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G Beaud

Publications and source records attributed to G Beaud.

At least 37 records · Page 2Linked to original sources
PubMed1986

Purification and characterization of a protein synthesis inhibitor associated with vaccinia virus.

A protein synthesis inhibitor, solubilized from vaccinia virus (Ben-Hamida, F., Person, A., and Beaud, G. (1983) J. Virol. 45, 452-455), has been purified to homogeneity, yielding a basic protein with molecular mass of 11 kDa. This purified protein migrates as a single spot in two-dimensional gel analysis (isoelectric point above 8.6). It is phosphorylated by the vaccinia-associated protein kinase, and it aggregates in the absence of reducing agents. This 11-kDa protein inhibits protein synthesis when added to a reticulocyte lysate at a stoichiometric ratio of approximately one protein molecule/ribosome, and it associates with the ribosome fraction after incubation in reticulocyte lysates or in Ehrlich ascites tumor cell lysates. As previously described for the inhibitor associated with vaccinia cores, the purified inhibitor inhibits the formation of the 40 S ribosomal subunit X Met-tRNAi ribosomal initiation complex. It has no detectable effect on the formation of the ternary complex (Met-tRNAi X GTP X eucaryotic initiation factor 2). This inhibitor associated with vaccinia virus particles may be involved in the shutoff of host protein synthesis and may also be responsible for the absence of virus replication in some cell-virus systems.

Animals↗
PubMed1985

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.

Base Sequence↗
PubMed1984

Translation in micrococcal nuclease-treated cell-free extracts from Ehrlich ascites tumor cells. Stimulation by initiation factor eIF-2B.

Translation of exogenous mRNAs in micrococcal nuclease-treated extracts from Ehrlich ascites tumor cells is greatly stimulated by the addition of crude initiation factors or initiation factors eIF-2B and eIF-2 containing eIF-2B. The requirement for exogenous eIF-2B in micrococcal nuclease-treated extracts does not result from either loss or enhanced phosphorylation of eIF-2 during incubation.

Animals↗
PubMed1983

Solubilization of a protein synthesis inhibitor from vaccinia virions.

The protein synthesis inhibitor previously demonstrated to be associated with vaccinia cores was quantitatively solubilized from vaccinia virions or cores after an endogenous protein kinase reaction at pH 10. The presence of the inhibitor in the soluble extract correlated with the presence of soluble virion proteins phosphorylated in vitro. Partially purified inhibitor blocked methionyl-tRNAfMet-40S initiation complex formation, as was the case in cell-free extracts derived from vaccinia virus-infected cells.

Hydrogen-Ion Concentration↗
PubMed1982

Expression of vaccinia virus early mRNA in Ehrlich ascites tumor cells. 1. Translation of cellular and viral early mRNA in cell-free systems from uninfected and virus-infected cells at the early stage.

Translation of cellular and early vaccinia RNA in nuclease-treated lysates, derived from uninfected and vaccinia-virus-infected cells at the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; this conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells, as suggested in the accompanying paper.

Animals↗
PubMed1982

Expression of vaccinia virus early mRNA in Ehrlich ascites tumor cells. 2. Part of the polysomes at an early stage of virus infection are not bound to the cytoskeleton.

A method for preparing detergent cytoskeletons from uninfected or vaccinia-virus-infected cells is described. This method resulted in the fractionation of the cytoplasmic compartment into soluble and cytoskeletal fractions. More than 85% of small molecules and tRNA were released from the cytoskeleton and recovered into the soluble fraction. The cytoskeletal fraction contained about 40% of the cytoplasmic proteins and 67% of total cytoplasmic RNA. Similar values were obtained for vaccinia-virus-infected cells. In contrast, whereas 85% of the cellular polysomes and poly(A)-rich RNA remained associated with the cytoskeleton in uninfected cells, more than 40% of vaccinia early mRNA engaged into polysomes was found to be not associated with the cytoskeleton. A similar partition was found when the viral RNA was labeled for 15 min at 5 min and 20 min after the infection or when varying the duration of the pulse. Soluble and cytoskeletal viral polysomes were found to synthesize a similar set of proteins after translation in vitro of the corresponding mRNA. The fate of rapidly labeled cellular poly(A)-rich RNA upon vaccinia virus infection was followed by a glucosamine/uridine chase procedure, and also that of relatively stable poly(A)-rich RNA after long-term labeling and chase. In both cases no release of poly(A)-rich RNA from the cytoskeleton occurred after vaccinia virus infection. These experiments reveal that cellular mRNA remains associated to the cytoskeleton in EAT cells infected with vaccinia virus (early period) whereas at least 40% of the vaccinia early polysomes are not associated with the cytoskeleton. A model for vaccinia early mRNA metabolism is presented, which may account for the rapid shut-off of host protein synthesis.

Animals↗
PubMed1981

The inhibition of vaccinia virus replication by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB): an effect at the assembly stage.

The step sensitive to DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) in vaccinia virus replication has been investigated. Ninety microM-DRB extensively inhibited the yield of vaccinia virus after infection of Ehrlich ascites tumour cells. DRB did not inhibit cytoplasmic vaccinia DNA replication. Cytoplasmic viral RNA synthesis (both early and late) was also apparently unaffected and the virus RNAs thus synthesized were normal sized. The expression of early, intermediate and late proteins was not detectably impaired by DRB in vaccinia virus-infected cells. DRB inhibited vaccinia virus replication at the assembly stage since most of the virus DNA remained in a DNase-sensitive form in the infected cells and the virus was therefore not normally coated with virus proteins.

Animals↗
PubMed1980

Inhibition of 40S--Met--tRNAfMet ribosomal initiation complex formation by vaccinia virus.

Infection with vaccinia virus (a poxvirus) quickly and efficiently shuts off host protein synthesis in the presence of actinomycin D (refs 3--5) or cycloheximide. The cellular messenger RNA apparently remains stable in the infected cells exposed to inhibitors of viral gene transcription. In some cases vaccinia viral RNA or poly(A) synthesis have been implicated in the establishment of this effect. However, in the presence of cordycepin (3-deoxyadenosine) which blocks viral gene transcription and cytoplasmic poly(A) synthesis, cellular protein synthesis is still efficiently inhibited in vaccinia virus-infected cells. This shutoff is also observed in vitro, in the corresponding cell-free extracts, and in a reticulocyte lysate. Therefore the shutoff of host protein synthesis is probably mediated by a factor associated with vaccinia virions. We now report that the formation of the 40S--Met-tRNAfMet initiation complex is inhibited in cytoplasmic extracts derived from vaccinia virus-infected cells exposed to cordycepin to block viral gene expression. A similar inhibition is found in reticulocyte lysates incubated with purified vaccinia cores, confirming the hypothesis that the factor associated with the viral cores is responsible for the inhibition observed in vaccinia virus-infected cells exposed to inhibitors of transcription.

Animals↗
PubMed1979

Phosphorylation in vivo of a vaccinia-virus structural protein found associated with the ribosomes from infected cells.

When vaccinia-virus-infected cells were labeled with radioactive phosphate in the absence of viral gene expression an additional phosphoprotein, containing phosphoserine, was found specifically associated with the ribosomes. The phosphoprotein was removed from the ribosomes following a 0.5 M KCl washing or after EDTA treatment. This additional phosphoprotein was found in infected cells after either a long (3-4 h) or a short (30 min) labeling period; it was detected when the infected cells were incubated in the presence or absence of an inhibitor of RNA or protein synthesis. This phosphoprotein originated from the phosphorylation of vaccinia virion structural protein VP11b (Mr 11,000) at a specific site since only a single major phosphopeptide was obtained after trypsin digestion. This phosphoprotein was also present in purified vaccinia virions labeled with radioactive phosphate. VP11b protein was phosphorylated in vitro by the protein kinase associated with the cores. When the reaction was carried out at an alkaline pH the phosphorylation in vitro occurred at different sites in the protein; at neutral pH the phosphorylation of VP11b was more specific and, as judged by tryptic peptide analysis, occurred mainly at the same site as in the phosphorylation in vivo. A role for the involvement of phosphoprotein VP11b in the establishment of the shut off of host protein synthesis by vaccinia virus is suggested.

Animals↗
PubMed1978

In vitro inhibition of protein synthesis by purified cores from vaccinia virus.

The mechanism of the shutoff of cellular protein synthesis in vaccinia virus-infected cells has been investigated by using in vitro systems. Purified vaccinia cores cause inhibition of endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems. Translation of viral mRNA from turnip yellow mosaic virus is also impaired in wheat germ cell-free extracts. The block induced by vaccinia cores in protein synthesis is not due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. No nucleolytic activity able of digesting mRNA could be detected in purified vaccinia cores with three sensitive tests. There is a lack of inhibition in the poly(Phe)-poly(U) system, which bypasses the normal initiation process. An almost complete disaggregation of polyribosomes in the reticulocyte lysate appears when vaccinia cores are present. These results indicate that mRNA translation in a cell-free system is affected predominantly at the level of polypeptide chain initiation.

Animals↗
PubMed1978

Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3'-deoxyadenosine).

Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.

Animals↗
PubMed1975

Messenger activity of RNA transcribed in vitro by DNA-RNA polymerase associated to vaccinia virus cores.

The coding properties of RNA transcribed in vitro by purified vaccinia cores have been investigated using Krebs ascites tumor cells, L cells, and reticulocyte lysates. Six to 10 proteins synthesized in vitro are separated on polyacrylamide gels by electrophoresis in the presence of sodium dodecyl sulfate. Their molecular weights vary from 10,000 to 44,000. The electrophoretic behavior of these proteins is similar to that of early proteins isolated from infected L cells. The tryptic peptide analysis of one of these proteins indicates similarity in amino acid sequences. These results show fidelity of both in vitro transcription and molecular weight above 44,000 are synthesized in vitro does not seem due to a competition between 12S mRNA synthesized in excess and RNA of a higher sedimentation coefficient present in a lower amount.

Animals↗