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The role of the negative-stranded virus accessory C proteins is difficult to assess because they appear sometimes as nonessential and thereby of no function. On the other hand, when a function is found, as in the case of Sendai virus, it... more
The role of the negative-stranded virus accessory C proteins is difficult to assess because they appear sometimes as nonessential and thereby of no function. On the other hand, when a function is found, as in the case of Sendai virus, it represents an enigma, in that the C proteins inhibit replication under conditions where the infection follows an exponential course. Furthermore, this inhibitory function is exerted differentially: in contrast to the replication of internal deletion defective interfering (DI) RNAs, that of copy-back DI RNAs appears to escape inhibition, under certain experimental conditions (in vivo assay). In a reexamination of the C effect by the reverse genetics approach, it was found that copy-back RNA replication is inhibited by C in vivo as well, under conditions where the ratio of C to copy-back template is increased. This effect can be reversed by an increase in P but not L protein. The "rule of six" was differentially observed in the presence or a...
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Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus... more
Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus (SeV) was recovered from cDNA via the initial expression of the viral N, P, and L genes from plasmids, the abrogation of C protein expression from the plasmid P gene was found to be necessary for virus recovery. We have investigated the effect of C coexpression on the amplification of an internally deleted defective interfering (DI) genome directly in the transfected cell, for which, in contrast to virus recovery experiments, genome amplification is independent of mRNA synthesis carried out by the SeV polymerase. We find that C protein coexpression also strongly inhibits the amplification of this DI genome but has little or no effect on that of a copy-back DI genome (DI-H4). We have also characterized the C protein from a mutant SeV and found that (i...
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The immunoreactivity of the Sendai virus HN and F0 glycoproteins was shown to mature before reaching the final form exhibited by the native mature proteins. The maturation process differed for the two proteins. The native F0... more
The immunoreactivity of the Sendai virus HN and F0 glycoproteins was shown to mature before reaching the final form exhibited by the native mature proteins. The maturation process differed for the two proteins. The native F0 immunoreactivity was shown to be defined cotranslationally, and the addition of high-mannose sugar residues may represent the final step in defining the maturation of immunoreactivity. On the other hand, native HN immunoreactivity was slowly fashioned during the hour after the completion of protein synthesis. Although addition of high-mannose sugar could constitute a necessary step in this slow maturation process, it was shown not to be sufficient. Processing of high-mannose sugars and HN self-association in homodimers and homotetramers were investigated as possible steps involved in the slow maturation of HN immunoreactivity. They were found not to play a significant role. On the other hand, conformational changes presumably took place during the maturation of ...
Research Interests: Biology, Virology, Medicine, Biological Sciences, Virus, and 15 moreAnimals, Membrane Fusion, P-glycoprotein, Tunicamycin, Glycoproteins, Immunoprecipitation, Hemagglutinin, Neuraminidase, Time Factors, Protein Conformation, Mannose, Epitopes, Protein Denaturation, Cricetinae, and Medical and Health Sciences
The addition of the hepatitis delta virus genomic ribozyme to the 3' end sequence of a Sendai virus defective interfering RNA (DI-H4) allowed the reproducible and efficient replication of this RNA by the viral functions expressed from... more
The addition of the hepatitis delta virus genomic ribozyme to the 3' end sequence of a Sendai virus defective interfering RNA (DI-H4) allowed the reproducible and efficient replication of this RNA by the viral functions expressed from cloned genes when the DI RNA was synthesized from plasmid. Limited nucleotide additions or deletions (+7 to -7 nucleotides) in the DI RNA sequence were then made at five different sites, and the different RNA derivatives were tested for their abilities to replicate. Efficient replication was observed only when the total nucleotide number was conserved, regardless of the modifications, or when the addition of a total of 6 nucleotides was made. The replicated RNAs were shown to be properly enveloped into virus particles. It is concluded that, to form a proper template for efficient replication, the Sendai virus RNA must contain a total number of nucleotides which is a multiple of 6. This was interpreted as the need for the nucleocapsid protein to con...
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The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of ∼15 kb, contains six transcription units flanked at the 3′ and 5′ ends by a short (∼ 50- to 60-nucleotide) extracistronic sequence, dubbed the positive... more
The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of ∼15 kb, contains six transcription units flanked at the 3′ and 5′ ends by a short (∼ 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3′ end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3′-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5′-3′ positi...
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Research Interests: Chemistry, Biology, Medicine, Clinical Microbiology, Biological Sciences, and 14 moreCell line, Humans, Animals, Polymerase Chain Reaction, Plasmids, Chickens, HeLa cells, Medical School, Vaccinia Virus, Genetic Recombination, Transfection, Base Sequence, Molecular Sequence Data, and Medical and Health Sciences
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Portions of the Sendai virus genome were randomly cloned by using virion 50S RNA and calf thymus DNA pentanucleotides as primers. The recombinant clones were probed first with radiolabeled products of an in vitro virion RNA polymerase... more
Portions of the Sendai virus genome were randomly cloned by using virion 50S RNA and calf thymus DNA pentanucleotides as primers. The recombinant clones were probed first with radiolabeled products of an in vitro virion RNA polymerase reaction to locate early message clones and then with a probe from the viral genome 3' end to locate the most 3'-proximal clones. Clones were then ordered from the 3' end of the genome and used to construct a genetic map of the 3'-proximal third of the genome by hybrid-selection of mRNAs. We report that the gene order for this region is 3'-NP - P + C - M-5' and that the genetic loci of the viral P and C proteins cannot be separated by these techniques.
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Members of the Paramyxoviridae such as measles, mumps, and parainfluenza viruses have pleomorphic, enveloped virions that contain negative-sense unsegmented RNA genomes. This is encapsidated by multiple copies of a viral nucleocapsid... more
Members of the Paramyxoviridae such as measles, mumps, and parainfluenza viruses have pleomorphic, enveloped virions that contain negative-sense unsegmented RNA genomes. This is encapsidated by multiple copies of a viral nucleocapsid protein N to form a helical ribonucleoprotein complex (termed the nucleocapsid), which acts as the template for both transcription and replication. Structure analysis of these viruses has proven challenging, owing to disordered regions in important constituent proteins, conformational flexibility in the nucleocapsid and the pleomorphic nature of virus particles. We conducted a low-resolution ultrastructural analysis of Sendai virus, a prototype paramyxovirus, using cryo-electron tomography. Virions are highly variable in size, ranging approximately from 110 to 540 nm in diameter. Envelope glycoproteins are densely packed on the virion surface, while nucleocapsids are clearly resolved in the virion interior. Subtomogram segmentation and filament tracing ...
Research Interests: Electron Tomography, Biology, Virology, RNA polymerase, Medicine, and 15 moreBiological Sciences, Virus, Animals, Genome, Structure Analysis, Gene Dosage, Particle Size, Newcastle disease, Protein Conformation, RNA Dependent RNA Polymerase, Peste des petits ruminants, Low Resolution, Nucleocapsid, Chick embryo, and Medical and Health Sciences
The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome... more
The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome are bound to 2,564 subunits of the N protein, each interacting with six nucleotides so tightly that the bases are poorly accessible to soluble reagents. With such a uniform structure, the question of template recognition by the viral RNA polymerase has been raised. In a previous study, the N-phase context has been proposed to be crucial for this recognition, a notion referring to the importance of the position in which the nucleotides interact with the N protein. The N-phase context ruled out the role of the template 3′-OH congruence, a feature resulting from the obedience to the rule of six that implies the precise interaction of the last six 3′-OH nucleotides with the last N protein. The N-phase context then allows prediction of the recognition by...
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Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M... more
Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3′ untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.
Research Interests: Molecular Biology, RNA, Biology, Virology, Medicine, and 15 moreGene Silencing, Biological Sciences, RNA interference, Cell line, Humans, Virus, Green Fluorescent Protein, Inner Core, HeLa cells, Transfection, Lipid Bilayer, Plasma Membrane, Viral Replication, short interfering RNA, and Medical and Health Sciences
The “rule of six” stipulates that the Paramyxovirus RNA polymerase efficiently replicates only viral genomes counting 6 n + 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at... more
The “rule of six” stipulates that the Paramyxovirus RNA polymerase efficiently replicates only viral genomes counting 6 n + 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at the RNA 3′-OH end (3′-OH congruence) may be required for recognition of an active replication promoter. Alternatively, assuming that the six positions for the interaction of N with the nucleotides are not equivalent, the nucleotide position relative to N may be critical (N phase context). The replication abilities of various minireplicons, designed so that the 3′-OH congruence could be discriminated from the N phase context, were studied. The results strongly suggest that the application of the rule of six depends on the recognition of nucleotides positioned in the proper N phase context.
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Two transmembrane glycoproteins form spikes on the surface of Sendai virus, a member of the Respirovirus genus of the Paramyxovirinae subfamily of the Paramyxoviridae family: the hemagglutinin-neuraminidase (HN) and the fusion (F)... more
Two transmembrane glycoproteins form spikes on the surface of Sendai virus, a member of the Respirovirus genus of the Paramyxovirinae subfamily of the Paramyxoviridae family: the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. HN, in contrast to F, is dispensable for viral particle production, as normal amounts of particles can be produced with highly reduced levels of HN. This HN reduction can result from mutation of an SYWST motif in its cytoplasmic tail to AFYKD. HN AFYKD accumulates at the infected cell surface but does not get incorporated into particles. In this work, we derived experimental tools to rescue HN AFYKD incorporation. We found that coexpression of a truncated HN harboring the wild-type cytoplasmic tail, the transmembrane domain, and at most 80 amino acids of the ectodomain was sufficient to complement defective HN AFYKD incorporation into particles. This relied on formation of disulfide-bound heterodimers carried out by the two cysteines present in t...
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For the non-segmented, negative-stranded RNA viruses, the mechanism controlling transcription or replication is still a matter of debate. To gain information about this mechanism and about the nature of the RNA polymerase involved, the... more
For the non-segmented, negative-stranded RNA viruses, the mechanism controlling transcription or replication is still a matter of debate. To gain information about this mechanism and about the nature of the RNA polymerase involved, the length of an intervening sequence separating the 3′ end of Sendai virus minigenomes and a downstream transcription-initiation signal was increased progressively. It was found that transcription, as measured by green fluorescent protein (GFP) expression, decreased progressively in proportion to the increase in length of the intervening sequence. GFP expression correlated well with the levels of GFP mRNA in the cells, as measured by quantitative primer extension and by RNase protection. Thus, mRNA transcription was inversely proportional to the length of the inserted sequence. These data are evidence that the RNA polymerase initiating transcription at the downstream transcription signal somehow sees the distance separating this signal and the template 3...
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Paramyxovirus genomes contain a linear array of five to ten genes sequentially transcribed by the viral RNA polymerase. mRNA synthesis initiates at a nucleotide signal (gs1) within the genomic promoter located at the genome 3′ end. To... more
Paramyxovirus genomes contain a linear array of five to ten genes sequentially transcribed by the viral RNA polymerase. mRNA synthesis initiates at a nucleotide signal (gs1) within the genomic promoter located at the genome 3′ end. To gain information about the mechanism involved in transcription initiation, a search was carried out for upstream nucleotides required for gs1 and the effects of the gs1 nucleocapsid protein (N) phase context on transcription regulation were determined. For both purposes, tandem promoter mini-genomes carrying a transcription signal ectopically positioned downstream of a replication-only signal were used. The requirement for hygromycin resistance gene expression was used in an attempt to select essential nucleotides within randomized stretches of nucleotides. Nucleotide insertions or deletions were also made on either side of the transcription signal to change its original N phase context in the five remaining possibilities and GFP expression from these ...
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Tetherin is an interferon-inducible factor that restricts viral particle production. We show here that Sendai virus (SeV) induces a drastic decrease in tetherin levels in infected HeLa cells. Using ectopic expression of tetherin in... more
Tetherin is an interferon-inducible factor that restricts viral particle production. We show here that Sendai virus (SeV) induces a drastic decrease in tetherin levels in infected HeLa cells. Using ectopic expression of tetherin in Madin–Darby canine kidney cells, we find that infectious SeV production is sensitive to restriction by tetherin, suggesting that SeV downregulates tetherin to counter this form of cellular restriction. By using radioactive tetherin in pulse–chase experiments, applying conditions that limit protein degradation, and by estimating tetherin mRNA levels, we find that tetherin degradation is the mechanism of downregulation. Suppression of the virus envelope proteins matrix, fusion (F) or haemagglutinin-neuraminidase protein (HN) during the course of infection demonstrates that F and HN, in concert, are responsible for tetherin degradation. The mechanism(s) by which these two viral glycoproteins participate in degrading tetherin remains to be determined.
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A protein kinase activity has been found associated with purified Sendai virions. Most of the virion proteins and exogenous protamine served as substrates for this enzyme.
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Substitution of Val113in Sendai virus (SeV) M protein generates non-functional polypeptides, characterized by their exclusion from virus particles and by their ability to interfere with virus particle production. These phenotypic traits... more
Substitution of Val113in Sendai virus (SeV) M protein generates non-functional polypeptides, characterized by their exclusion from virus particles and by their ability to interfere with virus particle production. These phenotypic traits correlate with a single-band PAGE migration profile, in contrast to wild-type M (Mwt), which separates into two species, one of which is a phosphorylated form. The single-band migration is likely to result from a conformational change, as evidenced by the lack of maturation of a native epitope and by a particular tryptic digestion profile, and not from the phosphorylation of all M molecules, an assumption consistent with the PAGE migration feature. One of the M mutants (HA–M30, an M protein carrying Thr112Met and Val113Glu substitutions tagged with an influenza virus haemagglutinin epitope) was characterized further in the context of SeV infection, i.e. under conditions of co-expression with Mwt. HA–M30is shown (i) to bind mainly to membrane fraction...
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Cytoplasmic actins have been found interacting with viral proteins and identified in virus particles. We analyzed by confocal microscopy the cytoplasmic β- and γ-actin patterns during the course of Sendai virus infections in polarized... more
Cytoplasmic actins have been found interacting with viral proteins and identified in virus particles. We analyzed by confocal microscopy the cytoplasmic β- and γ-actin patterns during the course of Sendai virus infections in polarized cells. We observed a spectacular remodeling of the β-cytoplasmic actin which correlated with productive viral multiplication. Conversely, suppression of M during the course of a productive infection resulted in the decrease of particle production and the absence of β-actin remodeling. As concomitant suppression of β- and γ-actins resulted as well in reduction of virus particle production, we propose that Sendai virus specifically induces actin remodeling in order to promote efficient virion production. Beta- and γ-cytoplasmic actin recruitment could substitute for that of the endosomal sorting complex required for transport (ESCRT) mobilized by other enveloped viruses but apparently not used by Sendai virus.
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Paramyxoviruses are pleomorphic particles consisting of a spike-covered, lipid-containing envelope enclosing a helical nucleocapsid. The viral genome is a single-stranded, negative-sense RNA molecule. The helical nucleocapsid consists of... more
Paramyxoviruses are pleomorphic particles consisting of a spike-covered, lipid-containing envelope enclosing a helical nucleocapsid. The viral genome is a single-stranded, negative-sense RNA molecule. The helical nucleocapsid consists of RNA, nucleocapsid protein (NP), and two other proteins (P and L), and is covered by an envelope consisting of a lipid bilayer as well as two virus-coded glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. Virus assembly occurs by a process of budding at the cellular plasma membrane. The viral glycoproteins are embedded as integral membrane proteins on the cell surface, and the internal viral components (M protein and nucleocapsid) associate with regions of the membrane which contain these glycoproteins. Virions are formed by a process of outfolding, or budding, at the cell surface, during which there is continuity between the plasma membrane of the host cell and that of the emerging virus particle (Compans et al., 1966). Stages in the assembly of a paramyxovirus are depicted in Fig. 1. The first recognizable change in the plasma membrane is the ability to detect viral antigens on the cell surface, as seen by immunoferritin labeling in Fig. 1A, in morphologically normal regions of the plasma membrane. A more striking change is seen in Fig. 1B, in which viral nucleocapsids are closely aligned under the plasma membrane. The external surface of the membrane exhibits an additional electron-dense layer in these regions, reflecting the concentration of viral glycoproteins on the external surface. Virions form by budding or outfolding of such modified areas of membrane, as seen in Fig. 1C. The virions acquirea lipid bilayer with a composition which closely reflects that of the plasma membrane of the host cell (Klenk and Choppin, 1970). In contrast, all the proteins of the virion are coded for by the viral genome, and host cell proteins are effectively excluded from the virus during the budding process (Compans and Klenk, 1979).
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In the course of a Sendai virus infection, short genome versions (called defective) are frequently produced. These compete with the full length genome for replication (hence called interfering) and decrease the severity of the infection.... more
In the course of a Sendai virus infection, short genome versions (called defective) are frequently produced. These compete with the full length genome for replication (hence called interfering) and decrease the severity of the infection. The portion of the defective genome required for this effect can be limited, much shorter than the size of the defective genome. We postulated then that defective genome size follows from conditions required for their production more than for exerting the protective effect. Therefore, we set up experiments to determine which of the steps involved in their production may require this larger size. We constructed a series of Sendai virus mini-genomes, increasing their size from the smallest possible and we followed their ability to replicate, to amplify through multiples cycles of infection and to be packaged into virus particles, all steps required for their efficient production. We found that the mini-genomes replicated and amplified efficiently regardless of their size. The uptake into virus particles, however, depended on a threshold length. The data suggest no limitation in the ability of the viral RNA polymerase to generate and amplify very short size defective genomes. They however, point to limitations in the ability of short genomes to be packaged into virus particles.
Research Interests: Biology, Genome Size, Medicine, Biological Sciences, Virus, and 4 moreAnimals, Genome, Viral Replication, and Cricetinae
Research Interests: Molecular Biology, RNA, Biology, RNA polymerase, Medicine, and 15 moreNon Coding Rna, Biological Sciences, Cercopithecus aethiops, Humans, Tissue culture, Sequence alignment, Animals, HeLa cells, Base Sequence, RNA Dependent RNA Polymerase, Nucleotides, Molecular Sequence Data, Cricetinae, Medical and Health Sciences, and In Vitro Techniques
To gain an understanding of the mechanism(s) by which Sendai virus generates a persistent infection, the expression of the hemagglutinin-neuraminidase (HN) and fusion (Fo) glycoproteins at the surfaces of BHK-21 cells infected with... more
To gain an understanding of the mechanism(s) by which Sendai virus generates a persistent infection, the expression of the hemagglutinin-neuraminidase (HN) and fusion (Fo) glycoproteins at the surfaces of BHK-21 cells infected with standard virus, a mixture of standard and defective interfering (DI) particles (mixed virus infection), and during persistent infection was investigated. The expression of HN and Fo was measured on the surfaces of infected cells by the binding of anti-HN and anti-Fo monoclonal antibodies. The results show that HN expression was restricted relative to Fo during mixed virus and persistent infections. The decreased levels of HN were investigated further by pulse-chase experiments which revealed that HN has an increased turnover rate in persistently infected cells and, to a lesser extent, in mixed virus infected cells. In analyzing the [35S]methionine-labeled protein composition of virus particles produced during the pulse-chase experiments, the increased turnover of newly synthesized HN was found to correlate with its decreased incorporation into virus particles. Interestingly, the poor HN incorporation also correlates with less efficient incorporation of the matrix M protein into virus particles.
Research Interests: Kinetics, Biology, Medicine, Gene expression, Biological Sciences, and 15 moreCell line, Kidney, Beta decay, Animals, Monoclonal Antibodies, Enzyme, Hemagglutinin, Neuraminidase, Enzyme activity, Amino Acid Profile, Monoclonal Antibody, Cell Surface Markers, Turnover Rate, Cricetinae, and Medical and Health Sciences
The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (i) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (ii) a set of overlapping... more
The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (i) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (ii) a set of overlapping peptides completely spanning the identified nucleoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous immunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunized with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.
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The genomic and antigenomic 3′ ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple... more
The genomic and antigenomic 3′ ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located between two essential elements of the replication promoter. Transcription initiation, then, is separated from the replication initiation site by only 56 nt on the genome, so that transcription and replication may sterically interfere with each other. In order to study the initiation of Sendai virus mRNAs without this possible interference, Sendai virus mini-genomes were prepared having tandem promoters in which replication takes place from the external one, whereas mRNA synthesis occurs from the internal one. Transcription now initiates at position 146 rather than position 56 relative to the genome 3′ end. U...
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The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also... more
The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy (‘stutter’ on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their...
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This paper presents a study which evaluates the use and efficiency of two computer learning tools for basic virology. The first, VIROLAB, is a simulation of a biology laboratory in which the learner uses virtual lab facilities to help... more
This paper presents a study which evaluates the use and efficiency of two computer learning tools for basic virology. The first, VIROLAB, is a simulation of a biology laboratory in which the learner uses virtual lab facilities to help defective viruses to multiply. The system also includes a hypertext that provides learners with some knowledge on virology. The second tool is the hypertext which has been extracted from VIROLAB and which is now considered as an independent learning environment. This study addressed two specific questions: (1) which of the two tools was the most efficient for knowledge acquisition, and (2) were there any differences in the use of the two hypertexts? Comparison of pre‐ and post‐test scores showed that neither of the two learning tools is better than the other. However, analysis of the navigation paths showed that VIROLAB users dissociated the reading of the integrated hypertext from the problem resolution activities. The implications of this finding for...
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Research Interests: Pediatrics, Magnetic Resonance Imaging, Electroencephalography, Medicine, Measles Virus, and 15 moreHumans, Female, Infant, European, Immune system, Disease Progression, Risk Factors, Age of Onset, Myoclonus, Antiviral Agents, Measles, Paediatrics and reproductive medicine, inosine pranobex, In Utero, and Fatal outcome
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Background Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now... more
Background Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs. Results Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared wi...