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L Roux

Publications and source records attributed to L Roux.

At least 37 records · Page 2Linked to original sources
PubMed1995

Functional characterisation of the genomic and antigenomic promoters of Sendai virus.

A natural Sendai virus internal deletion defective interfering (DI) RNA, previously shown to encode a truncated NP protein and previously cloned under the control of the T7 RNA polymerase promoter, was expressed from plasmid and shown to replicate in cell tissue culture when the viral proteins NP, P, and L were coexpressed from cloned genes. The efficient replication was dependent on the total length of the RNA to be a multiple of 6 nucleotides, showing that the "rule of six" applied for a DI RNA that has conserved the end sequences of the nondefective viral RNA. Compared to the copy-back H4 DI RNA, the replication efficiency of the internal deletion DI RNA was reproducibly 20-fold lower. Reciprocal exchanges between the minus-strand 3'-end primary sequences of the two DI RNAs showed that the replication efficiency of the derivatives obtained directly correlated with the origin and the extent of the primary sequence. Moreover, some of the derivatives exhibited a replication efficiency comparable to that of the copy-back DI RNA with, however, the ability to transcribe a functional mRNA similar to the internal deletion DI RNA. This indicated that the transcription ability of a viral RNA was not sufficient to explain a low replication efficiency.

Animals↗
PubMed1994

Presence of a truncated form of the Sendai virus P protein in a long-term persistent infection: implications for the maintenance of the persistent state.

In this report we have monitored viral gene expression, both at the RNA and protein level, after the establishment of a long-term persistent infection of Sendai virus. The persistent infection was initially established by infecting BHK cells with a viral stock containing a short (1.4 kb) copy-back DI (DIH4). After over 120 weeks in culture this short copy-back DI had been replaced by two large deletion DIs (approximately 7 and 12 kb) from which was expressed an N-terminally truncated form of the P protein. The mRNA for this protein was detected in cells and the deletion within the P gene was mapped by PCR cloning and sequencing of intracellular nucleocapsid RNA. This truncated P protein (derived by deleting the N-terminal half of the cloned Pwt gene) has already been shown to function as a dominant negative for DI replication when driven by cloned viral genes. Cloning and expression of the truncated P from the long-term persistent infection revealed that this protein had retained the dominant negative phenotype. The presence of such a protein would severely depress viral gene expression and may therefore play an important role in the maintenance of persistence.

Animals↗
PubMed1994

The Sendai virus matrix protein appears to be recruited in the cytoplasm by the viral nucleocapsid to function in viral assembly and budding.

The matrix (M) protein is viewed as the regulator of paramyxovirus particle assembly and budding. Accordingly it was observed to be mutated, and/or decreased in amount, in cases where virus particle production was significantly reduced. Here, a non-productive [non-defective and defective interfering (DI)] Sendai virus infection of COS cells is presented where virus particle production is abolished in the presence of a normal amount of intracellular M protein. In this infection the haemagglutinin-neuraminidase envelope glycoprotein is shown to be dispensable for virion production, and the fusion (F0) envelope glycoprotein behaves as in a productive infection. The M protein is shown to accumulate in perinuclear patches within the cytoplasm. In contrast, localization in the plasma membrane is observed in productive infections. However in both productive and non-productive infections a significant fraction of M protein is found in association with cellular membranes. The M protein-membrane association is shown to take place in the absence of any other viral component, and the M protein-membrane complex exhibits properties similar to those observed for the integral membrane protein F0. However these properties are distinct from those of the phosphoprotein, which is thought to associate with membranes in a non-specific manner. Concomitant with the cytoplasmic accumulation of M protein and the reduction of virus particle production in this non-productive infection, DI nucleocapsids are shown not to associate with cellular membrane fractions. This is a property which coincides with their poor envelopment in virus particles. Taken together, these data indicate the need for M protein to be recruited at the perinuclear membranes by the nucleocapsids to participate in viral assembly and budding. This view is consistent with a process of viral assembly taking place on internal cytoplasmic membranes rather than at the plasma membrane.

Animals↗
PubMed1994

Effects of cocaine on blood flow and prostaglandin metabolites in rat sciatic nerve.

To better understand the mechanisms of local anesthetic-reduced nerve blood flow and nerve blood flow regulation, the effects of cocaine on blood flow and vasoactive prostaglandins were tested in the sciatic nerve of anesthetized rats. After 30 min, nerve blood flow was significantly reduced from baseline by perineural injection of 160 mM cocaine [-29.4 +/- 4.0 (SD) laser-Doppler flow units (P < 0.001)] but not saline (1.6 +/- 11.3). These same nerves were removed and assayed for the stable metabolites 6-ketoprostaglandin F1 alpha and thromboxane B2 of the vasoactive eicosanoids prostacyclin and thromboxane A2, respectively. Both metabolites were reduced, but the ratio of thromboxane B2 to 6-ketoprostaglandin F1 alpha was greater (P < 0.05) in nerves pretreated with cocaine (6.1 +/- 3.2 vs. 2.4 +/- 1.1) and was inversely correlated (P < 0.01) with nerve blood flow. In a separate experiment, perineural injection of the prostaglandin synthesis inhibitor indomethacin (0.5 mg in 0.5 ml of either saline or 50% ethanol) reduced nerve blood flow as well (P < 0.05). These results are consistent with the proposal that cocaine inhibits nerve blood flow by effects on nerve prostaglandin metabolites.

6-Ketoprostaglandin F1 alpha↗
PubMed1994

[Computerization in intensive care].

Computers already constitute an integral part of intensive care units as almost all pieces of sophisticated equipment in intensive care contain a computerized component. However, a marked lack of computerization exists with regard to data collection, data analysis and in teaching support systems. This article presents a summary of the advantages of computerization of data in intensive care and cautions on possible pitfalls in the choice of equipment and in its implementation.

Hospital Information Systems↗
PubMed1993

Molecular cloning and characterization of a Sendai virus internal deletion defective RNA.

A small defective Sendai virus RNA was selectively amplified from a virus preparation obtained after serial undiluted passages in embryonated eggs. Preliminary characterization showed that this defective RNA was a true internal deletion defective RNA, containing the 5' and 3' ends of the non-defective viral genomic RNA. Cloning of this RNA after reverse transcription and polymerase chain reaction amplification was performed in such a way that an exact copy of the defective RNA could be obtained by transcription of the plasmid with T7 RNA polymerase. Sequence analysis of the plasmid allowed further characterization of the defective RNA. It was shown potentially to encode a C-terminally truncated nucleocapsid (NP) protein of 162 amino acids. This truncated NP protein was identified in cells naturally infected with the defective virus preparation. Moreover the protein produced was shown to correspond to the protein synthesized in vitro from the T7 polymerase transcript of the cloned defective genome.

Base Sequence↗
PubMed1993

The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.

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 contact exactly 6 nucleotides.

Animals↗
PubMed1992

Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA.

Using the unique sequence organization of copy-back defective interfering (DI) RNAs of paramyxoviruses, Sendai virus (SV), and measles virus copy-back DI RNAs were PCR amplified and cloned, without having to separate them from their helper nondefective genomes. The cloning was designed so that T7 polymerase transcription of the plasmids would generate DI RNAs with the exact 5' and 3' ends. The SV DI clone, transcribed from the plasmid in BHK cells using T7 polymerase produced by a vaccinia virus recombinant, was encapsidated and replicated by the SV-L, P/C, and NP proteins expressed from cloned genes. Such experiments open the possibility of examining the cis-acting sequences involved in viral multiplication directly, without using indirect markers such as CAT activity.

Animals↗
PubMed1992

Sendai virus M protein is found in two distinct isoforms defined by monoclonal antibodies.

The use of a monoclonal antibody defines a subset of Sendai virus M protein representing about 30% of total. This M protein acquires, during the hour following synthesis, an epitope not present on the bulk of M. This epitope maturation is observed in acutely as well as in persistently infected cells. It takes place in vivo in absence of other viral proteins, but it is not observed when the protein is synthesized in a reticulocyte lysate. Epitope maturation does not appear to result from phosphorylation, acylation or disulfide bond formation. If immunofluorescent staining seems to indicate a preferential association of this subset of M protein with nucleocapsids, this is not confirmed by immunogold staining or by nucleocapsid isolation. Incubation of cytoplasmic extracts or of purified M protein in conditions which do not favor M to M protein association results in a relative increase of M protein carrying the maturing epitope. It is concluded that M protein exists in two distinct isoforms.

Animals↗
PubMed1991

Protection against lethal Sendai virus infection by in vivo priming of virus-specific cytotoxic T lymphocytes with a free synthetic peptide.

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.

Animals↗
PubMed1990

Selective and transient association of Sendai virus HN glycoprotein with BiP.

From 10-min [35S]methionine pulse-labeled Sendai virus-infected BHK cells, an anti-BiP monoclonal antibody precipitated, along with the BiP protein, the hemagglutinin-neuraminidase protein (SV-HN) fivefold better than the fusion protein (SV-Fo). A minimal estimate of 30% of the newly made HN was complexed to BiP. The majority of the HN in the complex was endo-H sensitive and the molar ratio of BiP:HN was estimated to be 1:2. With time, HN dissociated from BiP, and the rate of dissociation was found to be inversely proportional to the rate at which HN acquired its native structure. It is proposed that association with BiP followed by slow release (i) is responsible for the HN slow maturation and (ii) represents a normal step in its maturation pathway.

Animals↗
PubMed1990

Intracellular stability of nonreplicating paramyxovirus nucleocapsids.

Using Northern blot analysis, we have demonstrated the ability of infectious measles and Sendai virus particles to rescue the intracellular replication of their homologous defective interfering (DI) nucleocapsids up to 3 days and 1 day, respectively, after initial DI infection. The half-life of the paramyxovirus DI nucleocapsids was therefore judged to be similar to that of rhabdoviruses, and to significantly differ from that of orthomyxoviruses. Moreover, we conclude that the intracellular half-life of measles virus DI nucleocapsids makes possible DI replication in the human body after vaccination with a DI-contaminated attenuated live virus, even when this vaccination represents a low multiplicity of infection.

Animals↗
PubMed1990

Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines.

Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.

Blotting, Northern↗