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Poly(A)-containing encephalomyocarditis virus RNA functions as an excellent template for cDNA synthesis in vitro with an RNA-dependent DNA polymerase in the presence of an oligothymidylate primer. Under appropriate conditions, discrete transcripts of increasing chain length were obtained, suitable for sequence analysis. A limited cDNA fragment of 36 nucleotides, primer (dT)10 included, was synthesized when dGTP was omitted from the reaction mixture and its primary structure was elucidated using direct DNA-sequencing methods. The complement corresponds to the 3' end of encephalomyocarditis RNA. The hexanucleotide (5'-3')(A-A-U-A-A-A) found in this sequence is also present in all 3' non-coding regions of poly(A)-containing eukaryotic mRNAs studied until now, in nearly identical positions relative to the poly(A) tail. The possible biological significance of this structural homology is discussed.

The processes of melting and reassociation of double-stranded RNA in dimethylsulfoxide were studied. The addition of a small amount of LiCl results in great results in great reduction of Tm (temperature of melting), whereas the NaCl produces the opposite effect. It is suggested, that LiCl coordinates the molecules of H2O, reducing their activity, and consequently destabilises dsRNA. Mild conditions for melting and reassociation of RNA can be created. It was found that under optimal conditions for dsRNA melting, the degree of strand separation depends on the overall concentration of RNA, irrespective of the type of RNA added to the dsRNA preparation. Reassociation of dsRNA of EMC virus proceeds much faster than that of dsRNA of a related poliovirus. Addition of poly(C) to an annealing mixture slows down the rate of reassociation of EMC dsRNA, producing no effect on the poliovirus dsRNA reassociation. It is suggested that the presence of large poly(C) and poly(G) tracts in the complementary strands of the RNA determines its anomalous fast reassociation. Upon incubation of completely separated strands of EMC dsRNA in a water solution with high ionic strength partially double-stranded aggregates are formed. The formation of aggregates is prevented by addition of poly(A), which indicates that they are produced by "zippening" of a molecule starting with poly(A):poly(U) region. The significance of homopolymeric regions for stability of dsRNA of the EMC virus as well as their role in viral multiplication are discussed.

All four capsid proteins of encephalomyocarditis virus and the precursor to two of these were resolved from purified virions with two-dimensional gel electrophoresis. In addition, all of the known stable virus-specific proteins found in infected cells, but not the primary and intermediate precursor proteins, could be resolved with these techniques.

Encephalomyocarditis (EMC) viral RNA was isolated from purified virus grown in Ehrlich ascites tumor cells. The viral RNA was found to contain polyadenylic acid [poly(A)] regions that were very heterogeneous in length. Chromatography of the EMC viral RNA on oligo(dT)-cellulose columns separated the RNA into three distinct fractions (peaks 1 to 3). Approximately 20% of the EMC viral RNA appeared as peak 1, 40% as peak 2, and 40% as peak 3. The RNA in each fraction appeared to be intact as shown by co-sedimentation with 35S unfractionated EMC viral RNA in SDS-sucrose density gradients. Approximately 95 to 100% of peaks 1 and 3, and 60 to 70% of peak 2, reappeared at the same elution position after rechromatography on oligo(dT)-cellulose. The RNA in peak 1 contained poly(A) with an average length of 16 nucleotides, peak 2 contained poly(A) with an average of 26 nucleotides, and peak 3 contained an average of 74 nucleotides in its poly(A) region. The distribution in the three fractions, as well as the average length of the poly(A) moieties, was relatively unaffected by changes in the cell suspension medium used during infection. Finally, each of the three viral RNA fractions was assayed for biological activity using an infectious RNA assay on L-cell monolayers. Infectivity of the viral RNA was found to increase with poly(A) length, with peak 3 viral RNA being approximately 10 times more infectious than peak 1 viral RNA.

The poly C tract in the RNA of the aphtho- and cardio viruses has been examined in several isolates of foot-and-mouth disease virus (FMDV) and encephalomyocarditis (EMC) virus. The length of the tract is variable, containing 100 to 170 bases in the FMDV isolates and 80 to 250 bases in the EMC virus isolates. Each poly C tract contains c. 10% A and U residues, located at the 5' end, i.e. most of the tract is a continuous run of C residues. The position of the tract on the genome was the same in each of the FMDV isolates, about 400 bases from the 5' end, whereas in the EMC virus isolates it was about 150 bases from the 5' end.

Poly(A) sequences were demonstrated in the RNA of encephalomyocarditis (EMC) virus by affinity chromatography on poly(U)-Sepharose, treatment with RNases, and determination of the ability of the RNA to prime for reverse transcription. The size of the poly(A) sequences was estimated as 10-20 nucleotides in length by determination of the fraction resistant to pancreatic RNases plus T1 RNases, followed by analysis of electrophoretic mobility on polyacrylamide gels and of the AMP-to-adenosine ratio after alkaline hydrolysis. The poly(A) sequences are located at the 3' end of the RNA as shown by mild digestion by snake venom phosphodiesterase.

A low-molecular-weight protein was found to be associated with intact 35S RNA isolated from purified encephalomyocarditis virus. This protein was positively charged at pH 3.5, sensitive to proteinase K treatment, and labeled with either 3H-amino acids or 32Pi.

The polypeptides of encephalomyocarditis, Mouse-Elberfeld and type 5 rhinoviruses behave similarly when chromatographed on calcium phosphate (brushite), each being eluted by a linear phosphate buffer gradient containing sodium dodecyl sulphate in three major peaks, CI, C2 and C3. Analysis of the peaks by polyacrylamide gel electrophoresis suggests that the major capsid polypeptides of these three picornaviruses elute in the order: delta (peak CI), gamma with (peak C2) and alpha (peak C3).

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.

Encephalomyocarditis (EMC) virus RNA contains a covalently bound sequence of polyriboadenylic acid (poly(A). This was determined by two-dimensional gel electrophoresis of complete T1 and pancreatic RNase digests of formamidesucrose gradient-purified RNA and subsequent analysis of the product by alkaline hydrolysis. The size of the EMC virus genomic poly(A) sequence was estimated by formamide-polyacrylamide gel electrophoresis of the RNase-resistant product, or by [3H-]poly(U) hybridization to freshly purified virion RNA, to be, on average, 40 nucleotides in length. The evidence obtained from [3H-]isoniazid labelling and other experiments would indicate that the poly(A) sequence is located at the 3'-terminus of EMC virus RNA.

Encephalomyocarditis (EMC) virus RNA, selected by its affinity for oligo(dT)-cellulose, contains poly(A) of size : (i) about 14 nucleotide residues long, based on the percentage of radioactivity in the RNA resistant to digestion by a mixture of pancreatic and T1 RNases; (ii) about 15 residues long, as measured by the ratio of the amount of terminal adenosine to internal adenylic acid in isolated poly(A); and (III) in the range 12 to 45 residues, the majority of tracts being about 16 to 18 residues long, based upon electrophoretic mobility on polyacrylamide gels using poly(A) molecules of known size as mol. wt. markers. The poly(A) appears to be located at the 3'-terminus of the virus genome since the tract, liberated by digestion with a mixture of pancreatic and T1 RNases, was shown by compositional analysis to contain a non-phosphorylated 3'-terminus and only adenine residues. The size heterogeneity in the poly(A) tracts revealed by gel electrophoresis is also consistent with a terminal location. Comparison of our data for EMC virus with published data for other picornaviruses suggests that the sizes of poly(A) tracts in polio- and Mengovirus RNA have been overestimated; poly(A) tracts in cardioviruses appear to be smaller than those in poliovirus; the minimum size of poly(A) required for full infectivity of picornavirus RNA has also been overestimated; a tract of at least 13 adenine residues long is required for full infectivity of EMC virus RNA.

Deproteinized encephalomyocarditis [32P]RNA, after digestion with a mixture of RNases A, T1 and T2, yields mononucleotides and a labelled compound, which is positively charged at pH 3.5. This product can be digested with pronase and has a close electrophoretic mobility to a protein with a molecular weight of 7000-8000. Covalently bound nucleotide-peptides were isolated from this compound after treatment with RNases and pronase. It was shown that the 5'-terminal uridylic acid is covalently linked with peptides. The phosphodiester bond between uridylic acid and peptides is discussed.

The two-dimensional mapping of tryptic peptides of encephalomyocarditis virus-specific proteins has demonstrated that the amino acid sequence of non-structural polypeptide G constitutes a portion of the molecule of a precursor of capsid proteins, polypeptide A. The results of pulse-chase in vitro translation experiments strongly suggest that polypeptide G corresponds to a C-terminal moiety of polypeptide A. Variations in the polyprotein cleavage maps of different picornaviruses are briefly discussed.

Zinc ion inhibits the posttranslational cleavages of human rhinovirus-1A, encephalomyocarditis virus, and poliovirus polypeptides. Each virus displayed a different susceptibility to zinc. However, in each case the cleavages of the capsid precursor and the cleavages analogous to the C --> D --> E conversion in encephalomyocarditis virus were most sensitive to zinc. Higher concentrations of zinc resulted in the buildup of even larger precursor polypeptides of a size between 106,000 and 214,000 daltons. The sizes of these polypeptides and the relative position of their gene loci on the viral RNA were determined. These data were used to place these polypeptides in the over-all scheme of viral protein processing.

About 80% of the RNA molecules extracted from encephalomyocarditis (EMC) virus were bound by oligo(dT)-cellulose under conditions which bind poly(A) but not poly(C) nor ribosomal RNA. This shows that most EMC virus RNA molecules contain a poly(A) tract. Both bound and unbound fractions contained RNA molecules of apparently the same size when examined by sucrose gradient sedimentation, but the bound fraction contained an adenylic acid-rich segment of about 20 nucleotides long, whereas the unbound RNA did not. The bound RNA had 200 times the specific infectivity of the unbound RNA which suggests that the poly(A) tract present in EMC virus RNA is required for infectivity.

The nature of the 5' terminus of encephalomyocarditis (EMC) virion RNA has been investigated. We have failed to detect any capped products or nucleoside polyphosphates arising upon complete digestion of the RNA with T1, T2, and pancreatic ribonucleases, and it would therefore appear that the 5' terminus of EMC virus RNA is not phosphorylated and not capped with m7G. EMC virions do contain, however, large amounts of all four 5'-monosubstituted nucleoside triphosphates (4.2M pppG; 16.4M pppA; 3.OM pppU and 2.5M pppC), of which at least a proportion (about 15-20%) appear to remain bound to fully denatured RNA in the presence of divalent cations.

Partial purification of the encephalomyocarditis protease synthesized in extracts from rabbit reticulocytes shows that the activity responsible for cleaving coat precursor protein cosediments with a previously unmapped virus-coded protein with an apparent molecular weight of 20,000. Tryptic analysis shows that this protein is derived from protein D, a virus-coded component of the encephalomyocarditis RNA polymerase.

Rapid, detailed, and accurate analysis of the length spectrum of 3' terminal poly(A) in an RNA population can be obtained by 3'-terminal 32P-labelling of RNA with T4 RNA ligase, digestion with ribonucleases T1 and A, and use of gel sequencing methods. Length distributions of 3'-terminal poly(A) of EMC virus, poliovirus, rhinovirus, RAV-61, and CPMV virion RNAs as well as mouse globin mRNA are presented.

The RNA-dependent RNA polymerase (replicase) of encephalomyocarditis (EMC) virus was found to be closely associated with the smooth membranes of infected BHK-21 cells. An RNA-dependent EMC replicase was extracted from the membranes with 0.15% sodium dodecyl sulfate (SDS) and 1,1,2-trichlorotri-fluoroethane (Genetron 113) and further purified by high-salt dextran-polyethylene glycol phase separation, sievorptive chromatography, and glycerol gradient sedimentation. The enzyme does not manifest strict specificity toward EMC RNA template. It can use also Qbeta RNA, rRNA of BHK cells, or poly(C). SDS-polyacrylamide gel electrophoresis of purified EMC replicase labeled with radioactive methionine revealed that, of all the stable EMC proteins, the enzyme contains predominantly the 56,000-dalton (E) polypeptide.

Radio-iodination causes encephalomyocarditis virus to behave aberrantly when examined by affinity chromatography and to sediment rapidly during analysis on sucrose density gradients suggesting that aggregation had taken place. The change in physical properties of the virus occurred whether iodination was carried out with 125I or 131I, with radio-iodine from two different sources, or using two different iodination procedures. The changes were not observed in virus subjected to an iodination procedure in the absence of radio-iodine suggesting that modification of tyrosine residues was involved rather than a side reaction such as amino acid oxidation. It is recommended that caution be exercised when following the fate of radio-iodinated virus in any particular study because its behaviour may not reflect that of normal, non-iodinated virus present.

Encephalomyocarditis virus contains approximately 200 molecules of putrescine, 100 molecules of spermidine, and 40 molecules of spermine which could neutralize 11% of the viral genome. The same polyamines are present in different proportions in the Krebs ascites tumor cell in which the virus was grown.

In poliomyelitis and encephalomyocarditis virus RNAs the relative frequencies of several short oligonucleotide sequences differ substantially from those expected on a random basis. The frequencies of CpGp-containing sequences are low, and the frequency of GpUpApGp is considerably lower than that of GpApUpGp. The relationship of these findings to those obtained from host cell RNAs is descussed.

The cross-reactivity of proteins coded for by the seven serotypes of foot-and-mouth disease virus (FMDV) was assessed by reaction of infected cell lysates with polyclonal and monospecific antisera against the structural and nonstructural proteins of FMDV type A12 strain 119ab. It was shown that the homologous polypeptides from most serotypes are antigenically related. The least cross-reactivity occurred between VP1, VP3, and the protease (3C) of type A12 and South African Territories types 1 and 3. There was also a reduced degree of reactivity of A12 VP1 serum with VP1 from some A subtypes and the other serotypes. Comparison of FMDV proteins with polypeptides from other picornaviruses by a radioimmune binding assay revealed a low level of reactivity of antisera against some A12 polypeptides with encephalomyocarditis virus (EMCV) infected cell lysates but no reactivity with bovine enterovirus type 1 and swine vesicular disease virus infected cells. The same EMCV proteins were immunoprecipitated by the various reactive A12 antisera, but the reaction was abolished if the lysate from EMCV infected cells was denatured prior to immunoprecipitation.

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of (35)S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.