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M Bouloy

Publications and source records attributed to M Bouloy.

At least 55 records · Page 3Linked to original sources
PubMed1990

Primer extension dideoxy chain termination nucleotide sequencing of partially purified RNA virus genomes: a technique for investigating low titre viruses with extensive genome secondary structure.

A modified version of the primer extension dideoxy chain termination nucleotide sequencing technique (Sanger et al., 1977) is described. This method has advantages over existing molecular cloning and primer extension techniques in that it allows the genome of RNA viruses to be directly sequenced from partially purified RNA preparations. Thus, viruses growing at unacceptably low titres in tissue culture can now be partially purified from infected mouse brain and sequenced. The technique also incorporates steps for the denaturation of secondary structure which has previously provided difficulties for primer extension sequencing.

Animals↗
PubMed1990

The 15 amino acid residues preceding the amino terminus of the envelope protein in the yellow fever virus polyprotein precursor act as a signal peptide.

The 15 amino acids which precede the sequence of the envelope (E) protein in the yellow fever virus (YFV) polyprotein precursor have been proposed to function as a signal peptide for the E protein (P. Desprès A. Cahour, C. Wychowski, M. Girard and M. Bouloy; Ann. Inst. Pasteur/Virol., 139, 59-67, 1988). To confirm this hypothesis, recombinant SV40 genomes were constructed in which the sequence of the E protein, or that of the poliovirus VP0 capsid polypeptide were placed immediately downstream of and in frame with the sequence of the putative signal peptide, under the control of the late SV40 promoter. The E protein expressed by the hybrid virus SV-E was recognized by two neutralizing monoclonal antibodies directed against the YFV envelope protein. In this construct, the E protein was deleted of its C-terminal transmembrane zone. Therefore, as expected, the protein appeared to be efficiently transported along the exocytic pathway and excreted into the cell culture medium. In addition, when the putative signal peptide was fused in frame with poliovirus polypeptide VP0, the expressed chimeric polypeptide was targeted to the endoplasmic reticulum where it underwent glycosylation.

Amino Acid Sequence↗
PubMed1989

Modulations of the in vitro translational efficiencies of Yellow Fever virus mRNAs: interactions between coding and noncoding regions.

As an approach to define the structural features within the 5' noncoding region of Yellow Fever virus (YFV) that modulate mRNA translational efficiency, we have studied how minor changes in this region affect the translational capacity in vitro of the corresponding mRNAs. A cDNA sequence coding for part of the YFV structural proteins was inserted into the vector pGEM3 containing the bacteriophage T7 promoter. This vector was engineered by site-directed mutagenesis to permit in vitro synthesis of transcripts containing only 5 vector nucleotides at their 5' end. The sequence of the YFV 5' untranslated region was further modified in order to alter the secondary structure of resulting T7 transcripts. The efficiency of these messengers in programming cell-free translation systems varied from 1- to 15-fold, correlating inversely with the potential of the 5' untranslated sequences to form stable secondary structures. A chimaeric messenger containing the YFV 5' noncoding (5' NC) region linked to a heterologous mRNA derived from Germiston virus, was tested for its in vitro translatability. We found a translational efficiency about 2-fold higher than that obtained with homologous transcripts, suggesting that YFV 5' NC region can function as a potential enhancer for gene expression. Data obtained with a series of plasmids constructed by linking the native YFV 5'NC region to various coding regions of the YFV genome indicated that interactions between the untranslated sequence and protein coding regions influence mRNAs translational efficiency.

Base Sequence↗
PubMed1989

Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins.

The yellow fever virus (YFV) cDNA segment coding for the part of the precursor polyprotein generating the structural proteins C (capsid), prM (precursor to the membrane protein M), and E (envelope) was expressed in vitro by using the T7 promoter-polymerase transcription system coupled to translation in rabbit reticulocyte lysates. A polypeptide of the expected molecular weight was observed to accumulate in the assay and was processed into proteins C, prM, and E only when dog pancreas microsomal membranes were added to the translation system. Proteins prM and E were translocated inside the endoplasmic reticulum, where prM underwent glycosylation. Regions essential for translocation of these proteins were localized to the 18- and 15-amino-acid C-terminal hydrophobic regions of proteins C and prM, respectively. Translocation of protein prM appeared to be less efficient than that of protein E. Maturation of these proteins followed different kinetics, indicating that the prM signal is probably cleaved off more slowly. A polypeptide composed of proteins C and prM, similar to the NVx polypeptide described in yellow fever virus-infected cells, was also produced in the in vitro system in the presence of membranes. No mature protein M was detected, suggesting that the cleavage of prM to M is a late processing event mediated by a protease different from endoplasmic reticulum signalases.

Biological Transport↗
PubMed1988

Nucleotide sequence of the M segment of Germiston virus: comparison of the M gene product of several bunyaviruses.

The complete nucleotide sequence of the M RNA segment of Germiston bunyavirus was determined from plasmids containing overlapping M cDNA inserts. The M segment is 4534 nucleotides long and contains a 50-base-long inverted terminal repeat which can form a stable hydrogen-bonded secondary structure with a delta G of -45.8 kcal/mol. The RNA molecule complementary to viral RNA contains a single large open reading frame that encodes a 1437 amino acid-long protein with hydrophobic amino and carboxy terminal regions, which could represent signal and anchor sequences, respectively. It is presumed that this gene product is the polyprotein precursor to glycoproteins G1 and G2 and to the nonstructural polypeptide NSM. The nucleotide and amino acid sequences of the M RNA of Bunyamwera virus (prototype of the serogroup) and snowshow hare and La Crosse viruses (California serogroup) (Lees et al., 1986; Eshita and Bishop, 1984; Grady et al., 1987) were compared to those of Germiston virus. An overall amino acid sequence homology of 44% was found between Germiston and snowshoe hare viruses and of 61% between Germiston and Bunyamwera viruses. Most of the cysteines, three out of seven of the potential glycosylation sites, as well as the N and C terminal hydrophobic domains, are conserved between the four viruses.

Amino Acid Sequence↗
PubMed1987

The S segment of the Germiston virus RNA genome can code for three proteins.

The complete sequence of the S segment of Germiston bunyavirus has been determined from plasmids containing S cDNA inserts. The S segment is 980 nucleotides long with the first 15 bases at the 3' end complementary to the first 15 bases at the 5' end. Three overlapping open reading frames (ORF) were identified in the viral complementary RNA strand. The first ORF codes for a polypeptide of 233 amino acids (Mr 26,600) which is the nucleoprotein N. The second ORF codes for a polypeptide of 109 amino acids (Mr 11,800) which corresponds to the NSS protein, also called p12. Following this ORF, in the same frame, a third ORF which could encode a polypeptide of 75 amino acids was identified. Such a polypeptide has not yet been detected in infected cells. The N and NSS proteins of Germiston virus were compared with the corresponding proteins of La Crosse, snowshoe hare, and Aino viruses, and show a high extent of homology.

Amino Acid Sequence↗
PubMed1987

High genetic stability of the region coding for the structural proteins of yellow fever virus strain 17D.

The genome of the Pasteur 17D-204 vaccine strain of yellow fever virus has been cloned into pBR327. The inserts of recombinant plasmids were analysed by restriction cleavage pattern and compared with that of the genome of another substrain previously cloned and sequenced. Ten of the overlapping inserts were found to contain the sequence of the complete genome. We have sequenced approximately 3680 bases of the 5' region which codes for the C, M and E structural proteins and the NS1 non-structural protein. This sequence is the same as that reported previously, indicating a remarkable stability of these two vaccine substrains.

Cloning, Molecular↗
PubMed1984

A transcript from the S segment of the Germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein.

Analysis of the RNAs present in BHK-21 cells infected with Germiston virus showed that the transcripts from the L and M segments have a size similar to that of their template, whereas two types of complementary RNA are transcribed from the S segment. One, S1, is a full-length "plus" RNA strand (antigenome), and the other, S2, is an incomplete plus RNA strand which serves as mRNA for at least the synthesis of the N protein and a virus-specific nonstructural polypeptide, p12. The 5' ends of these two transcripts appeared to be identical and complementary to the 3' ends of the viral RNA. Our results suggest that transcription of the S fragment either stops 100 to 150 nucleotides from the 5' end of the template, generating an S2 molecule, or continues, generating an S1 molecule. Neither the S1 antigenome nor the S2 mRNA molecules were polyadenylated at their 3' ends or capped at their 5' ends.

Animals↗
PubMed1981

A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription.

We propose a mechanism for the priming of influenza viral RNA transcription by capped RNAs in which specific 5'-terminal fragments are cleaved from the capped RNAs by a virion-associated endonuclease. These fragments would serve as the actual primers for the initiation of transcription by the initial incorporation by the initial incorporation of a G residue at their 3' end. We show that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5' methylated cap structure (m7GpppXm) preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3'-terminal hydroxyl groups. RNAs containing the 5'-terminal structure GpppG could not be cleaved to produce these specific fragments. Consistent with our proposed mechanism, those capped fragments that function as primers could be linked to a G residue in transcriptase reactions containing alpha-32P-GTP as the only ribonucleoside triphosphate. The pattern of G and C incorporation onto these primer fragments suggests that this incorporation is directed by the second and third bases at the 3' end of the virion RNA template, which has the sequence 3' UCG. Primer fragments with a 3'-terminal A residue were used more efficiently than those with a 3'-terminal G residue, indicating a preference for generating an AGC sequence in the viral mRNA complementary to the 3' end of the virion RNA. Cleavage of the RNA primer and initiation of transcription are not necessarily coupled, because a 5' fragment isolated from one reaction could be used as a primer when added to a second reaction. Uncapped ribopolymer inhibitors of viral RNA transcription inhibited the cleavage of capped RNAs.

Endonucleases↗
PubMed1980

RNA primers and the role of host nuclear RNA polymerase II in influenza viral RNA transcription.

Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs. Our recent experiments have proved these two hypotheses. Purified globin mRNAs were shown to stimulate viral RNA transcription in vitro very effectively. The resulting transcripts directed the synthesis of all the non-glycosylated virus-specific proteins in cell-free systems. Other eukaryotic mRNAs were also active as primers. The presence of a 5' terminal methylated cap structure in the priming mRNA was required for its priming activity. Thus, with globin mRNA, removal of the cap eliminated essentially all of its priming activity, and much of this activity could be restored by enzymically recapping the globin mRNA. Using globin mRNA containing 32P only in its cap, we demonstrated that the 5' cap of the globin mRNA primer was physically transferred to the viral RNA transcripts during transcription. Gel electrophoretic analysis suggested that, in addition to the cap, about 10-15 other nucleotides were also transferred from the globin mRNA to the viral RNA transcripts. A mechanism for the priming of influenza viral RNA transcription by globin mRNA is proposed. Initial experiments strongly suggest that priming by capped host mRNAs also occurs during the synthesis of viral mRNA in vivo.

Amanitins↗
PubMed1980

Both the 7-methyl and the 2'-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription.

The ability of eukaryotic mRNAs to serve as primers for influenza virus RNA transcription depends on the presence of a 5'-terminal methylated can structure, the absence of which eliminates essentially all priming activity [Plotch, S. J., Bouloy, M. & Krug, R. M. (1979) Proc. Natl. Acad. Sci. USA 76, 1618-1622]. The present study was undertaken to determine the extent to which each of the methyl groups in the cap influences the priming activity of a mRNA. To assess the importance of the 2'-O-methyl group on the penultimate base of the cap, we used several plant viral RNAs containing the monomethylated cap 0 structure, m7GpppG. Brome mosaic virus (BMV) RNA 4 stimulated influenza virus RNA transcription only about 10-15% as effectively as did globin mRNA, which has a cap with a 2'-O-methyl group. When the cap of BMV RNA 4 was enzymatically 2'-O-methylated, its priming activity was increased 14-fold. Qualitatively similar results were obtained with other plant virus RNAs. To assess the importance of the terminal 7-methyl group, BMV RNA 4 containing the cap structure GpppGm was prepared by a series of chemical and enzymatic steps. These molecules were found to be only about 15% as active in priming as BMV RNA 4 molecules containing the fully methylated cap, m7GpppGm, indicating that the terminal 7-methyl group also strongly enhances priming activity. These results indicate that the cap 1 structure (m7GpppXm) found in all mammalian cellular mRNAs is more stringently required for priming influenza virus RNA transcription than for translation in cell-free systems.

Methylation↗
PubMed1979

Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro.

We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Natl. Acad. Sci. USA 75, 4886-4890]. Here, we present direct evidence that the 5'-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (beta-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the beta-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing (32)P label only in the cap (m(7)G(32)pppm(6)A(m)-) were prepared by recapping beta-eliminated globin mRNAs with the vaccinia virus enzymes, [alpha-(32)P]GTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a (32)P-labeled 5'-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segments were 10-15 nucleotides longer at their 5' end than ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.

Base Sequence↗
PubMed1979

Cap and internal nucleotides of reovirus mRNA primers are incorporated into influenza viral complementary RNA during transcription in vitro.

Reovirus mRNA's containing a 5'-terminal methylated cap structure (m(7)GpppG(m)) were shown to be effective primers for influenza viral RNA transcription in vitro catalyzed by the influenza virion transcriptase. Priming activity required the presence of methyl groups in the cap since reovirus mRNA's with 5'-terminal GpppG were inactive as primers. Both the cap and internal nucleotides were physically transferred from radiolabeled reovirus mRNA to influenza viral complementary RNA (cRNA) during transcription in vitro. By using reovirus mRNA's with methyl-(3)H-labeled caps as primers, we showed that the influenza viral cRNA synthesized in the presence of unlabeled nucleoside triphosphates contained [methyl-(3)H]m(7)GpppG(m), identical to that found in the reovirus mRNA primer. To demonstrate transfer of internal residues, reovirus mRNA's synthesized in the presence of all four alpha-(32)P-labeled ribonucleoside triphosphates were used as primers. The resulting influenza viral cRNA was (32)P-labeled. Diethyl-aminoethyl-Sephadex chromatography of the RNase T2 digest of this cRNA demonstrated (32)P radiolabel in both internal residues (charge -2) and the cap (charge -4.6). Approximately 25 internal nucleotides along with the cap of reovirus mRNA were transferred to each chain of influenza viral cRNA. Gel electrophoretic analysis indicated that the segments of influenza viral cRNA primed by reovirus mRNA were approximately the same size as those primed by a different mRNA, globin mRNA, strongly suggesting that the influenza virion transcriptase complex transfers approximately the same number of nucleotides plus the cap from different mRNA primers to the 5' end of influenza viral RNA transcripts.

Base Sequence↗