Investigation of the thermal shift assay and its power to predict protein and virus stabilizing conditions.

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 Tm, 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 Tm showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on Tm for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing potential using cell culture infectivity assay (CCID50) and results showed lack of correlation with TSA results. Only weak correlation of Tm shift and protein polymerization measured by SEC-HPLC was obtained, meaning that polymerization cannot be predicted from Tm shifts.

Structure and organization of paramyxovirus particles.

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.

Características de la estructura molecular de las proteínas E del virus del Zika y E1 del virus de la rubéola y posibles implicaciones en el neurotropismo y en las alteraciones del sistema nervioso.

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.

Características de la estructura molecular de las proteínas E del virus del Zika y E1 del virus de la rubéola y posibles implicaciones en el neurotropismo y en las alteraciones del sistema nervioso / E-ZIKV and E1-RV proteins molecular structure and their potential implications in neurotropism and nervous system disorders

Resumen 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.

Abstract Introduction: Zika virus (ZIKV) is an enveloped flavivirus transmitted to humans mainly by Aedes aegypti. ZIKV infection has been associated with high neurotropism and neuropathic effects such as the Guillain-Barré syndrome in adults, and fetal and postnatal microcephaly and the congenital Zika virus syndrome similar to that produced by rubella virus (VR). Objective: To compare Zika virus membrane protein E (E-ZIKV) and rubella virus membrane protein E1 (E1-RV), and to propose possible implications for neurotropism and nervous system disorders associated with ZIKV infections. Materials and methods: The amino acid sequence of E-ZIKV protein (PDB: 5iZ7) was aligned to that of rubella virus glycoprotein E1 (PDB: 4ADG). The secondary structure elements were determined using the programs Vector NTI Advance®, DSSP, and POSA, and integrated data management tools (AlignX®). One of the main comparison and alignment criteria was the allocation of structurally equivalent residues with more than 70% identity. Results: E-ZIKV structural organization (PDB: 5iZ7) was similar to that of E1-RV (PDB: 4ADG) (70%-80% identity), and it was consistent with relevant structural features of viral membrane class II fusion glycoproteins. E-ZIKV and E1-RV exhibited highly conserved fusion structural elements at the distal region of domain II, which has been associated with the RV myelin oligodendrocyte glycoprotein and Axl cell receptors in ZIKV and other flaviviruses. Conclusion: The comparison of E-ZIKV and E1-RV proteins constitutes an essential step towards the definition of ZIKV neurotropism and pathogenesis molecular determinants, and for the adoption of diagnosis, prevention and treatment strategies against neurological complications induced by ZIKV infection.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

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.

A residue located at the junction of the head and stalk regions of measles virus fusion protein regulates membrane fusion by controlling conformational stability.

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 fusion machinery activated by sialic acid binding globular domain.

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.

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

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.

Base of the measles virus fusion trimer head receives the signal that triggers membrane fusion.

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 glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both SLAM and CD46 entry receptors.

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.

Conformational analysis of the partially disordered measles virus N(TAIL)-XD complex by SDSL EPR spectroscopy.

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) both free and in complex with the X domain (XD, aa 459-507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted N(TAIL) variants, we showed that the 490-500 region of N(TAIL) is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the alpha-helical sampling of the free form. In addition, we showed that the 505-525 region of N(TAIL) conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the N(TAIL)-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488-525 region of N(TAIL), in particular the unfolding of the alpha-helical region when the temperature was increased from 281 K to 310 K.

Measles virus: evidence for association with lung cancer.

In recent years the frequency of nonsmokers among lung cancer patients has increased to 10% to 15%. The measles virus has rarely been evoked as an etiological agent in malignant tumors and its role in carcinogenesis remains doubtful. It has been suggested that measles virus phosphoprotein may inhibit ubiquitination of Pirh2, which has been reported to be overexpressed in lung carcinoma and is responsible for degrading the cell cycle regulator p53. The authors conducted a clinicopathological study of newly diagnosed patients with non-small cell lung carcinoma of all stages seen in a 10-year period. Immunohistochemical studies for measles virus antigens, p53, and Pirh2 were performed using the avidin-biotin peroxidase complex. The authors found expression of measles virus antigens in 54 of 65 cases of non-small cell lung carcinoma. This finding is associated with the older age of the patients and with expression of Pirh2. The presence of Pirh2 itself was associated with improved survival.

Probing the spatial organization of measles virus fusion complexes.

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

Measles virus receptors.

Measles virus (MV) has two envelope glycoproteins, the hemagglutinin (H) and fusion protein, which are responsible for attachment and membrane fusion, respectively. Signaling lymphocyte activation molecule (SLAM, also called CD150), a membrane glycoprotein expressed on immune cells, acts as the principal cellular receptor for MV, accounting for its lymphotropism and immunosuppressive nature. MV also infects polarized epithelial cells via an as yet unknown receptor molecule, thereby presumably facilitating transmission via aerosol droplets. Vaccine and laboratory-adapted strains of MV use ubiquitously expressed CD46 as an alternate receptor through amino acid substitutions in the H protein. The crystal structure of the H protein indicates that the putative binding sites for SLAM, CD46, and the epithelial cell receptor are strategically located in different positions of the H protein. Other molecules have also been implicated in MV infection, although their relevance remains to be determined. The identification of MV receptors has advanced our understanding of MV tropism and pathogenesis.

Measles virus glycoprotein complex assembly, receptor attachment, and cell entry.

Measles virus (MV) enters cells by membrane fusion at the cell surface at neutral pH. Two glycoproteins mediate this process: the hemagglutinin (H) and fusion (F) proteins. The H-protein binds to receptors, while the F-protein mediates fusion of the viral and cellular membranes. H naturally interacts with at least three different receptors. The wild-type virus primarily uses the signaling lymphocyte activation molecule (SLAM, CD150) expressed on certain lymphatic cells, while the vaccine strain has gained the ability to also use the ubiquitous membrane cofactor protein (MCP, CD46), a regulator of complement activation. Additionally, MV infects polarized epithelial cells through an unidentified receptor (EpR). The footprints of the three receptors on H have been characterized, and the focus of research is shifting to the characterization of receptor-specific conformational changes that occur in the H-protein dimer and how these are transmitted to the F-protein trimer. It was also shown that MV attachment and cell entry can be readily targeted to designated receptors by adding specificity determinants to the H-protein. These studies have contributed to our understanding of membrane fusion by the glycoprotein complex of paramyxoviruses in general.

[The genetic characteristics analysis of fusion gene of 13 strains measles viruses in China].

OBJECTIVE: To study genetic characteristics of fusion gene (F gene) of H1 genotype of wild measles virus circulated in China from 1999 to 2003. METHOD: 13 H1 genotype representative strains including 8 subgenotype H1a strains and 5 subgenotype H1b strains isolated in mainland China during 1999-2003 were selected. Entire F gene were amplified by reverse transcript-polymerase chain reaction and sequenced, then they were compared with the nucleotide acid sequences of Chinese measles vaccine strains and the representative strains of A, D3, D6, D7 and E genotypes from other countries. The phylogenetic analysis were conducted. RESULTS: Among 13 representative strains circulated in China during 1999 to 2003, the homology of nucleotide acid and amino acid were 98.1%-99.0% and 98.1%-100% respectively, and were 95.7%-96.2% and 96.9%-97.2% respectively compared with Chinese vaccine strains,while compared with other genotype strains, the homology of nucleotide acid were 94.4%-96.2%. In F gene,the 3 glycosylation site (aa32, aa64 and aa70), aa112 and aa195 which played the important role to viral fusion had no changes. CONCLUSION: The F genes of H1 genotype measles virus circulated in China mainland during 1999-2003 had no sig-nificant vibration, and known important functional sites in Fusion protein didn't have deviation, it could be postulated that the structure and functions of F protein of wild Measles viruses circulated in mainland China from 1999 to 2003 were conserved. F protein, however, played an important role in Measles viruses, it need to conducted routine monitoring to F gene. which benefit for understanding of epidemic features and genetic variation pattern of wild Measles viruses.

[The genetic characteristic analysis of fusion gene measles wild viruses of China in 2006].

OBJECTIVE: To study genetic characteristic of fusion gene (F gene) of wild type measles virus circulated in 2006, analysis the variation regular of F gene through comparing with measles virus of 1999-2003. METHOD: 9 representative strains, which isolated from 8 provinces in 2006, were selected. The whole F gene were amplified by reverse transcript-polymerase chain reaction and were sequenced, then they were compared with the nucleotide acid sequences of Chinese measles vaccine strains and the representative strains of 1999-2003, finally the phylogenetic analysis were conducted. RESULTS: Among 9 representative strains circulated in China in 2006, the homology of nucleotide acid and amino acid were 98.5%-99.8% and 99%-100% respectively, and they were 95.4%-96.2% and 96.7%-97.2% respectively compared with Chinese Vaccine strains, the homology of nucleotide acid and amino acid were 97.9%-99.7% and 98.1%-100% respectively compared with strains of 1999-2003, In F gene, the 3 glycosylation site (aa32, aa64 and aa70), aa112 and aa195, which played the important role to viral fusion, that have no changes. CONCLUSION: The F genes of measles virus of china in 2006 had no significant variation, the genetic diversity was not predominance comparing with strains of 1999-2003, and important functional sites in Fusion protein didn't change, the measles epidemic was not correlate with the F gene variation. Because F protein play an important role in Measles viruses, it needs to perform the routine monitoring of F gene, which benefit for understanding of epidemic features and genetic variation pattern of wild type Measles viruses.

Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins.

A major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells such as primary T cells, which hampers their application for gene therapy. Here we generated high-titer LVs incorporating Edmonston measles virus (MV) glycoproteins H and F on their surface. They allowed efficient transduction through the MV receptors, SLAM and CD46, both present on blood T cells. Indeed, these H/F-displaying vectors outperformed by far VSV-G-LVs for the transduction of IL-7-prestimulated T cells. More importantly, a single exposure to these H/F-LVs allowed efficient gene transfer in quiescent T cells, which are not permissive for VSV-G-LVs that need cell-cycle entry into the G1b phase for efficient transduction. High-level transduction of resting memory (50%) and naive (11%) T cells with H/F-LVs, which seemed to occur mainly through SLAM, was not at cost of cell-cycle entry or of target T-cell activation. Finally, the naive or memory phenotypes of transduced resting T cells were maintained and no changes in cytokine profiles were detected, suggesting that T-cell populations were not skewed. Thus, H/F-LV transduction of resting T cells overcomes the limitation of current lentiviral vectors and may improve the efficacy of T cell-based gene therapy.

Attenuation of V- or C-defective measles viruses: infection control by the inflammatory and interferon responses of rhesus monkeys.

Patients recruited in virus-based cancer clinical trials and immunocompromised individuals in need of vaccination would profit from viral strains with defined attenuation mechanisms. We generated measles virus (MV) strains defective for the expression of either the V protein, a modulator of the innate immune response, or the C protein, which has multiple functions. The virulence of these strains was compared with that of the parental wild-type MV in a natural host, Macaca mulatta. Skin rash, viremia, and the strength of the innate and adaptive immune responses were characterized in groups of six animals. Replication of V- or C-protein-defective viruses was short-lived and reached lower levels in peripheral blood mononuclear cells and lymphatic organs compared to the wild-type virus; none of the mutants reverted to the wild type. The neutralizing antibody titers and MV-specific T-cell responses were equivalent in monkeys infected with the viral strains tested, documenting strong adaptive immune responses. In contrast, the inflammatory response was better controlled by wild-type MV, as revealed by inhibition of interleukin-6 and tumor necrosis factor alpha transcription. The interferon response was also better controlled by the wild-type virus than by the defective viruses. Since V- and C-defective MVs induce strong adaptive immune responses while spreading less efficiently, they may be developed as vaccines for immunocompromised individuals. Moreover, MV unable to interact with single innate immunity proteins may be developed for preferential replication in tumors with specific contexts of vulnerability.

Reversible inhibition of the fusion activity of measles virus F protein by an engineered intersubunit disulfide bridge.

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