Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo.

The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

An electroblotting technique for assessing the integrity of the major immunogenic protein in foot-and-mouth disease virus vaccines.

A method has been developed whereby VP1, the major immunogenic protein of foot-and-mouth disease virus can be detected after electroblotting on nitrocellulose paper. Proteins can be examined in unfractionated virus harvests and after formulation as aluminium hydroxide-adjuvanted vaccines. The limit of detection is approximately 10 ng of VP1 and up to 20 samples can be analysed simultaneously. The technique allows the integrity of VP1 to be examined in fully formulated vaccines.

Encephalomyocarditis virus antibodies in sera from apparently normal pigs.

Antibodies which neutralise and precipitate encephalomyocarditis virus habe been found in the serum of more than 28 per cent of normal pigs in Britain. Neutralising activity was found in association with several size classes of antibody.

Analysis of the terminal sequences of the genome segments of four orbiviruses.

The RNA sequences of the terminal regions of the genome segments of 4 different orbiviruses were analysed. In 3 of these [bluetongue virus (BTV) types 1 and 20 and Ibaraki virus, a member of the epizootic hemorrhagic disease (EHD) serogroup], 1 strand of all of the genome segments analysed ends in 3'CAAUUU...5' while the other strand ends in 3'CAUUCA...5'. These conserved sequences are identical to those reported for BTV types 10 and 11. The 3' terminal sequences of segments 3 and 10 of the BTV 1 genome (including the conserved regions) were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all of the genome segments of these viruses) that the dsRNA molecules are fully base-paired end to end. Using in vitro synthesised mRNA of BTV 1 in annealing experiments with the separated ssRNAs from the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA...5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU...5' is of the opposite (-ve) polarity. The 4th virus analysed (Tilligerry, a member of the Eubenangee serogroup) only had 5 conserved bases at the 3' termini of 1 strand of each genome segment (3'CAU-CA...5') and 3 conserved bases at the 3' termini of the other strand (3'CA--U...5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments, particularly between the different BTV types. There was little evidence however for absolute conservation of "segment specific" sequences in these regions of the RNA, as reported for rotaviruses. The percentage of sequence homology found in the terminal regions of the genome segments between members of the BTV, EHD and Eubenangee serogroups examined correlates with the serological interrelationships between these groups of viruses. The conserved terminal sequences and sequence homology in these regions of the RNA are discussed as to their role in virus replication and their significance to the reassortment of genome segments between different viruses during dual infections.

Analysis of the terminal sequences of the genome segments of four orbiviruses.

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.

Processing and assembly of foot-and-mouth disease virus proteins using subgenomic RNA.

Recombinant DNA clones were constructed in order to study the mechanisms of proteolytic processing and assembly in foot-and-mouth disease virus (FMDV). RNA transcripts from these clones were synthesized using SP6 polymerase and translated in rabbit reticulocyte lysates. Efficient translation occurred in the absence of all 5' untranslated sequences and processing of the structural proteins occurred in the presence of functional 3C protease which can function in trans. The specificity of 3C protease activity is not limited to Glu-Gly bonds. Translation of correctly processed structural proteins leads to assembly of subviral structures resembling 'empty' particles. Further studies on the processing of the FMDV genome show that the primary cleavage (P1-P2) is mediated neither by 3C nor the second FMDV protease L. Preliminary evidence suggests that an initial very rapid cleavage occurs between 2A and 2B with subsequent cleavage of the P1/2A junction probably being carried out by 3C.

A comparative chemical and serological study of the full and empty particles of foot-and mouth disease virus.

The chemical and serological properties of the full, naturally occurring empty and artificially produced empty particles of foot-and-mouth disease virus, serotype A(subtype 10, strain 16) have been studies. The full 146S particles comprised the virus RNA, three polypeptides (VP1 to VP3) mol. wt. about 30 X 10-3, one polypeptide (VP4) mol. wt. about 13-5 X 10-3, and a small amount of a polypeptide (VPo) mol. wt. about 43 X 10-3. The naturally occurring 75S empty particles contained no RNA and much less VP1 and VP4 than were found in the fall particles. However they contained a much greater proportion of VPo than the full particles. Dialysis of purified full particles against tris-EDTA, pH 7-6, produced artificial 75S empty particles which contained only a small amount of RNA and no VP4; otherwise the polypeptide composition was similar to that of the full particles. Immunological and serological tests showed that the full particles were antigenically similar to the naturally occurring empty particles but distinct from the artificial empty particles. The latter particles, however, had serological properties similar to those of the 12S protein subunit of the virus. Both the full and naturally occurring empty particles attached efficiently to susceptible cells, whereas the artificial empty particles attached only to a limited extent. The results are related to the function of the individual polypeptides of the virus particle and compared with published work on other picornaviruses.

Examination of poliovaccines for the presence of SV40 sequences.

Batches of monovalent oral poliovirus vaccine bulks previously screened for infectious SV40 by cell culture methods and used in the United Kingdom from 1971-96 in trivalent oral poliovirus vaccine have now been examined by a newly developed PCR for the presence of SV40 sequences. Up to April 1997, over 190 batches have been examined. SV40 sequences were not detected in any of the vaccines. This provides additional assurance that oral poliovirus vaccines used in the U.K. from 1971 are free of SV40 contamination.

Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode.

The purified genomic dsRNA of bluetongue virus type 1 (BTV 1) was separated into 10 size classes by polyacrylamide gel electrophoresis. These genome segments were recovered individually from the gel, denatured with methylmercuric hydroxide and translated in vitro in a rabbit reticulocyte lysate system. The virus-specific proteins synthesised in vitro were compared by polyacrylamide gel electrophoresis with proteins from purified virus particles and cores and with virus-specified proteins synthesized in BHK 21 cells (in vivo). The identify of those BTV 1 proteins synthesized in vivo and in vitro which showed similar electrophoretic mobilities, was confirmed by electrophoretic analysis of their partial protease digests. The results of these experiments have allowed the assignment of each of the genome segments of BTV 1 to the protein(s) which it encodes and consequently to the structural and nonstructural proteins found in the infected cell.

The nature and spatial distribution of amino acid substitutions conferring resistance to neutralizing monoclonal antibodies in human rhinovirus type 2.

A total of 38 neutralization escape mutant viruses have been selected from a cloned stock of human rhinovirus serotype 2 (HRV-2), using either of two monoclonal antibodies (MAbs) which recognize overlapping epitopes as judged by competition binding. The mutant viruses were analysed for their sensitivity to a panel of antiviral MAbs by antibody binding and virus neutralization assays. The position and nature of the selected mutations was determined by sequencing of the virus RNAs, and the location of the substituted amino acids on the three-dimensional structure of the virus predicted from the co-ordinates determined for the closely related HRV-1A. Escape from neutralization could be attributed to single amino acid substitutions in all but one case, which had a deletion of four amino acids. In all cases in which the same mutation was found more than once, these mutations were transitions. The ratio of transition to transversion mutations was about 5:1 overall or about 1.7:1 if only unique substitutions are considered. Each antibody selected for a discrete cluster of mutations and the area of these clusters was considerably less than that determined to be in contact with antibodies from X-ray crystallographic analyses of antibody/protein complexes. One mutation did not occur within the cluster of others selected with the same antibody. This substitution occurred at the base of a small loop and may cause conformational changes at the virus surface.

Parameters influencing the attachment of hepatitis A virus to a variety of continuous cell lines.

We have investigated the interactions of purified radiolabelled hepatitis A virus (HAV) with a variety of continuous cell lines. Virus labelled either in vitro with radiolabelled iodine or in vivo with radiolabelled uridine bound to cells with similar efficiency. Attachment to BS-C-1 cells was calcium ion-dependent and this correlated with infectivity assay results. The cell tropism of HAV attachment was examined using cell suspensions and confluent cell monolayers at both 4 degrees C and 37 degrees C. The maximum level of attachment was observed at 4 degrees C with cells in suspension, but was severely inhibited by 2% foetal calf serum; these results again correlated with infectivity assays. The components of serum which inhibit attachment have been characterized by gel filtration chromatography, sucrose density gradient analysis, immunoprecipitation and Western blotting. The data show that such components are of high Mr and that the serum glycoprotein, alpha 2-macroglobulin, can partly mimic the inhibitory effect of whole serum.

Removal of the genome-linked protein of foot-and-mouth disease virus by rabbit reticulocyte lysate.

Rabbit reticulocyte lysate cleaves the genome-linked protein VPg from foot-and-mouth disease virus (FMDV) RNA. This activity could be reliably monitored since removal of the protein resulted in a change in migration in polyacrylamide gels of the small specific 5' and fragment of the RNA (S fragment). The unlinking activity cleaved the bond between the tyrosine residue of VPg and the RNA to leave a 5' phosphate on the RNA. The 5' sequence of the RNA from which VPg had been removed by rabbit reticulocyte lysate was the same as that of FMDV mRNA isolated from infected cells. VPg released from the RNA was rapidly degraded by the rabbit reticulocyte lysate to material which eluted with the inclusion volume of a Sepharose 6B column and partitioned to the aqueous phase during phenol extraction. The unlinking activity was inhibited by heating the lysate to 56 degrees C, by sodium dodecyl sulfate (SDS), EDTA, and Zn2+ ions but was unaffected by reducing agents, a translation inhibitor, and a number of protease and RNase inhibitors.

Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA.

An mRNA-dependent reticulocyte lysate has been used to translate foot-and-mouth disease virus RNA in vitro. Polypeptides P16, P20a, and P88, which have been shown to be derived from the 5' end of the RNA by pactamycin mapping experiments with infected cells, were preferentially synthesized in vitro. Removal of VPg, the small protein covalently linked to the 5' end of the genome RNA, had no effect on the translation of the RNA. The two RNA fragments (L and S) produced by specific digestion of the polycytidylic acid [poly(C)] tract with RNase H were also translated in vitro. The L fragment, consisting of RNA to the 3' side of the poly(C) tract and including the polyadenylic acid [poly(A)] tract, directed the synthesis of the same products as those made by full-length RNA. However, no small defined products were produced when the S fragment, which contains the 5' end of the RNA, was translated. These results show that the major initiation site for protein synthesis on foot-and-mouth disease virus RNA is to the 3' side of the poly(C) tract. Furthermore, the use of N-formyl [35S]methionine tRNAfMet as a label for the initiation peptides showed that the major polypeptide labeled in lysates primed with both full-length RNA and the L fragment was P16, i.e., the protein nearest the initiation site for translation as deduced from pactamycin mapping experiments. Fragments of RNA were also translated in vitro. Those containing the poly(C) tract gave products similar to those produced when full-length RNA was translated. The polypeptides synthesized when fragments containing the poly(A) tract were used, however, did not resemble those made from full-length RNA.

Heterogeneity of the genome-linked protein of foot-and-mouth disease virus.

The genome-linked protein of foot-and-mouth disease virus was examined by electrofocusing in polyacrylamide gels. Two proteins of different charge and amino acid composition were found. The tryptic peptide maps of the proteins were dissimilar. The possible relationship between the two proteins is discussed.

A high density component in several vertebrate enteroviruses.

In addition to the major infective component, which bands at a density of 1:34 g/ml in caesium chloride ("light component"), a component with a density of 1:44 g/ml ("heavy component") has been found in harvests of poliovirus (type I), Coxsackie B5 virus, a bovine enterovirus (VG-5-27) and swine vesicular disease virus (SVDV). With SVDV about 98% of the infectivity equilibrated at 1 . 34 g/ml but approx. 2% was present as a peak at 1 . 44 g/ml. The morphology of the two forms was similar but the heavy component had a smaller diameter (28 nm) than the light component (30 nm). No inter-conversion of the two forms was observed on re-cycling in fresh caesium chloride gradients and the two components had the same proportions of RNA and protein and the same polypeptide composition. Each component gave a similar proportion of the light and heavy forms on replication, but the light component had a specific infectivity about fourfold higher than that of the heavy component and was also much more efficient in eliciting the formation of neutralizing antibodies in guinea pigs. Although these results suggest that the two particles are alternative stable configurations of the virus, iodination failed to reveal any differences in the extent or pattern of labelling of the polypeptides in the two forms.

A re-appraisal of the biochemical map of foot-and-mouth disease virus RNA.

The proteins induced by infection of BHK 21 cells with foot-and-mouth disease virus have been compared by tryptic peptide analysis. The results indicate that there are three primary products 5'--P88, P52, P100--3'. The polypeptide P56, which we considered previously to be a primary product, is derived from the region of the genome that codes for P100. The results indicate that there are alternative cleavage pathways of P100, the polypeptide coded for by the 3' end of the genome.

All foot and mouth disease virus serotypes initiate protein synthesis at two separate AUGs.

Translation of the foot and mouth disease virus genome in vitro and in vivo indicated that all seven serotypes initiate protein synthesis at two separate AUGs. Sequence analysis of the region surrounding these AUGs has shown that the efficiency with which the initiating AUG is recognized is dependent on the flanking nucleotides. However, in vitro, the major factor determining which AUG is used is the concentration of Mg2+.

Biochemical mapping of the foot-and-mouth disease virus genome.

Four primary cleavage products, mol. wt. 10(3) X 100, 88, 56 and 52 (P100, P85, P56 and P52 respectively) are present in BHK 2I cells infected with foot-and-mouth disease virus (FMDV). However, no precursor polyprotein equal to the sum of their mol. wt. was detected, even when amino acid analogues and proteolytic enzyme inhibitors were used. Three of the primary products were shown to cleave to smaller polypeptides, including the capsid polypeptides of the virus. Polypeptide P88, which was shown to be the precursor of the capsid polypeptides, is translated from the gene located at the 5'-end of the genome. The order of the structural polypeptides, determined by the use of emetine, is VP4, VP2, VP3, VP1. The order of the remaining primary cleavage products is P52, P56 and P100. P56 is a stable product, identical with the virus infection associated (VIA) antigen found in virus harvests. The function of the other two products P52 and P100 is not known. EMDV thus differs from other picornaviruses in that there is an extra primary cleavage product, apparently resulting from translation of more of the virus genome.

Immunogenic and cell attachment sites of FMDV: further evidence for their location in a single capsid polypeptide.

Chymotrypsin cleaves only one of the four major polypeptides of foot-and-mouth disease virus (FMDV serotype O) in situ. This polypeptide (VP1, mol. wt. 29 X 10(3) was first cleaved into fragments of mol. wt. 20 and 9 X 10(3) and further cleavage could be prevented by the addition of a large excess of bovine serum albumin. The infectivity of the virus particles at this stage was the same as that of the intact virus although the rate of attachment to BHK 21 cells was slower and the immunogenic activity was reduced. If hydrolysis was allowed to continue, VP1 was cleaved into fragments with mol. wt. 18 and less than 9 X 10(3), similar to those obtained with trypsin and the virus particles then had a greatly reduced infectivity and a lower immunogenicity. Treatment of strains from five other serotypes of the virus with the two enzymes cleaved only VP1 in each instance and there was a corresponding loss of infectivity. The results are discussed in relation to the location and biological activity of the virus polypeptides.

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.