dynamic nature even in the bound state, typically defined as "fuzziness".

In this study we evaluated the long-term stability of a microarray-based serological screening platform, containing antigens of influenza A, measles and Streptococcus pneumoniae, as part of a preparedness research program aiming to develop assays for syndromic disease detection. Spotted microarray slides were kept at four different storage regimes with varying temperature and humidity conditions. We showed that under the standard storage condition in a temperature-controlled (21°C) and desiccated environment (0% relative humidity), microarray slides remained stable for at least 22 months without loss of antigen quality, whereas the other three conditions (37°C, desiccated; Room temperature, non-desiccated; Frozen, desiccated) produced acceptable results for some antigens (influenza A, S.pneumoniae), but not for others (measles). We conclude that these arrays for multiplex antibody testing can be prepared and stored for prolonged periods of time, which aids laboratory-preparedness and facilitates sero-epidemiological studies., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The paramyxovirus family comprises major human and animal pathogens such as measles virus (MeV), mumps virus (MuV), the parainfluenzaviruses, Newcastle disease virus (NDV), and the highly pathogenic zoonotic hendra (HeV) and nipah (NiV) viruses. Paramyxovirus particles are pleomorphic, with a lipid envelope, nonsegmented RNA genomes of negative polarity, and densely packed glycoproteins on the virion surface. A number of crystal structures of different paramyxovirus proteins and protein fragments were solved, but the available information concerning overall virion organization remains limited. However, recent studies have reported cryo-electron tomography-based reconstructions of Sendai virus (SeV), MeV, NDV, and human parainfluenza virus type 3 (HPIV3) particles and a surface assessment of NiV-derived virus-like particles (VLPs), which have yielded innovative hypotheses concerning paramyxovirus particle assembly, budding, and organization. Following a summary of the current insight into paramyxovirus virion morphology, this review will focus on discussing the implications of these particle reconstructions on the present models of paramyxovirus assembly and infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introducción. El virus del Zika (ZIKV) es un flavivirus con envoltura, transmitido a los seres humanos principalmente por el vector Aedes aegypti. La infección por ZIKV se ha asociado con un gran neurotropismo y con efectos neuropáticos, como el síndrome de Guillain-Barré en el adulto y la microcefalia fetal y posnatal, así como con un síndrome de infección congénita similar al producido por el virus de la rubéola (RV).Objetivo. Comparar las estructuras moleculares de la proteína de envoltura E del virus del Zika (E-ZIKV) y de la E1 del virus de la rubéola (E1-RV), y plantear posibles implicaciones en el neurotropismo y en las alteraciones del sistema nervioso asociadas con el ZIKV.Materiales y métodos. La secuencia de aminoácidos de la proteína E-ZIKV (PDB: 5iZ7) se alineó con la de la glucopreteína E1 del virus de la rubéola (PDB: 4ADG). Los elementos de la estructura secundaria se determinaron usando los programas Vector NTI Advance®, DSSP y POSA, así como herramientas de gestión de datos (AlignX®). Uno de los criterios principales de comparación y alineación fue la asignación de residuos estructuralmente equivalentes, con más de 70 % de identidad.Resultados. La organización estructural de la proteína E-ZIKV (PDB: 5iZ7) fue similar a la de E1-RV (PDB: 4ADG) (70 a 80 % de identidad), y se observó una correspondencia con la estructura definida para las glucoproteínas de fusión de membrana de clase II de los virus con envoltura. E-ZIKV y E1-RV exhibieron elementos estructurales de fusión muy conservados en la región distal del dominio II, asociados con la unión a los receptores celulares de entrada del virus de la rubéola (glucoproteína de mielina del oligodendrocito, Myelin Oligodendrocyte Glycoprotein, MOG), y con los receptores celulares Axl del ZIKV y de otros flavivirus.Conclusión. La comparación de las proteínas E-ZIKV y E1-RV es un paso necesario hacia la definición de otros factores moleculares determinantes del neurotropismo y la patogenia del ZIKV, el cual puede contribuir a generar estrategias de diagnóstico, prevención y tratamiento de las complicaciones neurológicas inducidas por el ZIKV.

The fusion (F) protein of measles virus performs refolding from the thermodynamically metastable prefusion form to the highly stable postfusion form via an activated unstable intermediate stage, to induce membrane fusion. Some amino acids involved in the fusion regulation cluster in the heptad repeat B (HR-B) domain of the stalk region, among which substitution of residue 465 by various amino acids revealed that fusion activity correlates well with its side chain length from the Cα (P<0.01) and van der Waals volume (P<0.001), except for Phe, Tyr, Trp, Pro and His carrying ring structures. Directed towards the head region, longer side chains of the non-ring-type 465 residues penetrate more deeply into the head region and may disturb the hydrophobic interaction between the stalk and head regions and cause destabilization of the molecule by lowering the energy barrier for refolding, which conferred the F protein enhanced fusion activity. Contrarily, the side chain of ring-type 465 residues turned away from the head region, resulting in not only no contact with the head region but also extensive coverage of the HR-B surface, which may prevent the dissociation of the HR-B bundle for initiation of membrane fusion and suppress fusion activity. Located in the HR-B domain just at the junction between the head and stalk regions, amino acid 465 is endowed with a possible ability to either destabilize or stabilize the F protein depending on its molecular volume and the direction of the side chain, regulating fusion activity of measles virus F protein.

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides., Importance: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides., (Copyright © 2016 American Society for Microbiology.)

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region., Competing Interests: The authors have declared that no competing interests exist.

To study the genetic characterization and amino acid mutation of hemagglutinin protein of four genotypes of wild type measles Viruses isolated in 2013 and 2014year,in China, including H1,B3,D8 and D9.Four genotype isolates of H1,D8,D9 and B3measles viruses were selected, and RNA of MV isolates were extracted. The complete sequence of hemagglutinin (1854nt) were amplified by RT-PCR and sequenced and Sequencher5.0was used to splice sequence. Phylogenetic analysis and the diversity of gene and amino acid were done by MEGA version 5.0,compared with 24 representative strains, repectively 4 H a sub-genotype strains,8D8 genotype strains,2D9 genotype strains,8B3 genotype strains and 2Chinese vaccine Strains, which were downloaded from GenBank. The homology of H gene nucleotide sequence and amino acid between the four genotype measles virus strains including H1,B3,D8,D9 and A genotype strains were respectively 94.2%-96.7%and 95.4%-96.7%and there were 20-28 amino acids difference between them; And the homology of H gene nucleotide sequence between Beijing14-1(H1a)and A genotype vaccine strains was 97.7%-98.2%.The N-glycosylation site in 240th was losen because of mutation. The homology of H gene nucleotide sequence and amino acid between the four genotype measles virus strains including H1,B3,D8,D9 and A genotype strains were high, and the Chinese vaccine can effectively prevent infection caused by H1asub-genotype,D8,D9 and B3imported genotypes virus strains.

Objective: The hybridoma technology is one of the most important advances in clinical immunology. Little is known about the differences between the antibodies produced during the in vivo immune response and those recovered in hybridoma libraries. Here, we investigate a potential fusion bias inherent to the hybridoma production process., Methods: Transgenic rats carrying human Ig heavy and light chain loci were immunized with measles virus (MV) to generate human mAbs. Usin g high-throughput sequencing of IgH mRNA, we compared the IgH repertoire of lymph nodes and the derived hybridoma library using the sequences of the MV-specific hybridoma clones as a reference set with known specificity., Results: We observed that large clonotypes from the lymph nodes were not represented in the hybridoma library, but low-frequency B cell populations became highly enriched and most hybridoma clones were derived from these. Our data also showed that identical CDR3s evolved from diverse VDJ recombinations, indicating convergence of different B cells subpopulations towards expression of antibodies with similar paratopes., Conclusion: The efficient generation of mAbs results from a fusion process highly selective for rare antigen-specific B cells rather than in vivo expanded populations. Antibodies of particular interest may therefore be missed during classical hybridoma production.

Measles virus and mumps virus (MeV and MuV) are enveloped RNA viruses used for production of live attenuated vaccines for prophylaxis of measles and mumps disease, respectively. For biotechnological production of and basic research on these viruses, the preparation of highly purified and infectious viruses is a prerequisite, and to meet that aim, knowledge of their stability and biophysical properties is crucial. Our goal was to carry out a detailed investigation of the stability of MeV and MuV under various pH, temperature, shear stress, filtration and storage conditions, as well as to evaluate two commonly used purification techniques, ultracentrifugation and diafiltration, with regard to their efficiency and effect on virus properties. Virus titers were estimated by CCID50 assay, particle size and concentration were measured by Nanoparticle tracking analysis (NTA) measurements, and the host cell protein content was determined by ELISA. The results demonstrated the stability of MuV and MeV at pH <9 and above pH 4 and 5, respectively, and aggregation was observed at pH >9. Storage without stabilizer did not result in structural changes, but the reduction in infectivity after 24 hours was significant at +37 °C. Vortexing of the viruses resulted in significant particle degradation, leading to lower virus titers, whereas pipetting had much less impact on virus viability. Diafiltration resulted in higher recovery of both total and infectious virus particles than ultracentrifugation. These results provide important data for research on all upstream and downstream processes on these two viruses regarding biotechnological production and basic research.

Although most proteins fold by populating intermediates, the transient nature of such states makes it difficult to characterize their structures. In this work we identified and characterized the structure of an intermediate of the X domain of phosphoprotein (P) of measles virus. We obtained this result by a combination of equilibrium and kinetic measurements and NMR chemical shifts used as structural restraints in replica-averaged metadynamics simulations. The structure of the intermediate was then validated by rationally designing four mutational variants predicted to affect the stability of this state. These results provide a detailed view of an intermediate state and illustrate the opportunities offered by a synergistic use of experimental and computational methods to describe non-native states at atomic resolution., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: Imported measles virus (MV) outbreaks are maintained by poor vaccine responders and unvaccinated people. A convenient but more immunogenic vaccination strategy would enhance vaccine performance, contributing to measles eradication efforts. We report here the generation of alternative pediatric vaccines against MV with increased expression of the H protein in the background of the current MV vaccine strain. We generated two recombinants: MVvac2-H2, with increased full-length H expression resulting in a 3-fold increase in H incorporation into virions, and MVvac2-Hsol, vectoring a truncated, soluble form of the H protein that is secreted into the supernatants of infected cells. Replication fitness was conserved despite the duplication of the H cistron for both vectors. The modification to the envelope of MVvac2-H2 conferred upon this virus a measurable level of resistance to in vitro neutralization by MV polyclonal immune sera without altering its thermostability. Most interestingly, both recombinant MVs with enhanced H expression were significantly more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic after a single, human-range dose in genetically modified MV-susceptible mice., Importance: Measles incidence was reduced drastically following the introduction of attenuated vaccines, but progress toward the eradication of this virus has stalled, and MV still threatens unvaccinated populations. Due to the contributions of primary vaccine failures and too-young-to-be-vaccinated infants to this problem, more immunogenic measles vaccines are highly desirable. We generated two experimental MV vaccines based on a current vaccine's genome but with enriched production of the H protein, the main MV antigen in provoking immunity. One vaccine incorporated H at higher rates in the viral envelope, and the other secreted a soluble H protein from infected cells. The increased expression of H by these vectors improved neutralizing responses induced in two small-animal models of MV immunogenicity. The enhanced immunogenicity of these vectors, mainly from the MV that incorporates additional H, suggests their value as potential alternative pediatric MV vaccines., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N(0)) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N(0) assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N(0)₁₋₄₀₈ fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N(0)-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N(0) binding peptide, P₁₋₄₈, to the C terminus of an N(0)₂₁₋₄₀₈ fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N(0)-P complex. We solved the X-ray structure of the resulting N(0)-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N(0)-P interface and explains how P chaperones N(0), preventing both self-assembly of N(0) and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization., Importance: Measles virus is an important, highly contagious human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development to treat not only measles but also potentially other paramyxovirus diseases., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Modification of an enveloped measles virus was achieved by metabolic incorporation of azido sugars in host cells through the protein glycosylation process. Based on this, the resulting measles virus particles could be modified with azido groups on the surface glycoproteins, which could be further labeled with fluorescence dyes using a strain-promoted azide-alkyne cycloaddition reaction. We envision this metabolic labeling approach to be applicable to a wide variety of enveloped viruses, allowing the facile conjugation and surface modification.

Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus nucleocapsid offers important insights into the mechanism of the helical polymerization of nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design., (Copyright © 2015, American Association for the Advancement of Science.)

In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms.

Unlabelled: The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor., Importance: Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

(1)H-detected solid-state nuclear magnetic resonance (NMR) experiments are recorded on both intact and trypsin-cleaved sedimented measles virus (MeV) nucleocapsids under ultra-fast magic-angle spinning. High-resolution (1)H,(15)N-fingerprints allow probing the degree of molecular order and flexibility of individual capsid proteins, providing an exciting atomic-scale complement to electro microscopy (EM) studies of the same systems., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The structures of two constructs of the measles virus (MeV) phosphoprotein (P) multimerization domain (PMD) are reported and are compared with a third structure published recently by another group [Communie et al. (2013), J. Virol. 87, 7166-7169]. Although the three structures all have a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure and unveiled that the three structures suffer from significant structural deformation induced by intermolecular interactions within the crystal. These results show that crystal packing can bias conclusions about function and mechanism based on analysis of a single crystal structure, and they challenge to some extent the assumption according to which coiled-coil structures can be reliably predicted from the amino-acid sequence. Structural comparison also highlighted significant differences in the extent of disorder in the C-terminal region of each monomer. The differential flexibility of the C-terminal region is also supported by size-exclusion chromatography and small-angle X-ray scattering studies, which showed that MeV PMD exists in solution as a dynamic equilibrium between two tetramers of different compaction. Finally, the possible functional implications of the flexibility of the C-terminal region of PMD are discussed.

A quartet of attachment proteins and a trio of fusion protein subunits play the cell entry concert of parainfluenza viruses. While many of these viruses bind sialic acid to enter cells, wild type measles binds exclusively two tissue-specific proteins, the lymphatic receptor signaling lymphocytic activation molecule (SLAM), and the epithelial receptor nectin-4. SLAM binds near the stalk-head junction of the hemagglutinin. Nectin-4 binds a hydrophobic groove located between blades 4 and 5 of the hemagglutinin β-propeller head. The mutated vaccine strain hemagglutinin binds in addition the ubiquitous protein CD46, which explains attenuation. The measles virus entry concert has four movements. Andante misterioso: the virus takes over the immune system. Allegro con brio: it rapidly spreads in the upper airway's epithelia. 'Targeting' fugue: the versatile orchestra takes off. Presto furioso: the virus exits the host with thunder. Be careful: music is contagious., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Measles virus nucleoprotein is the most abundant viral protein and tightly encapsidates viral genomic RNA to support viral transcription and replication. Major phosphorylation sites of nucleoprotein include the serine residues at locations 479 and 510. Minor phosphorylation residues have yet to be identified, and their functions are poorly understood. In our present study, we identified nine putative phosphorylation sites by mass spectrometry and demonstrated that threonine residue 279 (T279) is functionally significant. Minigenome expression assays revealed that a mutation at the T279 site caused a loss of activity. Limited proteolysis and electron microscopy suggested that a T279A mutant lacked the ability to encapsidate viral RNA but was not denatured. Furthermore, dephosphorylation of the T279 site by alkaline phosphatase treatment caused deficiencies in nucleocapsid formation. Taken together, these results indicate that phosphorylation at T279 is a prerequisite for successful nucleocapsid formation.

Introduction: The European Regional Office of the World Health Organization (WHO/Europe) developed a strategic approach to stop the indigenous transmission of measles in its 53 Member States by 2015. This study describes the measles laboratory surveillance activity performed by the National Reference Laboratory for Measles and Rubella at the Italian National Institute of Health (Istituto Superiore di Sanità) during 2010., Methods: Urine, oral fluid and capillary blood samples from 211 suspected measles cases arrived to the NRL from different regions of Italy for confirmation of the clinical diagnosis. Serological and/or molecular assays were performed; after molecular detection, positive samples were sequenced and genotyped., Results and Discussion: 85% (180/211) of the specimens were confirmed as measles cases and 139 of these were analyzed phylogenetically. The phylogenetic analysis revealed a co-circulation of D4 and D8 genotypes for the reviewed period.

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Numerous relatively short regions within intrinsically disordered proteins (IDPs) serve as molecular recognition elements (MoREs). They fold into ordered structures upon binding to their partner molecules. Currently, there is still a lack of in-depth understanding of how coupled binding and folding occurs in MoREs. Here, we quantified the unbound ensembles of the α-MoRE within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein. We developed a multiscaled approach by combining a physics-based and an atomic hybrid model to decipher the mechanism by which the α-MoRE interacts with the X domain of the measles virus phosphoprotein. Our multiscaled approach led to remarkable qualitative and quantitative agreements between the theoretical predictions and experimental results (e.g., chemical shifts). We found that the free α-MoRE rapidly interconverts between multiple discrete partially helical conformations and the unfolded state, in accordance with the experimental observations. We quantified the underlying global folding-binding landscape. This leads to a synergistic mechanism in which the recognition event proceeds via (minor) conformational selection, followed by (major) induced folding. We also provided evidence that the α-MoRE is a compact molten globule-like IDP and behaves as a downhill folder in the induced folding process. We further provided a theoretical explanation for the inherent connections between "downhill folding," "molten globule," and "intrinsic disorder" in IDP-related systems. Particularly, we proposed that binding and unbinding of IDPs proceed in a stepwise way through a "kinetic divide-and-conquer" strategy that confers them high specificity without high affinity.

The atomic structure of the stable tetramerization domain of the measles virus phosphoprotein shows a tight four-stranded coiled coil. Although at first sight similar to the tetramerization domain of the Sendai virus phosphoprotein, which has a hydrophilic interface, the measles virus domain has kinked helices that have a strongly hydrophobic interface and it lacks the additional N-terminal three helical bundles linking the long helices.

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.

Measles virus (MV), one of the most contagious agents, infects immune cells using the signaling lymphocyte activation molecule (SLAM) on the cell surface. A complex of SLAM and the attachment protein, hemagglutinin (MVH), has remained elusive due to the intrinsic handling difficulty including glycosylation. Furthermore, crystals obtained of this complex are either nondiffracting or poorly-diffracting. To solve this problem, we designed a systematic approach using a combination of the following techniques; (1) a transient expression system in HEK293SGnTI(-) cells, (2) lysine methylation, (3) structure-guided mutagenesis directed at better crystal packing, (4) Endo H treatment, (5) single-chain formation for stable complex, and (6) floating-drop vapor diffusion. Using our approach, the receptor-binding head domain of MV-H covalently fused with SLAM was successfully crystallized and diffraction was improved from 4.5 Å to a final resolution of 3.15 Å . These combinational methods would be useful as crystallization strategies for complexes of glycoproteins and their receptors.

Interaction of polystirolsulphonate with polymerization degree of 8 (PSS-8) and polyallylamin PAA (molecular mass 60 kilodaltons) with viruses from bloodline of paramixo- and orthomixoviruses by the example of measles virus, parotitis and flu leads to the decreasing of infective activity. The possible mechanism of viral inhibitive action of these chemical compounds is damaging of interfacial antigenic proteins of paramixo- and orthomixoviruses. In this study it was detected the change of surface tension of bilayer lipid membrane in the presence of PSS-8 and PAA. The change of surface tension leads to disorder in viral proteins adsorption in bilayer lipid membrane. This process could lead to disorder of juncture and self-assembly of virions.

Transcriptional induction of beta interferon (IFN-β) through pattern recognition receptors is a key event in the host defense against invading viruses. Infection of cells by paramyxoviruses, like measles virus (MV) (genus Morbillivirus), is sensed predominantly by the ubiquitous cytoplasmic helicase RIG-I, recognizing viral 5'-triphosphate RNAs, and to some degree by MDA5. While MDA5 activation is effectively prevented by the MV V protein, the viral mechanisms for inhibition of MDA5-independent induction of IFN-β remained obscure. Here, we identify the 186-amino-acid MV C protein, which shuttles between the nucleus and the cytoplasm, as a major viral inhibitor of IFN-β transcription in human cells. Activation of the transcription factor IRF3 by upstream kinases and nuclear import of activated IRF3 were not affected in the presence of C protein, suggesting a nuclear target. Notably, C proteins of wild-type MV isolates, which are poor IFN-β inducers, were found to comprise a canonical nuclear localization signal (NLS), whereas the NLSs of all vaccine strains, irrespective of their origins, were mutated. Site-directed mutagenesis of the C proteins from an MV wild-type isolate and from the vaccine virus strain Schwarz confirmed a correlation of nuclear localization and inhibition of IFN-β transcription. A functional NLS and efficient nuclear accumulation are therefore critical for MV C to retain its potential to downregulate IFN-β induction. We suggest that a defect in efficient nuclear import of C protein contributes to attenuation of MV vaccine strains.

Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix-matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices.

Abstract vectors (LVs) that are targeted to APC using a chimeric measles virus (MV) hemagglutinin (H). The MV H protein is mutated to prevent binding to MV receptors and incorporates a single-chain antibody that recognizes murine major histocompatibility complex class II (MHC II). This targeted LV is highly efficient in transduction of freshly isolated mouse B cells and dendritic cells. MHC II-positive cells in spleen are transduced after intravenous injection, and a robust immune response to an antigen transgene is generated.

Tools that can aid in vitro and in vivo imaging and also noninvasively determine half-life and biodistribution are required to advance clinical developments. A Function-Spacer-Lipid construct (FSL) incorporating fluorescein (FSL-FLRO4) was used to label vesicular stomatitis virus (VSV), measles virus MV-NIS (MV) and influenza virus (H1N1). The ability of FSL constructs to label these virions was established directly by FACScan of FSL-FLRO4 labeled VSV and MV, and indirectly following labeled H1N1 and MV binding to a cells. FSL-FLRO4 labeling of H1N1 was shown to maintain higher infectivity of the virus when compared with direct fluorescein virus labeling. A novel tyrosine (125)I radioiodinated FSL construct was synthesized (FSL-(125)I) from FSL-tyrosine. This was used to label VSV (VSV-FSL-(125)I), which was infused into the peritoneal cavity of laboratory mice. Bioscanning showed VSV-FSL-(125)I to localize in the liver, spleen and bloodstream in contrast to the free labels FSL-(125)I or (125)I, which localized predominantly in the liver and thyroid respectively. This is a proof-of-principle novel and rapid method for modifying virions and demonstrates the potential of FSL constructs to improve in vivo imaging of virions and noninvasively observe in vivo biodistribution., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with the Edmonston measles virus (MV) hemagglutinin and fusion glycoproteins (Hgps and Fgps) (H/F-LVs), which, for the first time, allowed efficient transduction of quiescent human B and T cells. These target cells express both MV entry receptors used by the vaccinal Edmonston strain, CD46 and signaling lymphocyte activation molecule (SLAM). Interestingly, LVs pseudotyped with an MV Hgp, blind for the CD46 binding site, were completely inefficient for resting-lymphocyte transduction. Similarly, SLAM-blind H mutants that recognize only CD46 as the entry receptor did not allow stable LV transduction of resting T cells. The CD46-tropic LVs accomplished vector-cell binding, fusion, entry, and reverse transcription at levels similar to those achieved by the H/F-LVs, but efficient proviral integration did not occur. Our results indicate that both CD46 and SLAM binding sites need to be present in cis in the Hgp to allow successful stable transduction of quiescent lymphocytes. Moreover, the entry mechanism utilized appears to be crucial: efficient transduction was observed only when CD46 and SLAM were correctly engaged and an entry mechanism that strongly resembles macropinocytosis was triggered. Taken together, our results suggest that although vector entry can occur through the CD46 receptor, SLAM binding and subsequent signaling are also required for efficient LV transduction of quiescent lymphocytes to occur.