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E Kuechler

Publications and source records attributed to E Kuechler.

At least 55 records · Page 3Linked to original sources
PubMed1987

Comparison of the three-dimensional structure of two human rhinoviruses (HRV2 and HRV14).

An attempt has been made to build a model of human rhinovirus 2 (HRV2) based on the known human rhinovirus 14 (HRV14) structure. HRV2 was selected because its amino acid sequence is known and because it belongs to the minor rhinovirus receptor class as compared to HRV14, which belongs to the major class. Initial alignment of HRV2 with HRV14 based on the primary sequence and the knowledge of the three-dimensional structure of HRV14 showed that the most probable position of the majority of insertions and deletions occurred in the vicinity of the neutralizing immunogenic sites (NIm). Out of a total of 855 amino acids present in one copy of each of the capsid proteins VP1 through VP4 of HRV14, 411 are different between the two viruses. There are also 6 amino acid residues inserted and 14 residues deleted in HRV2 relative to HRV14. Examination of amino acid interactions showed several cases of conservation of function, e.g., salt bridges or the filling of restricted space. The largest variation amongst the residues lining the canyon, the putative receptor binding site, was in the carboxy-terminal residues of VP1.

Amino Acid Sequence↗
PubMed1987

Mechanism of entry of human rhinovirus 2 into HeLa cells.

Internalized human rhinovirus 2 (HRV2) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20 degrees but not at 34 degrees the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20 degrees prior to addition of monensin permits virus synthesis to occur, depending on the time of preincubation.

Capsid↗
PubMed1987

Evolutionary relationships within the human rhinovirus genus: comparison of serotypes 89, 2, and 14.

The complete nucleotide sequence of the genome of human rhinovirus type 89 was determined from the cDNA that had been cloned into Escherichia coli. The genome is 7152 nucleotides long and contains a single large open reading frame of 2164 codons. Translation commences at position 619 and ends 42 nucleotides before the poly(A) tract. The positions of three proteolytic cleavage sites in the polyprotein were determined by N-terminal amino acid sequencing of the capsid proteins; the remainder were predicted from comparisons with other picornaviruses. Extensive similarity between the derived amino acid sequences of human rhinovirus types 89 and 2 was found, whereas the similarity between human rhinovirus types 89 and 14 was considerably less. It is apparent that human rhinoviruses may be more closely related than has been previously thought.

Amino Acid Sequence↗
PubMed1987

Cleavage site between VP1 and P2A of human rhinovirus is different in serotypes 2 and 14.

The viral capsid protein VP1 of human rhinovirus serotype 2 (HRV2) was cleaved with cyanogen bromide. The peptides thus obtained were separated on an HPLC butyl reversed phase column. Their positions on VP1 were determined by amino-terminal sequencing using the known nucleotide sequence of the genomic RNA of HRV2. The putative carboxy-terminal peptide was further cleaved with trypsin and the resulting fragments were separated on a C18 reversed phase column. Amino-terminus of sequencing of the C-terminal peptide revealed alanine as being the carboxy terminus of VP1 in HRV2. This indicates that the processing of the polyprotein is different in HRV2 from the processing previously reported for HRV14 and poliovirus.

Amino Acid Sequence↗
PubMed1987

A neutralizing epitope on human rhinovirus type 2 includes amino acid residues between 153 and 164 of virus capsid protein VP2.

Use has been made of a monoclonal antibody (designated 8F5) to map a neutralizing epitope on the viral capsid protein VP2 of human rhinovirus 2 (HRV2). This antibody which was raised against the native virus, neutralizes HRV2 and is also capable of recognizing denatured VP2 on Western blots. To examine the binding site of 8F5, VP2 of HRV2 was expressed in Escherichia coli. Deletions starting at the 3' end were then introduced into the gene for VP2 using Bal-31 nuclease. Polypeptides shortened at the carboxy terminus of VP2 were obtained from the deletions and were blotted onto nitrocellulose. The samples were then probed with monoclonal antibody 8F5. Recognition by 8F5 was maintained as long as the expressed polypeptide contained the VP2 sequence up to amino acid 164 or beyond. However, when the VP2 sequence was truncated to amino acid 153 or less 8F5 was no longer able to bind. The neutralization epitope (or part of it) recognized by 8F5 on VP2 is therefore located between amino acids 153 and 164.

Amino Acid Sequence↗
PubMed1987

The molecular biology of human rhinoviruses.

A brief review of recent advances in the understanding of human rhinovirus molecular biology is presented. The importance of recent findings on the elucidation of serotypic diversity and their implications for the viral host-cell receptor site are emphasized. An introduction to the genome structure and to the pathway of gene expression in rhinoviruses leads on to a discussion of the crystal structure of human rhinovirus 14 (HRV14) and the antibody-inducing regions on the surface of the capsid. Evidence from these experiments indicates that four sites on HRV14 are responsible for inducing neutralizing antibodies. Amino-acid sequence comparisons reveal that these sites are different in other serotypes, strengthening the view that the sequences of these regions are fundamental in determining and defining rhinovirus serotypes. The crystal structure of HRV14 points to a depression in the viral capsid as being the site of binding to the host-cell receptor; however, residues involved in binding cannot yet be identified.

Amino Acid Sequence↗
PubMed1985

Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region.

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5' end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3' half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5' half (including the capsid proteins) are more heterogeneous.

Amino Acid Sequence↗
PubMed1984

Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA.

Wybutine (Ywye), situated next to the 3'-side of the anticodon of tRNAPhe from Saccharomyces cerevisiae, can be photo-crosslinked to mRNA when bound to Escherichia coli ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)6. In order to identify the site of reaction on the mRNA, 5'-[32P]-labelled pAUGUUU was crosslinked by irradiation at 320 nm with Phe-tRNAPhe from yeast bound to the acceptor-site. The photoproduct was subsequently digested with P1-nuclease and analyzed by electrophoresis followed by homochromatography in the second dimension. As a result of the photoreaction the wybutine was found to be crosslinked to the U at the 5'-position of the corresponding UUU-codon.

Base Sequence↗
PubMed1984

Relationship of human rhinovirus strain 2 and poliovirus as indicated by comparison of the polymerase gene regions.

cDNA clones representing the 3'-terminal region of the human rhinovirus strain 2 genome have been obtained. The sequence of 1425 nucleotides adjacent to the poly(A) tract is presented and contains an open reading frame of 1383 nucleotides. The derived amino acid sequence corresponding to the putative RNA polymerase-coding region is compared to those of poliovirus type 1 (Mahoney) and foot-and-mouth disease virus A12. A high degree of homology between human rhinovirus strain 2 and poliovirus type 1 (Mahoney) was found within the coding sequence but not within the 3'-untranslated region.

Amino Acid Sequence↗
PubMed1984

Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

3-(4'-Benzoylphenyl)propionyl[3H] Phe-tRNA bound to the peptidyl site of the ribosome is photo-crosslinked exclusively to 23S RNA on irradiation at 320 nm. The site of reaction has been identified both by hybridization and primer-extension experiments as uridine-2584 and uridine-2585, located within the central loop of domain V according to the secondary structure model of 23S RNA. The fact that the covalently crosslinked tRNA retains its ability to form a peptide bond, together with the proximity of this site to the position of several mutations leading to chloramphenicol or erythromycin resistance strongly argue that this region of the 23S-like rRNAs is an integral component of the peptidyl transferase site. On the basis of these results, and from comparative analysis of the 16 available large subunit rRNA sequences, we propose a model for the functional organization of the peptidyl transferase site involving interaction of domains II and V of 23S rRNA.

Acyltransferases↗
PubMed1983

Part of the 23S RNA located in the 11S RNA fragment is a constituent of the ribosomal peptidyltransferase centre.

Upon irradiation, 3-[4-benzoylphenyl]propionyl-PhetRNA bound to the P-site of poly(U)-primed ribosomes is exclusively cross-linked to 23S RNA. It is shown that the photoreaction only occurs with pyrimidine nucleotides. The site of the cross-link is located within an 11S RNA fragment, which comprises the 1100 nucleotides at the 3'-end of 23S RNA. The cross-linked Phe tRNA derivative is still functionally active in peptide bond formation. The site labelled on the 11S fragment is therefore an integral part of the peptidyltransferase centre.

Acyltransferases↗
PubMed1983

CAP binding proteins associated with the nucleus.

Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in initiation factors as well as in preparations of crude nuclei. It was released from nuclei by washing with buffer of moderate salt concentration. Three high molecular weight cap binding proteins (approximately 120 000, approximately 89 000, approximately 80 000 dalton) were found in the nuclear fraction and were only partly released upon nuclease digestion and high salt extraction.

Affinity Labels↗
PubMed1982

Identification of the cap binding protein of influenza virus.

The presence of a cap binding protein in influenza virus PR8 has recently been demonstrated by photoaffinity labelling with the cap-analogue (gamma [3 2P]-[4-(benzoylphenyl)methylamido]-7-methylguanosine 5'-triphosphate). This paper describes the identification of the labelled protein using two-dimensional gel electrophoresis. The protein is shown to be PB2, the smaller of the two basic P proteins in the polymerase complex.

Carrier Proteins↗
PubMed1982

Identification of the cap-binding protein of two strains of influenza A/FPV.

We have identified the cap-recognizing protein of two strains of influenza A fowl plague virus (FPV) by photoaffinity labelling of virion proteins with a photoreactive analogue of the 5'-methyl cap structure of messenger RNA. The cap-recognizing protein of influenza A/FPV/Rostock/34 is the P2 polypeptide, and that of influenza A/FPV/Dutch/27 (Dobson) is the P3 polypeptide. In each case the cap-recognizing protein is the product of RNA segment 1.

Affinity Labels↗
PubMed1980

Mechanism of translocation: relative arrangement of tRNA and mRNA on the ribosome.

AcPhe-tRNAPhe from yeast can be photocross-linked to poly(U) on Escherichia coli ribosomes. The photoreaction occurs at the wybutine base situated next to the 3' side of the anticodon. The kinetics and efficiency of crosslinking of AcPhe-Phe-tRNA are the same at both the acceptor site and the peptidyl site. Therefore, the orientation of wybutine with respect to the mRNA is similar in both the pretranslocational and posttranslocational states. AcPhe-Phe-tRNA crosslinked at the acceptor site can still be translocated to the peptidyl site, demonstrating that tRNA and mRNA are transported together. The experiments support a model of translocation in which the conformation of the anticodon loop of tRNA is similar in both the peptidyl site and the acceptor site.

Anticodon↗
PubMed1980

Photo-induced crosslinking between phenylalanine transfer RNA and messenger RNA on the Escherichia coli ribosome.

Crosslinking between phenylalanyl transfer RNA from brewer's yeast and poly(U) can be obtained when ribosomal complexes are irradiated at > 300 nm; the ribosomal complexes were formed by enzymatic binding of the tRNA. The crosslinked product was isolated on oligo(dA)-cellulose columns. The experiments suggest that the photo-crosslinking occurs via the wybutine in the anticodon loop of the tRNA. This system can be used to study interactions between tRNA and messenger RNA on the ribosome.

Escherichia coli↗