Pamela J. Hughes

Analysis of a New Human Parechovirus Allows the Definition of Parechovirus Types and the Identification of RNA Structural Domains

ABSTRACT Human parechoviruses (HPeV), members of the Parechovirus genus of Picornaviridae, are frequent pathogens but have been comparatively poorly studied, and little is known of their diversity, evolution, and molecular biology. To increase the amount of information available, we have analyzed 7 HPeV strains isolated in California between 1973 and 1992. We found that, on the basis of VP1 sequences, these fall into two genetic groups, one of which has not been previously observed, bringing the number of known groups to five. While these correlate partly with the three known serotypes, two members of the HPeV2 serotype belong to different genetic groups. In view of the growing importance of molecular techniques in diagnosis, we suggest that genotype is an important criterion for identifying viruses, and we propose that the genetic groups we have defined should be termed human parechovirus types 1 to 5. Complete nucleotide sequence analysis of two of the Californian isolates, representing two types, confirmed the identification of a new genetic group and suggested a role for recombination in parechovirus evolution. It also allowed the identification of a putative HPeV1 cis-acting replication element, which is located in the VP0 coding region, as well as the refinement of previously predicted 5′ and 3′ untranslated region structures. Thus, the results have significantly improved our understanding of these common pathogens.

Arginine-Glycine-Aspartic Acid Motif Is Critical for Human Parechovirus 1 Entry

ABSTRACT 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.

In Vivo and In Vitro Identification of Structural and Sequence Elements of the Human Parechovirus 5′ Untranslated Region Required for Internal Initiation

ABSTRACT 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.

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.

Terminal RNA Replication Elements in Human Parechovirus 1

ABSTRACT To define structural elements critical for RNA replication in human parechovirus 1 (HPeV1), a replicon with chloramphenicol acetyltransferase as a reporter gene and an infectious virus cDNA clone have been used. It was observed that there are cis-acting signals required for HPeV1 replication located within the 5′-terminal 112 nucleotides of the genome and that these include two terminal stem-loops, SL-A and SL-B, together with a pseudoknot element. Significant disruption of any of these structures impaired both RNA replication and virus growth. In view of the similarity in terminal structures to several picornaviruses, such as cardioviruses and hepatoviruses, the insights generated in this work are of wider significance for understanding picornavirus replication.

The sequence of the coxsackievirus A21 polymerase gene indicates a remarkably close relationship to the polioviruses

SummaryWe have sequenced the coxsackievirus A21 polymerase gene and 3′ noncoding region. The sequence is 98 per cent homologous at the amino acid level to the three poliovirus serotypes. This is comparable to the relationship between polioviruses and indicates a recent evolutionary divergence of the viruses.

A novel method of typing rhinoviruses using the product of a polymerase chain reaction

SummaryAt present rhinoviruses are detected and serotyped in tissue cultures, a slow and laborious process. Previously we have described how the polymerase chain reaction can be used as a rapid method for detecting the presence of a rhinovirus, or enterovirus, in clinical samples without the need to culture. Here we describe a new method which uses the product of the polymerase chain reaction to determine the type of the rhinovirus. The technique is rapid and simple and should eventually greatly facilitate studies on rhinovirus infections.

Rhinovirus detection using probes from the 5′ and 3′ end of the genome

SummaryThis study investigated the abilities of cDNA probes from the 5′ and 3′ ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5′ end of the genomes of HRV-14, 9, and 1B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3′ end probes were specific for the homologous virus. However, along HRV-9 probe detected a large number of serotypes.It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5′ non coding region may overcome this problem.