Tuija Pöyry

Molecular Typing of Enteroviruses: Current Status and Future Requirements

SUMMARY Human enteroviruses have traditionally been typed according to neutralization serotype. This procedure is limited by the difficulty in culturing some enteroviruses, the availability of antisera for serotyping, and the cost and technical complexity of serotyping procedures. Furthermore, the impact of information derived from enterovirus serotyping is generally perceived to be low. Enteroviruses are now increasingly being detected by PCR rather than by culture. Classical typing methods will therefore no longer be possible in most instances. An alternative means of enterovirus typing, employing PCR in conjunction with molecular genetic techniques such as nucleotide sequencing or nucleic acid hybridization, would complement molecular diagnosis, may overcome some of the problems associated with serotyping, and would provide additional information regarding the epidemiology and biological properties of enteroviruses. We argue the case for developing a molecular typing system, discuss the genetic basis of such a system, review the literature describing attempts to identify or classify enteroviruses by molecular methods, and suggest ways in which the goal of molecular typing may be realized.

A perspective on mammalian upstream open reading frame function

Abstract Post-transcriptional control makes a major contribution to the overall regulation of gene expression pathway. Within the cytoplasm this is mediated by a combination of regulatory RNA motifs within the 5′ and 3′ untranslated regions of mRNAs and their interacting protein/RNA partners. One of the most common regulatory RNA elements in mammalian transcripts (present in approximately 40% of all mRNAs) are upstream open reading frames (uORFs). However, despite the prevalence of these RNA elements how they function is not well understood. In general, they act to repress translation of the physiological ORF under control conditions, and under certain pathophysiological stresses this repression can be alleviated. It is known that re-initiation following the translation of an uORF is utilised in some situations however there are numerous alternative mechanisms that control the synthesis of a protein whose mRNA contains uORFs. Moreover, the trans-acting factors that are also involved in this process are not well defined. In this review we summarise our current understanding of this area and highlight some common features of these RNA motifs that have been discovered to date.

Rapid molecular evolution of wild type 3 poliovirus during infection in individual hosts

A panel of nine neutralizing monoclonal antibodies was used to analyse the antigenic properties of 188 plaque-purified type 3 poliovirus strains from 17 faecal specimens, derived from eight people during a 2 month observation period. Most poliovirus specimens consisted of a mixture of antigenically distinct variants and the composition of the mixture was found to change between sequential specimens in many individuals, indicating antigenic evolution. Thirty-five strains representing different antigenic patterns were selected for partial sequencing of genomic RNA. Mutations leading to amino acid substitutions, as well as silent mutations, were seen at and close to the known antigenic sites. The frequency of silent mutations was used to estimate the evolutionary potential of the virus. The largest difference in silent changes between strains isolated from one person was 0.8%, which corresponds to a minimum of about 60 mutations per genome within a period of 3 weeks. The observed incidence of silent mutations between isolates from different persons was usually between 0.8 and 2%. These figures agree with the previously reported overall mutation rates of poliovirus, determined by other methods.

Antigenic properties of human parechovirus 1.

Human parechoviruses 1 and 2 (HPEV1 and HPEV2, respectively), formerly known as echoviruses 22 and 23, have been assigned to a novel picornavirus genus on the basis of their distinct molecular and biological properties. To study the immunological characteristics of HPEV1 capsid proteins, antigenic analysis was carried out by a peptide scanning technique, which can be used to identify the immunogenic peptide sequences of a protein. Partially overlapping peptides, representing the capsid of HPEV1, were synthesized using a 12 aa window in a three residue shift and reactivity of rabbit and murine HPEV1 antisera against these peptides were tested. Using this method, an antigenic site in the VP0 polypeptide, recognized by both rabbit and murine antisera, was identified. The sequence of this region was conserved among HPEV1 clinical isolates obtained from Finland and the United States. Antiserum against this peptide region showed neutralizing activity against HPEV1 in cell culture. Because the C-terminal region of HPEV1 VP1 contains a functional RGD motif, the antigenicity of this region was also tested. By using the corresponding peptide antiserum, neutralization of HPEV1 was observed. Cross-neutralization between HPEV1 and coxsackievirus A9, an enterovirus with a similar RGD motif in VP1, was also detected.

Rhinovirus RNA in the Maxillary Sinus Epithelium of Adult Patients with Acute Sinusitis

We used in situ hybridization for the detection of rhinovirus in maxillary sinus biopsy specimens obtained from 14 adult patients with acute sinusitis. In 7 specimens, rhinovirus RNA could be demonstrated in the maxillary sinus epithelium, thereby confirming the etiology of rhinovirus and the clinical suspicion of acute sinusitis.

Poliovirus internal ribosome entry segment structure alterations that specifically affect function in neuronal cells: molecular genetic analysis.

ABSTRACT Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5′ noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5°C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES.

Specific Interaction between Human Parechovirus Nonstructural 2A Protein and Viral RNA

The functional properties of the nonstructural 2A protein are variable among different picornaviruses. The 2A protein of the human parechovirus 1 (HPEV1) has been shown to lack the proteolytic activity found in many other picornaviruses, but no particular function has been identified for HPEV1 2A. To obtain information about the role of HPEV1 2A in the viral life cycle, the protein was expressed in Escherichia coli. A polyclonal antibody was then raised against the protein and employed to investigate its subcellular localization in the infected cells by immunofluorescence microscopy. Typically, a diffuse cytoplasmic staining pattern, concentrated to the perinuclear area, was observed in the infected cells. However, at late stages of infection some infected cells also exhibited diffuse nuclear staining. Viral RNA, visualized by fluorescent in situ hybridization, partly colocalized with 2A in the perinuclear region. Three experimental approaches including Northwestern blot, UV cross-linking, and gel retardation demonstrated that 2A possesses RNA binding activity. Competition experiments with various single-stranded RNA molecules addressed the specificity of 2A binding. These studies revealed that the 2A protein bound RNA corresponding to the 3′-untranslated region (UTR) of the viral genome with highest affinity. At the N- and C-terminal ends of the protein, two regions, necessary for RNA binding, were identified by mutagenesis. In addition, we demonstrated that 2A has affinity to double-stranded RNA containing 3′UTR(+)-3′UTR(-). In conclusion, our experiments showed that HPEV1 2A binds to viral 3′UTR RNA, a feature that could be important for the function of the protein during HPEV1 replication.

Evolution of influenza B/Victoria/2/87-like viruses: occurrence of a genetically conserved virus under conditions of low epidemic activity

Nucleotide sequence analysis of the gene region coding for the HA1 domain of the influenza B virus haemagglutinin was performed on seven field strains isolated during the 1989 to 1990 season and two field strains isolated in 1985 and 1988 in Finland. All isolates were antigenically and genetically related to B/Victoria/2/87 virus and distinct from B/Yamagata/16/88 virus. The three strains isolated at the beginning of the 1989 1990 season in Turku were almost identical to an American variant (B/Texas/37/88-B/Ohio/10/88) of the previous season, whereas the four strains isolated later in the 1989 to 1990 season in Helsinki formed a new group of heterogeneous viruses. The phylogenetic tree compiled suggests that the two branches had evolved from a common origin, probably in 1987.

Activation of a microRNA response in trans reveals a new role for poly(A) in translational repression

Here, we report that the untreated rabbit reticulocyte lysate contains over 300 different endogenous microRNAs together with the major components of the RNA-induced silencing complex and thus can be used as a model in vitro system to study the effects of microRNAs on gene expression. By using this system, we were able to show that microRNA hybridization to its target resulted in a very rapid and strong inhibition of expression that was exerted exclusively at the level of translation initiation with no involvement of transcript degradation or deadenylation. Moreover, we demonstrate that the magnitude of microRNA-induced repression can only be recapitulated in the context of a competitive translating environment. By using a wide spectrum of competitor cellular and viral RNAs, we could further show that competition was not exerted at the level of general components of the translational machinery, but relied exclusively on the presence of the poly(A) tail with virtually no involvement of the cap structure.

Genetic divergence of poliovirus strains isolated in the Karachi region of Pakistan

Seventy-seven wild poliovirus strains isolated from poliomyelitis cases in the Civil Hospital of Karachi in Pakistan in 1989-1993 were selected for partial sequence analysis covering the VP1/2A junction region of the viral genome to study the genetic relationships and epidemiological links between strains. Viral RNA was partially amplified by RT-PCR and sequenced by a solid phase method. Computer analysis revealed genetic divergence of the strains within each serotype. Most of the nucleotide differences between strains were silent: only a few specific amino acid substitutions were seen in the sequenced region. Three genotypes of poliovirus type 1 and two of poliovirus type 3 were co-circulating, while type 2 strains were represented by a single genotype. Representatives of all the genotypes present have been found among previously or concurrently characterized stains isolated elsewhere, but direct epidemiological links were found only in the case of serotype 1. Many of the epidemics caused by poliovirus type 1 in other countries were genetically linked to Pakistan. This study clearly shows the endemic circulation and wide variation of all three poliovirus serotypes in southern Pakistan and indicates the need for more effective vaccination programmes to prevent the further spread of these wild viruses.