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H O Voorma

Publications and source records attributed to H O Voorma.

At least 19 recordsLinked to original sources
PubMed1992

Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency.

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.

Base Sequence↗
PubMed1992

Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation.

The adenovirus tripartite leader (TPT) 5' untranslated region (5'UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5'UTR. The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct. Surprisingly, the TPT 5'UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap-independent initiation. In vitro protein-synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT-5'UTR-directed translation and poliovirus-induced host cell shut-off is discussed.

Adenoviridae↗
PubMed1992

Eukaryotic initiation factors-4E and -4F stimulate 5' cap-dependent as well as internal initiation of protein synthesis.

Two mechanisms of initiation of protein synthesis are known. The 5' cap-dependent model requires the activity of cap-binding eukaryotic initiation factors (eIF)1-4E and -4F, inducing unwinding of mRNA secondary structures. The internal initiation model is 5' cap-independent and requires a ribosomal entry site formed by higher order structures of the mRNA. It has been proposed that this mechanism does not need eIF-4E and eIF-4F. We prepared bicistronic transcripts on which both mechanisms of initiation occur, allowing the determination of the initiation factor dependence of these two mechanisms simultaneously. The unwinding factors eIF-4A, eIF-4B, and eIF-4F were found to be necessary for 5' cap-dependent initiation as well as for internal initiation. Surprisingly, efficient translation of both cistrons on the bicistronic mRNA required eIF-4E. A model is presented in which assembly of eIF-4E into a functional eIF-4F complex is a prerequisite for both types of initiation.

Bacterial Proteins↗
PubMed1992

Interaction of protein synthesis initiation factors with the mRNA cap structure.

The mechanism of mRNA recognition by proteins interacting with the mRNA cap structure was investigated by photochemical cross-linking of proteins with 32P-labelled reoviral RNAs. Using ribosomal washes as a source of eukaryotic protein synthesis initiation factors, we identified the well-known cap binding proteins eIF-4B and -4E, but eIF-2 and eIF-3 as well. The interplay of purified eIF-4A, -4B, and -4F was studied in relation to ATP dependence and cap analogue sensitivity of cap binding. Next to their well-known roles in the initiation process, eIF-2 and eIF-3 also cross-linked to the 5' cap. eIF-2 stimulated eIF-4B and -4E cross-linking, an observation that has been previously described more extensively. The interaction of eIF-2 with the 5' end of mRNA was extremely sensitive to K(+)-ions and was resistant to a high concentration of Mg(2+)-ions; this influence of mono- and divalent ions was in contrast with the cross-linking of eIF-4B and -4E. Optimal interaction of these factors was obtained at moderate K+ concentration and low Mg(2+)-ion concentrations. eIF-2 cross-linking was sensitive to high protein to mRNA ratios indicating a weak affinity as compared to eIF-4E and -4B. The interaction of eIF-3 with the cap of mRNA is also weak as it was counteracted by all other cap binding proteins, leading to an inability to detect the cross-linking of this protein in crude eIF preparations. Time kinetics of formation of complexes suggested eIF-2 to be one of the first factors to interact with mRNA. Preformed RNA-protein complexes were dissociated after cap analogue addition, suggesting reversible interactions between RNA and proteins.

Adenosine Triphosphate↗
PubMed1992

RNA unwinding by eukaryotic initiation factor 4A and nucleotide modification.

Unwinding of double-stranded RNA by nuclear helicases can lead to modification of adenosine-residues, resulting in inosine. During initiation of protein synthesis the 5' untranslated region of an mRNA is unwound by eukaryotic initiation factors (eIF) -4A and -4B. In this work we investigated the possible nucleotide modification after unwinding by eIF-4A and eIF-4B of in vitro synthesized, labeled RNA. The products of unwinding were analyzed by gel-electrophoresis and, after nuclease digestion, by thin layer chromatography of the mononucleotides. Crude protein fractions unwound the duplex RNA and converted part of the AMP-residues into IMP-residues. However, unwinding by purified factors was not linked to this conversion, the deamination of AMP residues. Concluding, unwinding of RNA during initiation of protein synthesis does not lead to conversion of adenosine into inosine.

Base Sequence↗
PubMed1991

The 5' untranslated region of encephalomyocarditis virus contains a sequence for very efficient binding of eukaryotic initiation factor eIF-2/2B.

The mechanism by which internal ribosomal binding on the picornaviral RNA takes place is still not known. An important role has been suggested for eukaryotic initiation factors eIF-4A, eIF-4B, as well as for some not yet defined trans-acting factors like p52 for poliovirus and p58 for encephalomyocarditis virus (EMCV). In this paper we describe the competition between the 5' untranslated region (UTR) of EMCV and globin mRNA for the translational apparatus in rabbit reticulocyte lysates and show that the factor that is competed for is eIF-2/2B. The EMC 5' UTR is a very strong inhibitor of globin synthesis in the rabbit reticulocyte lysate because of a 30-fold higher eIF-2/2B binding capacity. Mutations 100 to 140 nucleotides upstream of the initiation codon led to a decreased efficiency to initiate translation and to a decreased ability to inhibit globin mRNA translation. The results suggest an important role for eIF-2/2B binding in EMC RNA translation and therefore in internal initiation.

Base Sequence↗
PubMed1991

Evidence that eukaryotic initiation factor (eIF) 2 is a cap-binding protein that stimulates cap recognition by eIF-4B and eIF-4F.

We studied the mRNA-binding properties of eukaryotic initiation factor (eIF) 2. This Met-tRNA-binding factor interacts with the cap structure of reoviral mRNA in an ATP-independent manner. Both the beta- and gamma-subunit of eIF-2 are involved in the UV-induced cross-linking of eIF-2 to the cap. The interaction of eIF-2 with a messenger is sensitive to the cap analogue 7-methyl-guanosine 5'-triphosphate as measured by cross-linking and by mRNA retention on nitrocellulose filters. The cap-binding property of eIF-2 does not conflict with the current mRNA-binding model of initiation factors eIF-4A, -4B, and -4F: cross-linking of eIF-4E and of eIF-4B is stimulated by eIF-2. The eIF-2-mediated increase of eIF-4E interaction results in a decrease of the cross-linking of the beta- and gamma-subunits of eIF-2. The presence of GTP in the cross-linking assay interferes with the interaction of eIF-2 with the cap structure but does not inhibit the eIF-2 stimulated eIF-4E and -4B cross-linking. These observations indicate a role for eIF-2 in the mRNA recognition.

Animals↗
PubMed1991

Separation of translationally active mRNAs by reversed-phase ion-pair high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method on C4 columns was developed for the separation of mRNAs. The addition of methylmercuric hydroxide markedly influenced the separation according to length of these molecules. A method is given to recover minute amounts of translatable mRNA from the organic phase. The resolution of mRNAs improved with increasing pore size of the column support.

Animals↗
PubMed1991

Cowpea mosaic virus middle component RNA contains a sequence that allows internal binding of ribosomes and that requires eukaryotic initiation factor 4F for optimal translation.

Cowpea mosaic virus (CPMV) middle component RNA (M-RNA) encodes two proteins of 105 and 95 kDa, of which translation starts at nucleotide (nt) 161 and nt 512, respectively. In vitro translation of both proteins directed by T7 transcripts of M-RNA was stimulated fourfold by eukaryotic initiation factor 4F (eIF-4F), the cap-binding protein complex. The ratio of the synthesis of both proteins after translation was not influenced by eIF-4F or by any known eIF. Part of the CPMV 5' sequence was cloned downstream of the 5' untranslated region of ornithine decarboxylase (ODC); the latter untranslated sequence has a highly stable secondary structure, preventing efficient translation of ODC. Insertion of nt 161 to 512 of CPMV M-RNA upstream of the ODC initiation codon resulted in a marked increase in ODC translation, which indicates that the CPMV sequence contains an internal ribosome-binding site. The insertion conferred stimulation by eIF-4F on ODC translation, showing that eIF-4F is able to stimulate internal initiation.

Base Sequence↗
PubMed1990

An initiation signal in the 5' untranslated leader sequence of encephalomyocarditis virus RNA.

A 593 nucleotide fragment of the 5' leader of encephalomyocarditis virus RNA (EMCV-RNA) was linked to the SP6 promoter and inserted upstream of the reporter gene chloramphenicol acetyltransferase (CAT). The presence of the 5'-UTR of EMCV-RNA in the RNA transcripts, made in vitro with the SP6 polymerase, resulted in a strong translational enhancement when tested in the micrococcal nuclease-treated reticulocyte lysate. The transcripts were equally active with or without a 5' methylated capstructure as expected, since EMCV-RNA is one of the mRNAs capable of internal initiation. We searched for a signal in the 5' leader that allows the 43S preinitiation complex to bind internally and localized a hairpin containing a unique nucleotide sequence, CUUUA, present in a domain conserved among cardio- and aphtoviral RNAs. Replacing this sequence into AGCU resulted in a 50% loss of translational activity. A second mutation involving a U-G change in the stem of that hairpin resulted in an almost complete loss of initiation.

Animals↗
PubMed1989

Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones.

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.

Antibodies, Monoclonal↗
PubMed1988

Secondary structure and expression in vivo and in vitro of messenger RNAs into which upstream AUG codons have been inserted.

We wanted to discover whether the conformation of the mRNA leader sequence is involved in translational fidelity. For this purpose we constructed several mutants of Semliki Forest virus 26S mRNA and inserted AUG codons into the leader sequence. We then analyzed the results of in vitro and in vivo translation of these mRNAs, probed enzymatically the secondary structure and performed minimal energy folding of the transcripts. Our results indicate that the position of a hairpin in the leader sequence determines at which AUG codon downstream from that hairpin translation is initiated.

Base Sequence↗
PubMed1988

Measles virus fusion protein presented in an immune-stimulating complex (iscom) induces haemolysis-inhibiting and fusion-inhibiting antibodies, virus-specific T cells and protection in mice.

Immune-stimulating complexes (iscoms), which have recently been shown to be highly effective for the antigenic presentation of membrane proteins of viruses, were prepared with affinity-purified fusion (F) protein of measles virus (MV), using an adaptation of the standard method for iscom preparation. Immunization of monkeys with the F iscom preparation induced biologically active anti-F protein antibodies as was shown in haemolysis inhibition and cell-cell fusion inhibition tests. A whole MV iscom preparation, which also contained the haemagglutinin protein, induced not only also haemolysis-inhibiting antibodies, but, in contrast to the F iscom preparation, also haemagglutination-inhibiting and virus-neutralizing antibodies. In addition the F iscom preparation was shown to activate measles virus-specific T cells in mice. This was demonstrated by the generation of an MV-specific delayed type hypersensitivity response in F iscom-immunized animals and by the isolation of T cell clones specific for MV F protein with the T helper phenotype. Vaccination of mice with MV iscom or F iscom protected them from MV-induced fatal encephalopathy. The data concerning the immunogenicity of MV proteins presented in iscoms are discussed in relation to their potential for the development of an inactivated measles vaccine.

Animals↗
PubMed1988

Affinity labeling by a photoreactive GTP analogue of the delta-subunit of eukaryotic initiation factor eIF-2 in different initiation complexes.

The interaction of GTP with initiation factor eIF-2 in different complexes was studied by affinity labeling using a derivative of [3H]GTP carrying a photoreactive group in the alpha-phosphate moiety. In the binary complex [eIF-2.GTP analogue], in the ternary complex [eIF-2.GTP analogue.Met-tRNAf] as well as in the eIF-2. eIF-2B complex the alpha-subunit of eIF-2 was found to be specifically labeled. GTP is concluded to interact during polypeptide chain initiation with the alpha-subunit of eIF-2 at least by its alpha-phosphate group.

Affinity Labels↗
PubMed1987

Minimal energy foldings of eukaryotic mRNAs form a separate leader domain.

We have investigated the minimal energy foldings of 38 mature mRNAs, including the globin family, the insulins, the growth hormones and interleukin-2, and have compared these foldings with those of fully and partly randomised sequences. The mRNAs differ from the random sequences in that they form a separate leader hairpin of 40-60 nucleotides, with the initiation codon typically located downstream of this hairpin, followed by a main fold in which a region flanking the initiation codon is basepaired with the trailer: resulting in a close proximity of the 5' and 3' end of the mRNA. The formation of this conformation depends not only--or primarily--on the structure of the leader, but on both the leader and trailer sequence and their interaction with the coding sequence. Thus if, as the frequent occurrence of this pattern suggests, the secondary structure of the leader regions plays a role in the initiation of translation, possibly accounting for the specificity of initiation and the different translational efficiencies of various mRNAs, we expect that these features may be influenced both by leader and trailer mutants.

Adenoviridae↗