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Biomedical subjects

E Kuechler

Publications and source records attributed to E Kuechler.

At least 37 records · Page 2Linked to original sources
PubMed1993

Rhinoviral receptor discrimination: mutational changes in the canyon regions of human rhinovirus types 2 and 14 indicate a different site of interaction.

Amino acid sequence comparisons between the capsid proteins of several human rhinovirus (HRV) serotypes identified residues potentially involved in the discrimination between the major and the minor group receptors. Amino acids conserved within minor group HRVs were substituted in a full-length cDNA clone of HRV2 for those found at equivalent positions in major group HRVs. Transfection of HeLa cells with RNAs transcribed from seven individual mutated cDNAs gave rise to only two viable viruses; growth characteristics and affinity for the minor group receptor of both were unchanged compared to wild-type. Similar mutations in HRV14 were previously shown to alter the affinity for its receptor; the contact sites between the minor group viruses and the respective receptor may therefore be different.

Amino Acid Sequence↗
PubMed1992

Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A.

Proteinase 2A of human rhinovirus serotype 2 (HRV2 2A) was expressed in Escherichia coli and partially purified; the preparation was used to study various enzymatic parameters. Using a 16-amino acid peptide representing the native cleavage region of HRV2 2A, an apparent Km value of 5.4 x 10(-4) mol/liter was determined. A minimum of 9 amino acids (comprising residues P8 to P1') was necessary for cleavage to occur. Proteolysis of substituted peptides was highly tolerant toward changes at P1, P2', and P3' but an absolute requirement for glycine P1' and a high preference for threonine P2 was found. Furthermore, HRV2 2A only cleaved peptide substrates derived from other rhinovirus serotypes and poliovirus that possessed P2 Thr and P1' Gly. Thus, the sequence Thr-X-Gly may form the basis of the cellular cleavage site processed by rhinoviral 2As during viral replication. Studies with various inhibitors support the hypothesis that HRV2 2A belongs to a new class of cysteine proteinases.

Amino Acid Sequence↗
PubMed1992

Shedding of a rhinovirus minor group binding protein: evidence for a Ca(2+)-dependent process.

Soluble rhinovirus minor group binding activity was found to be shed into the medium upon incubation of HeLa cells at 37 degrees C. Although substantial amounts of this protein were released, no decrease of virus binding to the cell surface was seen. When the membrane-associated receptor was stripped from the cells with trypsin, virus binding was rapidly restored from an intracellular pool even in the absence of de novo protein synthesis. The release of this 85K virus-binding activity was inhibited by metal chelators such as EDTA, EGTA or 1,10-phenanthroline. The potential involvement of a Ca(2+)-dependent protease and/or a phospholipase in this process is discussed.

Calcium↗
PubMed1992

Using proteinase trapping to detect revertants of inactive rhinoviral 2A proteinase mutants.

The 2A proteinase of human rhinovirus 2 cleaves itself off the growing polyprotein at its own N terminus during translation; this property was used to develop an in vivo screening system with the lacZ gene fragment of M13mp18. The fusion of an active 2A proteinase to the C-terminus of the alpha-fragment did not affect alpha-complementation, as the proteinase cleaved itself off the alpha-fragment. However, an inactive 2A proteinase remained fused to the alpha-fragment hindering alpha-complementation. Random mutations were then introduced into the 2A gene site by PCR amplification. Mutants defective in alpha-complementation (thus containing an inactive 2A proteinase) were obtained at an efficiency of 5%, mutants showing reduced 2A activity at an efficiency of 1%. Mutants showing reduced or no 2A activity were then subjected to PCR mutagenesis. Three mutants reactivating an inactive 2A proteinase were examined and the compensatory changes determined.

Bacteriophages↗
PubMed1991

Proteinase trapping: screening for viral proteinase mutants by alpha complementation.

Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment. However, fusion of an inactive 2A prevented alpha complementation, as the 2A polypeptide remained fused to the alpha fragment. After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an alpha fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector. alpha complementation indicated intermolecular processing of the 2A cleavage site on the alpha fragment-inactive proteinase fusion protein. This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity.

Amino Acid Sequence↗
PubMed1991

Substrate requirements of a human rhinoviral 2A proteinase.

The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2. This enzyme was shown to have an unusual preference at the P1 site; out of eight amino acid substitutions made, only the branched amino acids Val and Ile were not readily accepted. The HRV2 2A was shown to process poorly the HRV89 2A cleavage site and to be unable to cleave at sites which included the P' region of poliovirus or HRV14. Furthermore, the 2A of HRV89 preferred the cleavage site of HRV2 to its own.

Amino Acid Sequence↗
PubMed1991

Human rhinovirus mutants resistant to low pH.

Mutants of human rhinovirus serotype 14 (HRV14) with increased resistance to treatment at low pH were obtained by repeated cycles of exposure to pH 4.5 and propagation in HeLa cells. Whereas wild-type virus lost more than 5 logs of infectivity upon incubation at pH 4.3, the three isolates examined were essentially unaffected. Conformational change of the viral capsid upon exposure to low pH was assessed as an increase of hydrophobicity by partition between an aqueous phase and a Triton X-114 phase; the mutants required exposure to a much lower pH to accumulate in the Triton phase than wild-type HRV14. The sequence of the capsid region was determined for three isolates; two isolates were found to have the changes Thr17 to lie in VP2 and Asn 100 to lie in VP1. The third isolate also had the change Thr17 to Ile in VP2; however, in VP1, Asp101 was replaced by Glu. Separate introduction of the mutations into full length cDNA clones of the wild-type sequence of HRV14 showed that only the changes in VP1 were necessary for the increased stability at pH 4.5. The implications of the mutations for the three-dimensional structure of the viral capsid are discussed.

Capsid↗
PubMed1991

Stabilization of human rhinovirus serotype 2 against pH-induced conformational change by antiviral compounds.

Four WIN compounds with anti-picornavirus activities were tested for their ability to stabilize human rhinovirus serotype 2 (HRV-2) against low pH-induced conformational changes in vitro, as determined by specific immunoprecipitation. These results were compared to the minimal inhibitory concentration (MIC) as measured in a plaque reduction assay. A direct relationship was observed between the concentration of the compound that prevented the low pH-induced conformational changes and the MIC, indicating that stabilization is an important element in the mode of action of these drugs against HRV-2.

Antiviral Agents↗
PubMed1990

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

Affinity Labels↗
PubMed1990

Identification of rhinoviruses by cDNA probes.

We have used nucleic acid hybridization for the detection and grouping of human rhinoviruses (HRV) according to their genetic relationships. Fifteen rhinovirus reference strains, seventy-one clinical isolates and four enteroviruses were propagated in cell cultures, spotted onto membrane filters and hybridized with radioactively labelled cDNA probes covering different parts of the genomes of HRV-1B, HRV-2, HRV-14, HRV-85 and HRV-89. When the rhinovirus and enterovirus reference strains were tested, the 5' probe of HRV-2 hybridized with thirteen of the fifteen HRV reference strains, with poliovirus type 3 and with ECHO virus 11. The HRV-14 5' probe reacted with eleven HRV reference strains and with all the enteroviruses studied. Sixty-nine of the 71 clinical isolates were recognised by the HRV-2 5' probe, whereas the HRV-14 probe from the same part of the genome hybridized with 54 field isolates. One of the two isolates that remained negative with the HRV-2 5' probe was detected with the HRV-2 probe that derived from the P2 region of the genome, and the other isolate was not detected by any of the probes. Probes from other parts than the 5' end of the genome were generally more specific, and clusters could be formed based on the reactivity of the HRV strains with these probes.

DNA Probes↗
PubMed1989

Human rhinovirus serotype 2: in vitro synthesis of an infectious RNA.

A complete cDNA copy of human rhinovirus serotype 2 RNA was placed under the control of a T7 RNA polymerase promoter. An in vitro transcribed RNA containing two extra G residues at the 5' end gave rise to plaques on transfection into HeLa cells. The efficiency was approximately half that obtained with viral RNA. On the contrary, an in vitro synthesized RNA containing 16 additional nucleotides at the 5' end was not infectious. This ability to make an infectious in vitro transcribed RNA will be useful in studying the characteristics of viruses using the human rhinoviral minor group receptor.

Cloning, Molecular↗
PubMed1988

Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA.

Photo-reactive 3-(4'-benzoylphenyl)propionyl-Phe-tRNA bound to the A- or the P-site was crosslinked to 23S RNA upon irradiation at 320 nm. The sites of reaction were identified as U-2584 and U-2585 at the A-site and A-2451 and C-2452 at the P-site. Minor crosslinks from both sites were observed at nucleotides A-2503 to U-2506. All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V. Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo-crosslinking. In addition, tetracycline was also shown to photo-crosslink to this region. These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A- and P-site.

Acyltransferases↗
PubMed1988

Characteristics of the minor group receptor of human rhinoviruses.

The receptor for the minor group of human rhinoviruses was solubilized from HeLa cell membranes with various detergents. Virus binding activity was determined in a filter binding assay using 35S-labeled human rhinovirus 2 (HRV2) as a probe. The receptor protein was enriched on Lens culinaris lectin columns and the active fractions were further purified by gel permeation and anion exchange chromatography. The receptor has an apparent molecular weight of 450 kDa in the presence of detergent. The binding activity is sensitive to trypsin, sulfhydryl modifying agents, but insensitive to neuraminidase. Divalent cations are essential for virus binding.

Acetylglucosaminidase↗
PubMed1987

Assembly of pre-mRNA splicing complex is cap dependent.

To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a functional splicing complex, the in vitro splicing of a truncated human metallothionein pre-mRNA was examined in the presence of the cap analogue m7GTP. Significant inhibition of splicing was observed at a concentration as low as 5 microM m7GTP. Analysis of the splicing reaction on glycerol density gradients showed two complexes sedimenting at 45S and 22S. When the reaction was carried out in presence of m7GTP a marked decrease of the material sedimenting at 45S, representing the active splicing complex, was observed. When capped pre-mRNA was replaced by uncapped pre-mRNA, complex formation was significantly reduced. These data indicate that the cap structure plays an important yet unknown role in the assembly of spliceosomes.

Exons↗