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G Stanway

Publications and source records attributed to G Stanway.

At least 19 recordsLinked to original sources
PubMed2002

A reliable RT-PCR/RFLP assay for the molecular classification of enterovirus reference and wild type strains to either of the two genetic clusters on the basis of 5'-UTR.

The available sequence data from a large segment of the 5'-UTR of all enterovirus reference and wild type strains were analysed in an attempt to discover possible restriction sites for reliable, serotypic identification of wild type isolates. No combination of restriction endonucleases, though, was found to produce serotype-, or group-specific haplotypes. Thirteen restriction enzymes were predicted to differentiate between representatives of the two enterovirus genetic clusters on the basis of 5'-UTR. One of these enzymes, BstOI, was tested in practice for the differentiation of 61 enterovirus reference strains from 56 different serotypes and 82 wild type strains which belong to the two genetic clusters on the basis of 5'-UTR. All the representatives of the two clusters were successfully differentiated with the specific restriction enzyme. Consequently, the simple RFLP-based assay presented here could be used as a very rapid and reliable means for the initial determination of whether a clinical isolate of unknown identity belongs to either of the 'poliovirus-like', or the 'CBV-like' genetic cluster on the basis of 5'-UTR classification of human enteroviruses.

5' Untranslated Regions↗
PubMed2001

Molecular detection and identification of an enterovirus during an outbreak of aseptic meningitis.

Stool samples from sixteen cases of children with meningitis originating from four different and geographically isolated parts of Greece were investigated for enteroviruses. The conventional method of cell culture in four different cell lines was initially used for the isolation of enteroviruses. The results showed a cytopathic effect (CPE) in all cases after two, or even more successive passages in only one cell line (RD), although a less-than-satisfactory CPE was obtained in many cases. Seroneutralization with RIVM mixed hyperimmune antisera followed and the isolates were typed as Coxsackie B viruses. The method of RT-PCR with enterovirus-specific primers targeted to the highly conserved 5'-UTR of the genome was initially used for the detection of enteroviruses from the inoculated cell cultures. A positive RT-PCR result was obtained for all of the clinical samples rapidly and accurately and the isolates were further characterized with the aid of Restriction Fragment Length Polymorphism (RFLP) analysis and Single Strand Conformation Polymorphism analysis (SSCP) of the amplicons. The RFLP analysis showed first of all that the isolates had an identical restriction pattern with Coxsackie B5 Faulkner reference strain with 4 out of 5 restriction enzymes and secondly, both RFLP and SSCP analysis indicated the epidemiological association of the isolates. The speed of the molecular methodology that was used in comparison with the conventional methods and its possible significance for the description of virus evolution and circulation in the populations is discussed.

Animals↗
PubMed2001

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry.

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.

Amino Acid Sequence↗
PubMed2000

The 2A proteins of three diverse picornaviruses are related to each other and to the H-rev107 family of proteins involved in the control of cell proliferation.

The 2A protein appears to be diverse among picornaviruses, in contrast to the other non-structural proteins, which have homologous structures and functions. In enteroviruses and rhinoviruses, 2A is a trypsin-like protease involved in protein processing and in shut-off of host-cell macromolecular synthesis. The aphthovirus and cardiovirus 2A is associated with an unusual processing event at the 2A/2B junction. It is shown here that the 2A protein of several diverse picornaviruses, the human parechoviruses, Aichi virus and avian encephalomyelitis virus, possess previously unrecognized conserved motifs and are likely to have a common function. Moreover, these motifs, a conserved histidine and flanking amino acids, an asparagine-cysteine dipeptide and a putative transmembrane domain, are characteristic of a family of cellular proteins, at least two of which are involved in the control of cell growth. These observations have important implications for an understanding of picornavirus genome structure and evolution, as well as pointing to possible functions of 2A in these viruses.

Amino Acid Sequence↗
PubMed2000

In vivo and in vitro identification of structural and sequence elements of the human parechovirus 5' untranslated region required for internal initiation.

Sequence analysis of the picornavirus echovirus 22 led to its classification as the first member of a new genus, Parechovirus, and renaming as human parechovirus type 1 (HPeV1). Although distinct from other genera in most of the genome, the 5' untranslated region (5'UTR) shows similarities to that of cardio/aphthoviruses in some of its structural domains (A to L). The 5'UTR plays an important role in picornavirus translation initiation and in RNA synthesis. To investigate translation in HPeV1, we engineered an extensive range of mutations (including precise deletions and point mutations) into the 5'UTR. Their effects were studied both by in vitro transcription-translation using a bicistronic construct and by in vivo studies using an infectious, full-length HPeV1 cDNA. These approaches allowed the HPeV1 internal ribosome entry site (IRES) to be mapped. Deletions within the first 298 nucleotides had little impact in the in vitro system, while deletions of nucleotides 298 to 538 had a significant effect. Precise removal of domains H and L (nucleotides 287 to 316 and 664 to 682, respectively) did not significantly reduce translation efficiency in vitro, while domains I, J, and K (nucleotides 327 to 545, 551 to 661, and 614 to 645, respectively) appeared to have much more important roles. Mutation of a phylogenetically conserved GNRA motif (positions 421 to 424) within domain I severely reduced translation. We also confirmed the identity of the AUG (positions 710 to 712) which initiates the open reading frame, the positive identification of which has not been possible previously, as the N terminus of the polyprotein is blocked and not amenable to sequence analysis. This is therefore important in understanding parechovirus genome organization. Mutation of the AUG or an upstream polypyrimidine tract leads to aberrant translation, suggesting they both form part of the parechovirus Yn-Xm-AUG motif. In vivo experiments confirmed the importance of domains I, J, and K, the conserved GNRA motif, polypyrimidine sequences, and AUG, as mutations here were lethal. These features are also important in the IRES elements of cardio/aphthoviruses, but other features reported to be part of the IRES of some members of these genera, notably domains H and L, do not appear to be critical in HPeV1. This adds weight to the idea that there may be functional differences between the IRES elements of different picornaviruses, even when they share significant structural similarity.

5' Untranslated Regions↗
PubMed1999

The crystal structure of coxsackievirus A9: new insights into the uncoating mechanisms of enteroviruses.

BACKGROUND: Coxsackievirus A9 (CAV9), a human pathogen causing symptoms ranging from common colds to fatal infections of the central nervous system, is an icosahedral single-stranded RNA virus that belongs to the genus Enterovirus of the family Picornaviridae. One of the four capsid proteins, VP1, includes the arginine-glycine-aspartate (RGD) motif within its C-terminal extension. This region binds to integrin alpha v beta 3, the only receptor for CAV9 to be conclusively identified to date. RESULTS: The crystal structure of CAV9 in complex with the antiviral compound WIN 51711 has been solved to 2.9 A resolution. The structures of the four capsid proteins, VP1 to VP4, resemble those of other picornaviruses. The antiviral compound is bound in the VP1 hydrophobic pocket, and it is possible that the pocket entrance contains a second WIN 51711 molecule. Continuous electron density for the VP1 N terminus provides a complete picture of the structure close to the fivefold axis. The VP1 C-terminal portion is on the outer surface of the virus and becomes disordered five-residues N-terminal to the RGD motif. CONCLUSIONS: The RGD motif is exposed and flexible in common with other known integrin ligands. Although CAV9 resembles coxsackie B viruses (CBVs), several substitutions in the areas implicated in CBV receptor attachment suggest it may recognise a different receptor. The structure along the fivefold axis provides new information on the uncoating mechanism of enteroviruses. CAV9 might bind a larger natural pocket factor than other picornaviruses, an observation of particular relevance to the design of new antiviral compounds.

Amino Acid Sequence↗
PubMed1999

Parechoviruses.

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5' Untranslated Regions↗
PubMed1998

Sequence, exon-intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans.

The prnA gene codes for a transcriptional activator that mediates proline induction of four other genes involved in proline utilization as a nitrogen and/or carbon source in Aspergillus nidulans. In this paper, we present the genomic and cDNA sequence and the transcript map of prnA. The PrnA protein belongs to the Zn binuclear cluster family of transcriptional activators. The gene shows a striking intron-exon organization, with the putative nuclear localization sequence and the Zn cluster domain in discrete exons. Although the protein sequence presents some interesting similarities with the isofunctional protein of Saccharomyces cerevisiae Put3p, a higher degree of similarity is found with a functionally unrelated protein Thi1 of Schizosaccharomyces pombe. A number of mutations mapping in the prnA gene were sequenced. This comprises a deletion that results in an almost complete loss of the prnA-specific mRNA, a mutation in the putative nuclear localization signal, a proline to leucine mutation in the second loop of the zinc cluster and a cold-sensitive mutation in the so-called 'central region'. Other complete or partial loss of function mutations map in regions of unknown function. We establish that the transcription of the gene is neither self-regulated nor significantly affected by carbon and/or nitrogen metabolite repression.

Aspergillus nidulans↗
PubMed1998

Molecular analysis of human parechovirus type 2 (formerly echovirus 23).

Picornaviruses have been divided into five genera until recently, when a sixth genus, Parechovirus, was defined. Human parechovirus type 1 (HPeV1; formerly echovirus 22) was the first recognized member of this genus and preliminary sequence analysis of echovirus 23 [now renamed human parechovirus type 2 (HPeV2)] suggested that it is also a parechovirus. Here we describe the complete nucleotide and predicted amino acid sequences of HPeV2, which indicate a close relationship to HPeV1 throughout the genome. Sequence covariance in the 5' untranslated region allows a prediction of the secondary structure, which indicates that these parechoviruses have a type 2 internal ribosome entry site, most closely related to that of cardioviruses. Overall, HPeV2 has 87.9% amino acid identity with HPeV1, most divergence being seen in regions of the capsid proteins that probably define antigenic sites. The N-terminal sequence extension to VP3, seen only in parechoviruses, is highly basic in both viruses, but has a variable sequence, suggesting that it does not have a sequence-specific role. There is an RGD motif near the C terminus of VP1, in an analogous location to that in HPeV1 which is believed to be functionally significant. The results confirm that both viruses are parechoviruses and give insights into the molecular features of this genus.

Amino Acid Sequence↗
PubMed1997

An RNA tertiary structure in the 3' untranslated region of enteroviruses is necessary for efficient replication.

RNA tertiary structures, such as pseudoknots, are known to be biologically significant in a number of virus systems. The 3' untranslated regions of the RNA genomes of all members of the Enterovirus genus of Picornaviridae exhibit a potential, pseudoknot-like, tertiary structure interaction of an unusual type. This is formed by base pairing between loop regions of two secondary structure domains. It is distinct from a potential, conventional pseudoknot, studied previously in poliovirus, which is less conserved phylogenetically. We have analyzed the tertiary structure feature in one enterovirus, coxsackievirus A9, using specific mutagenesis. A double mutant in which the potential interaction was destroyed was nonviable, and viability was restored by introducing compensating mutations, predicted to allow the interaction to reform. Phenotypic pseudorevertants of virus mutants, having mutations designed to disrupt the interaction, were all found to have acquired nucleotide changes which restored the potential interaction. Analysis of one mutant containing a single-base mutation indicated a greatly increased temperature sensitivity due to a step early in replication. The results show that, in addition to secondary structures, tertiary RNA structural interactions can play an important role in the biology of picornaviruses.

Base Sequence↗
PubMed1996

Genetic and phylogenetic clustering of enteroviruses.

Genetic and phylogenetic analysis of enteroviruses showed that in the 5'NCR enteroviruses formed three clusters: polioviruses (PVs), coxsackievirus A type 21 (CAV21), CAV24 and enterovirus type 70 (ENV70) formed one cluster; coxsackievirus B isolates (CBVs), CAV9, CAV16, ENV71, echovirus type 11 (EV11), EV12 and all partially sequenced echoviruses and swine vesicular disease virus (SVDV) belonged to another cluster and bovine enteroviruses (BEVs) formed the third cluster. In the capsid coding region five clusters were seen: PVs, CAV21 and CAV24 formed one cluster (PV-like); ENV70 formed a cluster of its own; all CBVs, CAV9, EV11, EV12 and SVDV formed the third cluster (CBV-like); CAV16, CAV2 and ENV71 belonged to the fourth cluster (CAV16-like) and BEVs formed their own cluster (BEV-like). In the 3'NCR the same clusters were seen as in the coding region suggesting a close association of the 3'NCR with viral proteins while the cellular environment may be more important in the evolution of the 5'NCR. Secondary structures were predicted in the 3'NCR, which showed two different patterns among the five clusters. A potential pseudoknot region common in all five clusters was identified. Although the BEV-like viruses formed a separate cluster in all genomic regions, in the coding region they seem to be phylogenetically related to the CAV16-like viruses.

Animals↗
PubMed1995

The genome of echovirus 11.

Echoviruses are the largest enterovirus subgroup consisting of 32 serotypes. They are common human pathogens causing, for example, meningitis, encephalitis and exanthema, but in spite of their clinical importance, relatively little is known about their biology. To illuminate the molecular characteristics of echoviruses, we have completed the genomic sequence of serotype 11. The RNA genome is 7438 nucleotides in length and it codes for a 2195 amino acid long polyprotein. When compared to other sequenced enteroviruses, echovirus 11 (EV11) shows remarkable similarity with coxsackie B viruses (CBVs) and coxsackievirus A9 (CAV9). On the basis of amino acid sequence homology in the capsid region, CAV9 is the virus most closely related to EV11. These two viruses have an apparent insertion sequence located at the C-terminus of the VP1 polypeptide. EV11, however, lacks the RGD motif found in the corresponding region of CAV9. The organization of the 5' end noncoding region resembles that of other enteroviruses, but contains a 12 nucleotides long poly-U stretch not seen in any other enterovirus sequenced to date.

Amino Acid Sequence↗
PubMed1995

Molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis.

A subset of infants dying suddenly and unexpectedly have myocarditis with or without pericarditis found at autopsy. To address whether viruses known to cause infantile myocarditis and pericarditis might be present in such infants, we examined myocardium, liver and skeletal muscle for the presence of genomic sequences from adenovirus, cytomegalovirus, enterovirus and echovirus 22/23 in infants enrolled in a comprehensive evaluation protocol. We studied eight infants who died suddenly and unexpectedly with histologic evidence of myocarditis and/or pericarditis detected at postmortem examination. One infant with myocarditis and pericarditis had adenovirus genome detected in the myocardium. In an additional infant with severe pericarditis alone, enterovirus genome was detected in the liver. Although echovirus 22/23 has been associated with myopericarditis in young infants, no previous studies have used molecular methods to search for the genomic sequences of these viruses in clinical samples. No echovirus 22/23 genome was detected in the patients reported here. The significance of enterovirus and adenovirus genome in the tissues of two patients dying suddenly and unexpectedly remains speculative but raises the possibility that pathogenic viruses may cause little or no clinical symptoms and yet be contributory to sudden death in young infants.

Adenoviridae↗
PubMed1995

Molecular relationships between 21 human rhinovirus serotypes.

We have analysed, by PCR using consensus primers followed by sequencing, 12 human rhinoviruses (HRVs) in a genomic region including that corresponding to the immunogenic site NIm-II. Together with published information, 21 sequences are available for comparison. In the region analysed, which encodes 112 amino acids, the majority (18) of the serotypes exhibited at least 70% amino acid identity to one another and some serotypes are very closely related. These include HRV-36, -58 and -89, known to exhibit antigenic cross reactivity, which were shown to differ at only three amino acid positions. Three serotypes, HRV-3, -14 and -72, share at least 84% identity with one another but are less than 66% identical to the majority group. Interestingly, membership of these two molecular clusters correlates with the groupings determined by sensitivity to antivirus drugs, suggesting that they reflect a fundamental division of HRVs. In contrast, there is no correlation with receptor grouping, since the majority group contains members belonging to both HRV receptor groups.

Amino Acid Sequence↗
PubMed1995

The coxsackievirus A9 RGD motif is not essential for virus viability.

An RGD (arginine-glycine-aspartic acid) motif in coxsackievirus A9 has been implicated in internalization through an interaction with the integrin alpha v beta 3. We have produced a number of virus mutants, lacking the motif, which have a small-plaque phenotype in LLC-Mk2 and A-Vero cells and are phenotypically normal in RD cells. Substitution of flanking amino acids also affected plaque size. The results suggest that interaction between the RGD motif and alpha v beta 3 is not critical for virus viability in the cell lines tested and therefore that alternative regions of the CAV-9 capsid are involved in internalization.

Amino Acid Sequence↗
PubMed1994

Molecular analysis of coxsackievirus A16 reveals a new genetic group of enteroviruses.

Coxsackievirus A16 (CAV16) a member of the Enterovirus genus of Picornaviridae, is associated with hand-foot-and-mouth disease, a febrile papulovesicular rash of childhood. We have determined the complete nucleotide sequence of the genome of the prototype strain of CAV16 which consists of 7413 nucleotides plus the poly(A) tail. Alignment of the sequence with the previously studied enteroviruses showed that the genome organization is typical for a member of this virus genus. However, the predicted amino acid sequence of individual CAV16 proteins differed from those of all previously sequenced enteroviruses by 25-62%. The genomic sequence of CAV2 in the capsid and 2A polypeptide regions was also determined. It was found to differ from that of CAV16 by no more than 5-43%. The partial nucleotide sequence of enterovirus 71 in the VP1-2A region suggested that it is also closely related to CAV16. The results indicate that CAV16, CAV2, and enterovirus 71 represent a distinct genetic group of enteroviruses.

Amino Acid Sequence↗