Correction to: Recommendations for the nomenclature of enteroviruses and rhinoviruses.

Unfortunately, one of the affiliations of author "A. E. Gorbalenya" was missed in original version. The affiliation is updated here.

Recommendations for the nomenclature of enteroviruses and rhinoviruses.

Enteroviruses (EVs) and rhinoviruses (RVs) are significant pathogens of humans and are the subject of intensive clinical and epidemiological research and public health measures, notably in the eradication of poliovirus and in the investigation and control of emerging pathogenic EV types worldwide. EVs and RVs are highly diverse in their antigenic properties, tissue tropism, disease associations and evolutionary relationships, but the latter often conflict with previously developed biologically defined terms, such as "coxsackieviruses", "polioviruses" and "echoviruses", which were used before their genetic interrelationships were understood. This has created widespread formatting problems and inconsistencies in the nomenclature for EV and RV types and species in the literature and public databases. As members of the International Committee for Taxonomy of Viruses (ICTV) Picornaviridae Study Group, we describe the correct use of taxon names for these viruses and have produced a series of recommendations for the nomenclature of EV and RV types and their abbreviations. We believe their adoption will promote greater clarity and consistency in the terminology used in the scientific and medical literature. The recommendations will additionally provide a useful reference guide for journals, other publications and public databases seeking to use standardised terms for the growing multitude of enteroviruses and rhinoviruses described worldwide.

ICTV Virus Taxonomy Profile: Picornaviridae.

The family Picornaviridae comprises small non-enveloped viruses with RNA genomes of 6.7 to 10.1 kb, and contains >30 genera and >75 species. Most of the known picornaviruses infect mammals and birds, but some have also been detected in reptiles, amphibians and fish. Many picornaviruses are important human and veterinary pathogens and may cause diseases of the central nervous system, heart, liver, skin, gastrointestinal tract or upper respiratory tract. Most picornaviruses are transmitted by the faecal-oral or respiratory routes. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Picornaviridae, which is available at www.ictv.global/report/picornaviridae.

Vaccinia virus G1 protein: absence of autocatalytic self-processing.

Vaccinia virus relies on a series of proteolytic cleavage events involving two viral proteins, I7 and G1, to complete its life cycle. Furthermore, G1 itself is cleaved during vaccinia virus infection. However, convincing evidence is lacking to show whether G1 participates in autoproteolysis or is a substrate of another protease. We employed both biochemical and cell-based approaches to investigate G1 cleavage. G1, when expressed in bacteria, rabbit reticulocyte lysates or HeLa cells, was not processed. Moreover, G1 was cleaved in infected cells, but only in the presence of virus late gene expression; cleavage was strongly inhibited by proteasome inhibitors. Thus, these results imply a more complex G1 cleavage reaction than previously envisaged.

Structural and biochemical features distinguish the foot-and-mouth disease virus leader proteinase from other papain-like enzymes.

The structures of the two leader protease (Lpro) variants of foot-and-mouth disease virus known to date were solved using crystals in which molecules were organized as molecular fibers. Such crystals diffract to a resolution of only approximately 3 A. This singular, pseudo-polymeric organization is present in a new Lpro crystal form showing a cubic packing. As molecular fiber formation appeared unrelated to crystallization conditions, we mutated the reactive cysteine 133 residue, which makes a disulfide bridge between adjacent monomers in the fibers, to serine. None of the intermolecular contacts found in the molecular fibers was present in crystals of this variant. Analysis of this Lpro structure, refined at 1.9 A resolution, enables a detailed definition of the active center of the enzyme, including the solvent organization. Assay of Lpro activity on a fluorescent hexapeptide substrate showed that Lpro, in contrast to papain, was highly sensitive to increases in the cation concentration and was active only across a narrow pH range. Examination of the Lpro structure revealed that three aspartate residues near the active site, not present in papain-like enzymes, are probably responsible for these properties.

Prevalence of neutralizing antibodies to Equine rhinitis A and B virus in horses and man.

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.5 A and diffracted to, at most, 5 A resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 X 0.3 X 0.7 mm3, diffracted to approximately 4.0 A resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 X 0.3 X 1.2 mm3 diffracted beyond 3.5 A resolution and were stable to X-ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group I222 (or I2(1)2(1)2(1)) and unit cell dimensions a = 65.9 A, b = 104.3 A, and c = 124.0 A, and appear well suited for high-resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.

Three-dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2.

The crystal structure of the antigen-binding fragment of a monoclonal antibody (8F5) that neutralizes human rhinovirus serotype 2 has been determined by X-ray diffraction studies. Antibody 8F5, obtained by immunization with native HRV2 virions, cross-reacts with peptides of the viral capsid protein VP2, which contribute to the neutralizing immunogenic site B in this serotype. The structure was solved by the molecular replacement method and has been refined to an R-factor of 18.9% at 2.8 A resolution. The elbow angle, relating the variable and constant modules of the molecule is 127 degrees, representing the smallest elbow angle observed so far in an Fab fragment. Furthermore, the charged residues of the epitope can be well accommodated in the antigen-binding site. This is the first crystal structure reported for an antibody directed against an icosahedral virus.

Extremely efficient cleavage of eIF4G by picornaviral proteinases L and 2A in vitro.

Certain picornaviruses encode proteinases which cleave the translation initiation factor eIF4G, a member of the eIF4F complex which recruits mRNA to the 40S ribosomal subunit during initiation of protein synthesis in eukaryotes. We have compared the efficiency of eIF4G cleavage in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (Lpro) or the human rhinovirus 2Apro. Under standard translation conditions, Lpro cleaved 50% of eIF4G within 4 min after initiation of protein synthesis, whereas 2Apro required 15 min. At these times, the molar ratios of proteinase to eIF4G were 1:130 for Lpro and 1:12 for 2Apro, indicating a much more efficient in vitro cleavage than previously observed. The molar ratios are similar to those observed during viral infection in vivo.

Characterization of the active site thiol group of rhinovirus 2A proteinase.

Picornains 2A are cysteine proteases of picornaviruses, a virus family containing several human and animal pathogens. The pH dependencies of the alkylations of picornain 2A of rhinovirus type 2 with iodoacetamide and iodoacetate show two reactive thiol forms, namely the free thiolate ion at high pH and an imidazole assisted thiol group at low pH. Kinetic deuterium isotope effects do not support general base catalysis by the imidazole group, but rather the existence of a catalytically competent thiolate-imidazolium ion-pair. The nature of the ion-pair differs from that of papain, the paradigm of cysteine proteases. The ion-pair is confined to the same, unusually narrow pH range in which the enzyme exhibits catalytic activity.

Sulphation of hirudin in BHK cells.

Hirudin, a thrombin inhibitor of the leech, was expressed in BHK cells; the alpha 1-antitrypsin signal peptide was used to direct secretion into the culture medium. The recombinant hirudin so produced inhibited thrombin and was shown by labelling experiments with [35S]sulphate to have been posttranslationally modified.

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.

Rhinovirus 2A proteinase mediated stimulation of rhinovirus RNA translation is additive to the stimulation effected by cellular RNA binding proteins.

The internal ribosome entry site (IRES) of enteroviruses, and especially human rhinoviruses (HRV), functions very inefficiently in rabbit reticulocyte lysates, but can be stimulated by addition of HeLa cell extracts. Two HeLa cell activities have been identified: the A-type activity is due to polypyrimidine tract binding protein and the B-type to unr. In addition HRV and enterovirus IRES function requires a third RNA binding protein, poly(rC) binding protein 2, but this is present in reticulocyte lysates in non-limiting amounts. IRES activity can also be stimulated by the cleavage of initiation factor eIF4G mediated by either HRV 2A protease, or foot-and-mouth disease virus (FMDV) L protease. This raises the question of whether this stimulation is independent of that effected by the three RNA binding proteins, or whether cleaved eIF4G functionally mimics one or more of these proteins. It is shown here that the stimulation of HRV IRES activity resulting from cleavage of eIF4G is additive with the stimulation effected by HeLa cell A- and B-type activities. It is proposed that the role of the RNA binding proteins is to maintain or attain the appropriate 3-dimensional structure of the IRES RNA element, whereas the function of eIF4G is to deliver the 40S ribosomal subunit to the correct site on the IRES, a function which, for reasons not yet fully understood, is fulfilled more efficiently by the C-terminal cleavage product of eIF4G than by the intact factor.

The structures of picornaviral proteinases.

Picornaviruses are a family of positive-strand RNA viruses the members of which include poliovirus, hepatitis A virus, rhinovirus, foot-and-mouth disease virus and encephalomyocarditis virus. The genetic information contained in the single-stranded, positive sense RNA genome is expressed as a single protein of around 2000 amino acids. This primary product of protein synthesis, designated the polyprotein, is subsequently cleaved into the mature viral proteins by proteinases present within it. The properties of the three defined proteolytic activities present in the picornaviruses are reviewed and the three-dimensional structures of the hepatitis A 3C proteinase and the leader proteinase of foot-and-mouth disease virus as well as a model of the structure of the HRV2 2A proteinase are compared with those of chymotrypsin, papain and streptomyces griseus A proteinase, respectively.

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.

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.

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.

Foot-and-mouth disease virus leader proteinase: involvement of C-terminal residues in self-processing and cleavage of eIF4GI.

The leader proteinase (L(pro)) of foot-and-mouth disease virus frees itself from the nascent polyprotein, cleaving between its own C terminus and the N terminus of VP4 at the sequence Lys-Leu-Lys- downward arrow-Gly-Ala-Gly. Subsequently, the L(pro) impairs protein synthesis from capped mRNAs in the infected cell by processing a host protein, eukaryotic initiation factor 4GI, at the sequence Asn-Leu-Gly- downward arrow-Arg-Thr-Thr. A rabbit reticulocyte lysate system was used to examine the substrate specificity of L(pro) and the relationship of the two cleavage reactions. We show that L(pro) requires a basic residue at one side of the scissile bond to carry out efficient self-processing. This reaction is abrogated when leucine and lysine prior to the cleavage site are substituted by serine and glutamine, respectively. However, the cleavage of eIF4GI is unaffected by the inhibition of self-processing. Removal of the 18-amino acid C-terminal extension of L(pro) slowed eIF4GI cleavage; replacement of the C-terminal extension by unrelated amino acid sequences further delayed this cleavage. Surprisingly, wild-type L(pro) and the C-terminal variants all processed the polyprotein cleavage site in an intermolecular reaction at the same rate. However, when the polyprotein cleavage site was part of the same polypeptide chain as the wild-type Lb(pro), the rate of processing was much more rapid. These experiments strongly suggest that self-processing is an intramolecular reaction.

A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase.

The leader (L) proteinases of aphthoviruses (foot-and-mouth disease viruses) and equine rhinovirus serotypes 1 and 2 cleave themselves from the growing polyprotein. This cleavage occurs intramolecularly between the C terminus of the L proteinases and the N terminus of the subsequent protein VP4. The foot-and-mouth disease virus enzyme has been shown, in addition, to cleave at least one cellular protein, the eukaryotic initiation factor 4G. Mechanistically, inhibitor studies and sequence analysis have been used to classify the L proteinases as papain-like cysteine proteinases. However, sequence identity within the L proteinases themselves is low (between 18% and 32%) and only 14% between the L proteinases and papain. Secondary structure predictions, sequence alignments that take into account the positions of the essential catalytic residues, and structural considerations have been used in this study to investigate more closely the relationships between the L proteinases and papain. In spite of the low sequence identities, the analyses strongly suggest that the L proteinases of foot-and-mouth disease virus and of equine rhinovirus 1 have a similar overall fold to that of papain. Regions in the L proteinases corresponding to all five alpha-helices and seven beta-sheets of papain could be identified. Further comparisons with the proteinase bleomycin hydrolase, which also displays a papain topology in spite of important differences in size and amino acid sequence, support these conclusions and suggest how a C-terminal extension, present in all three L proteinases, and predicted to be an alpha-helix, might enable C-terminal self-processing to occur.