Biomedical subjects

T Skern

Publications and source records attributed to T Skern.

46 records · Page 3Linked to original sources

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

Low cost apparatus for primer-directed DNA amplification using Thermus aquaticus-DNA polymerase.

An apparatus is described which permits the incubation of samples at three different temperatures in a cyclic fashion. The parts for the incubator are either present in every biochemical laboratory (water baths) or can be easily obtained at a low price (timers and magnetic valves). Thus the new DNA amplification procedure employing Thermus aquaticus-DNA polymerase can be carried out automatically without major investments.

DNA-Directed DNA Polymerase↗

PubMed1989

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis.

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

Amino Acid Sequence↗

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↗

PubMed1989

Typing of human rhinoviruses based on sequence variations in the 5' non-coding region.

Unambiguous assignment of restriction enzyme patterns to six individual serotypes of human rhinovirus was accomplished after amplification of a 380 bp DNA fragment derived from the 5' non-coding region. This was possible even though serotypes 1A and 1B and serotypes 2 and 49 differed only at 10 and 15 positions respectively. The method utilizes the conserved and variable components of this part of the genome and provides the basis for a simple and rapid method for typing of human rhinoviruses.

Base Sequence↗

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

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

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↗