Article

Ribonucleic Acid Content of Encephalomyocarditis Virus

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Abstract

SUMMARY The RNA content of encephalomyocarditis virus was found to be 31-7 %, a figure obtained by combining results from nitrogen and phosphorus determinations which gave 32.0 ~o, from extinction measurements at 260 nm. on perchloric acid hydrolysates which gave 31.3% and from orcinol determinations which gave 31"8 %. Specific extinction coefficients obtained were E~n. at 260 nm.=77"4 for the virus in o.I M-KCI+o.o2 M-phosphate buffer, pH 8% and EI~;. at 260 nm. =221 for its RNA in o-I M-tris+ HC1, pH 7"2.

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... The group the murine hepatitis viruses (MHV), turincludes strains OC-43, 2293, and 692 of key bluecomb disease virus, and the avian man, all of which cause common colds but infectious bronchitis viruses (IBV) (Bradare antigenically distinct (Kapikian et al., burne, 1970;McIntosh, 1974). Few biologi-1973), possibly other strains associated cal or biophysical data are available for with gastroenteritis, hepatitis, or nephrimost of these viruses, so that, as a group, tis in man (Holmes et al., 1970; Zuckerman the coronaviruses are not well understood et al., 1970;Wright, 1972;Ackermann et and cannot be adequately compared with al., 1974;Apostolov et al., 1975;Caul and other virus groups. Clark, 1975) (Sheboldov et al., 1973;Pokorny et al., 1975). ...
... at 256 nm. Values were corrected for light scattering by the 360-320 nm baseline subtraction method (Burness, 1970). ...
Article
Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 109.0–109.5 TCID50/ml and plaque titers from 1010.2–1010.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E1cm1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S20,v0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.
... If this assumption is correct, it follows that the ratio of the 32P-radioactivity in the 5'-nucleo- tide to that in the 3'-nucleotide is a reflection of the specific radioactivity of the 5'-nucleo- tide. The assumption that the 32P-radioactivity in 3'-nucleotides is representative of the number of each species of nucleotide present appears justified since the result given above for T2 RNase hydrolysates of EMC virus RNA is similar to the base composition obtained by direct determination of the E260 of the separated components (Burness, 1970). However, due to the non-random nature of a homopolymer tract, the same assumption may not hold unless the specific radioactivity of the 5'-nucleotide comprising the tract is about the same as the average for all nucleotides in the RNA. ...
Article
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.
Chapter
The picornavirus group numbers over 200 distinct serotypes, isolated from a wide variety of human and animal sources, and it is likely that many others exist in nature (1), as Vet unrecognised. Various sub-classifications of this large range of viruses have been proposed (2, 3). The most recent, and probably the most generally satisfactory classification (4, 5) arranges the picornaviruses into five genera (a) Cardioviruses, including encephalomyocarditis (EMG) and mengoviruses; (b) Human Rhinoviruses; (c) Equine Rhinoviruses; (d) Foot-and-Mouth Disease viruses; (e) Enteroviruses, including polio, Coxsackie viruses etc. The members of these different genera display differences in virus particle stability in the pH range 3–7, and in their particle density in caesium chloride, as well as in RNA base composition (see chapters 1 and 3).
Article
Preparations of purified virions of human rhinoviruses type 2 and type 14 and of an equine rhinovirus are each homogeneous by the criteria of electron microscopy and rate-zonal and isopycnic gradient centrifugation. The rhinoviruses differ from each other in intrinsic infectivity and buoyant density in CsCl, while their sedimentation rates in sucrose gradients are similar, about 4% less than poliovirus. Purification in CsCl gradients produces an artifactual increase of 7% in the rhinovirus sedimentation constant, which is reversed with time after removal of CsCl from the preparation. Rhinovirus types differ in part in their four size classes of major polypeptides: VP1, VP2, VP3, and VP4. Crude preparations also contain empty capsids which sediment at half the rate of the virions, contain VP1 and VP3 and polypeptides larger than any in the virions (VPO). There is another naturally occurring component, the slow component, which sediments 90% as fast as intact virions. The slow component gives rise to a dense component in CsCl gradients which is 0.04–0.05 g/ml more dense than intact virions. The dense component contains RNA, VP1, and VP3. On the basis of indirect evidence, only the smallest polypeptide, VP4, is missing from the slow component, and VP2 is lost during CsCl centrifugation. Components which resemble the slow and dense components can be produced from purified virions in vitro by careful acidification.
Article
The synthesis of rhinovirus type 20 specific RNA in infected HeLa cells was examined in the presence of actinomycin D and uridine-5-3H. The virus induced in the infected cells the synthesis of two major components of RNA sedimenting at 32 S and 18 S, respectively, and a heterogeneous component with a sedimentation coefficient of 24–26 S. Treatment of virus-specific RNA components with ribonuclease resulted in the complete degradation of 32 S and 24–26 S components, while the 18 S component showed partial resistance to ribonuclease. Poliovirus type 1 under identical conditions induced the synthesis of two components of RNA sedimenting at 35 S and 18 S, respectively. Rhinovirus specific RNA in infected HeLa cells was first detectable between 4 and 5 hr postinfection at about the same time that infectivity first appeared intracellularly. The maximal synthesis of virus specific RNA took place between 6 and 7 hr postinfection. The purification of rhinovirus in cesium chloride resulted in the loss of infectivity and appearance of phosphotungstic acid-penetrable virus particles. RNA extracted from the purified virions sedimented with a major peak at 32 S but with a heterogeneous skewed type of sedimentation along the trailing edge.
Article
SUMMARY A study was made of the virus-specific proteins present in Krebs 2 ascites cells infected with encephalomyocarditis (EMC) virus. About I5 virus-specific poly- peptides, tool. wt. varying from 13 ooo to I47OOO, were detected by acrylamide gel analysis of extracts from infected cells briefly pulsed with (l~C)-amino acids. Three of the components were probably capsid proteins, while the fourth capsid protein (73oo mol. wt.) was not detected in the infected cell and possibly arose after maturation by cleavage of the largest capsid protein. Pulse-chase experiments showed that only two medium-sized (mol. wt. 36000 and 54ooo) and some smaller (tool. wt. i350o to 1700o) non-capsid proteins were stable in vivo; the remaining larger non-capsid proteins lost radioactivity during the chase with cold amino acids, and were probably intermediates in the formation of smaller polypeptides by some post-translational cleavage process. Various methods were employed to block this cleavage process, utilizing elevated temperatures, protease inhibitors and amino acid analogues.
Article
Electron microscopy of the purified replicative form RNA of encephalomyocarditis virus revealed three categories of molecules: linear (2.33 ± 0.21 μm long), circular (2.42 ± 0.13 μm) and compact entities of unknown structure. The circular molecules were either in an extended form or had several superhelical twists. At least two components could be discerned in preparations of replicative form RNA by analytical velocity band centrifugation in 3 m-CsCl; the uncorrected sedimentation coefficients of these components were 24.7 s and 17.4 s, respectively. The effect of an intercalating dye, ethidium bromide, on the sedimentation rate of these components was studied. The plot of the sedimentation coefficient of the fastest component versus ethidium bromide concentration was shown to be qualitatively similar to that reported for supercoiled covalently closed circular DNA, i.e. it had a minimum at a certain concentration of ethidium bromide. This fact suggests that a portion of the replicative form RNA is probably represented by closed circular double-stranded molecules.Thermal denaturation-renaturation experiments suggested that both chains, at least in a portion of the isolated replicative form RNA molecules, were connected to each other in such a way that complete separation could not be achieved under the denaturing conditions. The binding between chains appeared to be due, at least to a large extent, to some kind of cross-linking, rather than to the existence of covalently closed circular molecules, since the fragmented linear molecules (fragmented by hydrodynamic shearing) possessed essentially the same ability to renature upon rapid cooling as did non-fragmented molecules of the replicative form RNA.
Article
SUMMARY The possibility that the haemagglutinin of encephalomyocarditis (EMC) virus might be a glycoprotein was investigated by looking for sugars and amino sugars in purified EMC virus particles grown in Krebs ascites tumour cells in vitro in the presence of isotopicatly labelled glucosamine, acetyl glucosamine, galactosamine, mannosamine or fucose. With either isotopically labelled glucosamine or galactos- amine the virus became significantly radioactive, whereas radioactivity was low in virus grown in the presence of mannosamine, or fucose, possibly because the latter was poorly taken up by the cells; acetyl glucosamine was completely excluded by Krebs cells, so its ability to be incorporated into virus could not be tested. Radioactivity derived from glucosamine remained firmly attached to the purified virus through both rate-zonal and equilibrium density centrifugation and chromato- graphy on calcium phosphate. This radioactivity did not appear due to simple adsorption of either glucosamine or (after synthesis) acetyl glucosamine, or to contamination of the preparation with infected host cell membranes. Estimates of the number of glucosamine molecules incorporated into the virus had values which varied with the position of the isotope in the glucosamine added, suggesting that it was not incorporated unchanged. Polyacrylamide gel electrophoresis of disrupted whole virus showed radio- activity derived from glucosamine to be present in all virus polypeptides, indicating that glucosamine was perhaps metabolized, amongst other things, to amino acids. Disruption of the virus with phenol revealed that 70 % of the radioactivity was in the virus RNA, mostly in ribose, the remaining 3o % being in virus protein, pre- sumably as amino acids. The total amount of radioactivity that could be in the form of glucosamine did not allow for more than o.6 glucosamine residues per virus particle. From this it was concluded that glucosamine is not a virus constituent per se, that the EMC virus particle does not contain glycoprotein and, consequently, that the virus haemagglutinin is not a glycoprotein.
Article
The in vivo synthesis of encephalomyocarditis-specific proteins was studied by labeling the viral proteins with radioactive amino acids under conditions where host-protein synthesis was almost completely inhibited. To assure recovery of all proteins, intact cells were lysed in hot 1% sodium dodecyl sulfate. These lysates were analyzed by quantitative high-resolution electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This technique allowed the detection and estimation of the molecular weight of 15 virus-specific polypeptides: A, 100,000; B, 90,000; C, 84,000; D, 75,000, D1, 65,000; E, 56,000; epsilon, 40,000; F, 38,000; alpha, 34,000; beta, 30,000; gamma, 23,000; G, 16,000; H, 12,000; I, 11,000; and delta, 9,000. Pulse-chase experiments, in conjunction with cyanogen bromide and tryptic mapping of the isolated polypeptides, indicate that at least three primary gene products (A,F,C), with a cumulative weight of about 220,000, are generated during translation of the RNA genome. Chains A and C then undergo post-translational cleavages, while F remains uncleaved. The proteins generated by the cleavage of A include all of the capsid chains (alpha, beta, gamma, delta, epsilon). Those generated by the cleavage of C include D and E. The chains alpha, beta, gamma, delta, E, F, G, H, I, with a cumulative molecular weight of about 230,000, are stable and are produced in about equimolar amounts. A model for the synthesis of, and a cleavage sequence that accounts for, all of the viral polypeptides is proposed.
Article
SUMMARY Electrophoretic analysis of native and denatured RNA of feline leukaemia virus (FeLV) has revealed small but significant size differences between the RNAs of a number of FeLV isolates. The mol. wt. of the denatured or subunit RNA has been estimated to range from 2.2 to 2.6 × 106 in the different isolates, each of which belonged to one or more FeLV subgroups (A, B and C). The RNA of subgroup A virus was smaller than that of subgroup B virus, while the RNA of subgroup C virus appeared to be intermediate in size between the RNAs of A and B subgroup viruses. Size differences were, however, also found between RNAs of FeLV isolates of a single subgroup (A), indicating that no simple relationship exists between RNA size and virus subgroup.
Article
Full-text available
SUMMARY A count of specifically labelled tryptic peptides from purified poliovirus (strain Mahoney) showed that the amino acid sequence comprising its structural protein contains at least 2I and 25 uniquely placed residues ofhistidine and methionine, re- spectively. Amino acid analysis after performic acid oxidation showed a content of, respectively, 21 and 27 moles of these residues per lOOO amino acids recovered. Accordingly, poliovirus structural protein must comprise a unique sequence of about I ooo amino acids, representing about 4o ~ of the coding potential of the poliovirus genome. The correspondence of this percentage with the proportion of the polio- virus genetic map occupied by structural protein genes (48 ~o) suggests that the map represents a major part of the genome. A value of about lOOO amino acids, or lOO to 1 lO ooo daltons of protein, for the repeating structural unit, coupled with a total content of 6.o to 6-4 x Io G daltons of protein in the poliovirus particle, indicates that it contains 60 such structural units and that its triangulation number = I.
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The genome of Mengo virus, “35 S” RNA, is a single polynucleotide chain having a molecular weight of 2.44 ± 0.08 × 106 daltons. This value has been determined from studies using the methods of polyacrylamide gel electrophoresis, sedimentation in dimethyl sulfoxide, sedimentation after reaction with formaldehyde and electron microscope measurements of the viral replicative form. SDS-polyacrylamide gel electrophoresis of solubilized purified virus revealed the presence of four major polypeptides (α, β, γ, and δ) having estimated molecular weights of 32,500, 29,600, 23,700, and 10,600 daltons, respectively. In addition, two minor components were detected. The first (D2) has a molecular weight of 57,700 daltons and the second (ϵ), a molecular weight of 39,000 daltons.
Article
The rate of polypeptide chain elongation in vitro has been estimated by analyzing the products synthesized under the direction of encephalomyocarditis virus (EMC) RNA. In typical reaction conditions, at 100 mM K+, amino acids are polymerized at a rate of 25 residues/min. The translation rate is dependent on the K+ concentration, ranging from 35 residues/min at 150 mM K+ to 15 residues/min at 50 mM K+. These rates are considerably slower than those obtaining in vivo, and this may curtail the translation of very long mRNAs.
Article
SUMMARY The encephalomyocarditis (EMC) virus particle appears to contain about 6o chains each of four major polypeptides and 2 chains of a minor component. Two major polypeptides were assumed to have the same mol. wt. of 32 ooo since they were not separated by electrophoresis on polyaerylamide gels containing SDS, although they are separable by calcium phosphate chromatography. The two other major polypeptides appeared to be homogeneous when examined by calcium phosphate chromatography; they have tool. wt. of 25 ooo and ~ I ooo, based upon gel electrophoresis, although examination of the latter polypeptide by gel filtration in the presence of 6 M-guanidine gave a mol. wt. of 67oo. The minor component has a tool. wt., determined by gel electrophoresis, of about 42000. Similar results were obtained for the closely related Mouse-Elberfeld (ME) virus except that two polypeptides of mol. wt. 33o0o and 3o8oo, were found rather than two of 32o00. Since the ME and EMC viruses studied were both grown in Krebs II ascites tumour cells, the differences in mol. wt. of the polypeptides probably reflected differences either in the specificity of the cleavage enzyme or in the amino acid sequence in the cleavage region.
Article
BMV is a small spherical plant virus whose genetic complement is divided among four single-stranded RNA species (RNAs 1-4) of approximate molecular weights 1.1, 1.0, 0.7, and 0.3×10 respectively13. The two smallest RNAs (3 and 4) each contain a copy of the gene for the single viral coat protein5"3. The high RNA content of the virus (21 %) and its high yield in the leaves of infected plants make BMV RNA potentially available in gramme quantities'4. More- over, the duplication of the coat protein gene offers a unique opportunity to study the influence of the structure of an mRNA on its translation. TRV (CAM isolate'5) is a cylindrical virus whose genetic complement is divided between two RNA species (long and short RNA), of molecular weights 2.5 and 0.7 X 10, respectively"6. Complementation experiments indicate that the short RNA contains the gene for the single viral coat protein'7.Figure 1 shows the incorporation of 35S-methionine by a cell-free extract of mouse L cells in the absence and presence of unfractionated BMV RNA. A strong stimulation of incorporation was observed which continued for at least 2 h.
Article
ME-virus, in the presence of 0.1 m-chloride ions at pH 5.7, can be thermally dissociated into 14 s subunits, free RNA and an insoluble precipitate (I-protein). The 14 s is a pentamer that can be further dissociated with 1 to 2 m-urea into five 5 s subunits. By sedimentation equilibrium measurements the 14 s subunits weighed about 425,000 daltons; the 5 s subunit weighed about 86,000 daltons. The latter subunit is a protein molecule (protomer) composed of one each of three non-identical polypeptide chains α, β and γ and is believed to be generated by dissociation of a δ chain from each of 60 structural subunits in the capsid.The I-protein contains the two minor components δ and ε which are always found in virus preparations; it also contains a definite proportion of the α, β and γ chains. The stoichiometry of the non-δ portion of the I-protein is consistent with that expected for a structure (ε-pentamer) analogous to a 14 s pentamer containing one ε chain in place of one of the five β chains; the amount of precipitate implies that there are two such ε-pentamers per average virion. Thus I-protein is believed to consist of two separate components (a) ε-pentamers and (b) δ chains.The requirement for 12 fivefold vertices in an icosahedral particle and the five-co-ordinated nature of the pentamers suggest that the ME-virion is composed of 12 × 5 or 60 protomers; the composition of the I-protein is consistent with the hypothesis that there are 58 (α, β, γ, δ) protomers for every 2 (α, ε, γ) protomers.The ME-virion behaves as if its 60 subunits were held together by two sets of bonding sites; (a) type 1 sites binding 12 pentamers together to form the 60 subunit shell and (b) type 2 sites binding 5 protomers together to form pentamers. It is proposed that mild acid dissociation of ME-virus is due to specific rupture of type 1 bonding sites and that dissociation of pentamers into protomers by molar concentrations of amide is due to dissociation of type 2 bonding sites.
Article
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.
Article
Ribonucleic acid (RNA) from encephalomyocarditis (EMC) virus stimulates the incorporation of amino acids into protein in cell-free protein-synthetic systems derived from Krebs mouse ascites tumor cells and chick embryo fibroblasts; the mouse system is the more responsive to the viral RNA. The greater part of this difference in activity can be ascribed to the cell sap, but the origin of the ribosomes also has a marked effect. The nature of the polypeptides formed in these cell-free systems was investigated by electrophoresis on polyacrylamide gels and by fingerprint analysis of tryptic digests. The same product in part appears to be synthesized in response to the EMC RNA in both systems. It was not detected if the EMC RNA was partly degraded (</=4S) or replaced by other species of RNA, including that from influenza virus. The results suggest that EMC RNA is partially translated in these systems to yield virus-specific polypeptides.
Article
The procedure used in this laboratory for the purification of encephalomyocarditis (EMC) virus has been described by Burness (1969a). Contamination with host cell material was shown to be absent by the lack of radioactivity in purified virus when homogenates of uninfected host cells containing protein and nucleic acid labelled with [3H]amino acids and 32P, respectively, were added to the crude virus before purification (Burness, 1969a). Although several methods were employed to check the purity of the product, contamination with proteins unique to infected cells, such as non-structural virus proteins was not excluded. We describe here a procedure to investigate and virtually exclude such possible contamination, by using EMC virus variants separable from one another by calcium phosphate chromatography. In the original description of the procedure it was suggested that ribosomes would be the most likely subcellular contaminants of picornavirus preparations since both kinds of particles have similar biophysical characteristics (Burness, 1969a).
Article
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.
Article
Vergleichende Untersuchungen mit Hilfe des CA-Testes an Frenkel- und BHK-allergischen Rindern bestätigen, daß für die nach einer mehrmaligen MKS-Schutzimpfung auftretende Allergie vom Spättyp sowohl das MKS-Virus allein als auch in Kombination mit allergenen BHK-Zellbestandteilen verantwortlich sein kann. Das MKS-Virus allein wirkt nur schwach allergen. Enthalten MKS-Vakzinen allergene BHK-Zellanteile, so bringen sie das Virus-Allergen im Sinne eines Adjuvans-Effektes stärker in Reaktion. Durch Aufspaltung des Virus gewonnene Virus-RNS war nicht allergen. Das isolierte Virus-Protein dagegen besaß eine stark allergene Wirkung. Modellversuche an sensibilisierten Meerschweinchen bestätigten diese Beobachtung. Meerschweinchen konnten sowohl mit komplettem, gereinigtem MKS-Virus, mit abgespaltenem MKS-Virus-Protein als auch mit BHK-Zellsubstanzen sensibilisiert werden. Die Allergie ließ sich durch Makrophagen übertragen. Die MKS-Allergie war in der Regel schwächer ausgebildet als die BHK-Allergie. Eine Ausnahme bildeten die Meerschweinchen, welche mit hochgereinigtem und konzentriertem Ultrazentrifugen-Virus-Pellet sensibilisiert worden waren. Hier traten im CA-Test praktisch nur noch virusspezifische Reaktionen auf. Mit reiner Virus-RNS konnten die Meerschweinchen nicht sensibilisiert werden. Das aus hochgereinigtem MKS-Virus abgespaltene Protein sensibilisierte die Meerschweinchen in stärkerem Maße als das komplette Virus. Im Kreuz-CA-Versuch an Meerschweinchen, die gegen die Virustypen O1, A5 oder C1 sensibilisiert worden waren, reagierten A5 und C1 stärker miteinander als mit O1. Dieser Typ besaß dagegen die stärkste sensibilisierende Wirkung. Aluminiumhydroxyd und DEAE-Dextran zeigten an nichtsensibilisierten Meerschweinchen eine stark toxische, komplettes Freund'sches Adjuvans dagegen eine schwach toxische Wirkung. In sensibilisierten Meerschweinchen führte komplettes Freund'sches Adjuvans zur Immunzellbildung, die sich mit Makrophagen passiv übertragen ließen. Further Investigations on FMD-Allergy of the Delayed Type Comparative investigations on Frenkel- and BHK-allergic cattle by means of the CA-Test verified that an allergy of the delayed type as appears after repeated vaccinations is due to the FMD-virus alone as well as to FMD-virus in combination with allergenic BHK-cell components. The FMD-virus alone gives only a weak reaction. FMD-vaccines, containing allergenic BHK-cell components, cause the virus allergen to react more strongly in a sort of adjuvant effect. The virus RNA, obtained by splitting the virus, appeared not to be allergenic. The isolated virus-protein however had a strong allergenic action. Tests with sensitized guinea pigs supported this observation. Guinea pigs could be sensitized with the complete, purified FMD-virus as well as with split FMD-virus-protein or with BHK-cell substances. This allergy can be transmitted by macrophages. Generally the FMD-allergy was weaker than the BHK-allergy. An exception was in guinea pigs which were sensitized by a highly purified and concentrated virus pellet. In these animals in the CA-Test the reaction was almost entirely virus specific. It was not possible to sensitize the guinea pigs with pure virus-RNA. On the other hand the protein obtained from intensively purified FMD-virus caused a much higher degree of sensitization in guinea pigs than the complete virus. In the cross-CA-Test with guinea pigs sensitized against the virus types O1, A5, or C1, types A5 and C1 had a stronger reaction with one another than with O1. This type, however, had the strongest sensitizing effect. Aluminium hydroxide and DEAE dextran appeared to be highly toxic for non-sensitized guinea pigs, whereas Freund adjuvant, in contrast, was only slightly toxic. It appeared that in sensitized guinea pigs complete Freund adjuvant caused the production of immune cells which were transmissible passively by macrophages. Suite des recherches sur la fièvre aphteuse-allergie cellulaire Des analyses comparées à l'aide du test CA sur les boeufs allergiques au Frenkel et BHK confirment que, pour une allergie cellulaire (delayed type) après une vaccination repetée, le virus-FA seul ainsi que combiné avec des éléments cellulaires-allergeniques-BHK provoque l'allergie. Le virus-FA seul n'a qu'un faible effet allergenique. Si les vaccines-FA contiennent des éléments cellulaires-allergeniques-BHK ils amènent le virus-allergen à une réaction plus forte dans le sens d'un effet-adjuvant. La ribonucleine acide, gagnée par la division du virus n'a pas agie d'une façon allergenique. Par contre la proteine isolée avait un fort effet allergenique. Expériences servant d'exemple sur des cobayes sensibilisés constatèrent cette observation. Des cobayes purent ětre sensibilisés non seulment par le virus-FA complet et depuré ainsi que par la proteine divisé du virus-FA, mais encore avec des éléments cellulaires-BHK. Cet allergie se transmit grǎce aux macrophages. En règle l'allergie-FA avait une constitution plus faible que l'allergie-BHK. A l'éxception des cobayes qui avaient été sensibilisés par le virus-pellet intensivement dépuré et concentré. Dans le cas présent n'apparurent pratiquement que dés réactions de virus specifique. Les cobayes ne purent ětre senibilisés par l'acide Ribonucleine pure du virus. La proteine, separée du virus-FA fortement dépureé avait sensibilisé les cobayes avec plus d'intensité que le virus complet. Au test CA de croisement avec de cobayes qui etaient sensibilisés contre les virus de type O1, A5, ou C1, les types A5 et C1 réagirent ensemble plus forte au test-CA qu'avec le type O1. Ce type, par contre, avait le plus fort de sensibilisation. Aluminiumhydroxyd et DEAE-dextrane avaint montré un fort effet toxique sur des cobayes non sensibilisés, par contre, le Freund adjuvant avaient montré un faible effet toxique. Il apparait donc des cobayes sensibilisés que le Freund adjuvant mène à la formation de cellules immunisées (immun-cells) qui etaient passivement transmissibles par les macrophages. Nuevas investigaciones sobre la alergia aftosa del tipo diferido Los estudios comparativos con ayuda de la prueba de anafilaxia cutánea (AC) en reses bovinas alérgicas frente a la vacuna según Frenkel o la elaborada en células renales de hamsters lactantes (BHK) confirman que la alergia del tipo diferido, registrada a continuación de vacunaciones repetidas contra la glosopeda, se puede deber tanto al virus aftoso solo como en combinación con los componentes celulares BHK alérgenos. El virus aftoso solo no tiene más que una acción alérgena muy débil. Cuando las vacunas antiaftosas contienen fracciones celulares BHK alérgenas, llevan al alérgeno del virus a una reacción más intensa en el sentido de un efecto coadyuvante. El ácido ribonucleico obtenido por desdoblamiento del virus no era alérgeno, mientras que la proteína aislada del virus poseía una acción alérgena muy marcada. Las observaciones experimentales en cobayas sensibilizados confirman este lance. Los cobayas se pudieron sensibilizar con el virus aftoso completo y purificado, con proteína disociada del mismo virus y también con substancias celulares BHK. La alergia pudo transmitirse a través de macrófagos. Por regla general, la reacción alérgica aftosa se manifestaba de forma más débil que la alergia BHK. Una excepción la constituyeron los cobayas que previamente se sensibilizaron con concentrados de virus altamente purificados en la ultracentrífuga. En la prueba de AC no se obtuvieron prácticamente más que reacciones específicas de virus. Con ácido ribonucleico puro del virus no resultó posible sensibilizar cobayas. La proteína disociada a partir de un virus aftoso harto purificado sensibilizaba los cobayas de manera más efectiva que el virus completo. En la prueba cruzada de AC en cobayas, los cuales habían sido sensibilizados frente a los tipos de virus O1, A5 ó C1, el A5 y C1 reaccionaban entre sí con intensidad mayor que con O1. Pero este último tipo poseía el efecto sensibilizante más intenso. El hidróxido de aluminio y el DEAE-dextrano manifestaban en cobayas no sensibilizados una acción tóxica fuerte, a la vez que el coadyuvante completo de Freund una acción tóxica débil. En cobayas sensibilizados, el coadyuvante completo de Freund ocasionaba la formación de células inmunes, las cuales se podían transmitir pasivamente por medio de los macrófagos.
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The particles of Chara corallina virus (CCV) and those of tobamoviruses share many properties, but differ in some. CCV particles are tubular, 532 nm long, and 18 nm wide, and are helically constructed with a basic pitch of 2.75 nm; their isoelectric point is pH 3.4–3.7; their sedimentation coefficient (s20, w) is 230 S; they contain 5% RNA with a molecular weight of 3.6 × 106 daltons, and with a base ratio of G 24.5, A 28.0, C 20.0, U 27.5; their coat protein is similar in size to that of tobamoviruses (about 17.5 × 103 daltons). CCV is considered to be a tobamovirus. CCV may be transmitted experimentally to uninfected Chara corallina cells by injection of virus particles, and causes chlorosis and death in about 10–12 days. A classification of CCV and 68 other tobamovirus isolates, whose coat protein composition is known, showed that CCV is distant from all, but is most closely related to the cucurbit tobamoviruses.
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The presence in encephalomyocarditis (EMC) virus RNA of homonucleotide tracts 10 nucleotides or more in length has been investigated by testing the ability of homo-oligodeoxynucleotides to prime DNA synthesis in the reverse transcriptase from avian myeloblastosis virus. Neither (dC)10 nor (dA)10 promoted incorporation of [3H]deoxynucleotides into acid-insoluble material but (dG)10 and (dT)12-18 were effective primers and produced DNA products approximately 2000 nucleotides in length. We conclude that there are single-stranded oligo(rC) and oligo(rA) tracts in native EMC virus RNA at 37 degrees C. Kinetic analysis indicated that oligo(dT) priming is similar to priming on ovalbumin mRNA and that it gives rise to only one DNA product per template molecule. Oligo(dG) priming appears to be complicated by self-aggregation of the primer. Oligo(dT)-primed and oligo(dG)-primed DNA have both been separated on alkaline-sucrose gradients into two peaks of which only the 'heavier' will hybridise to EMC virus RNA. Competitive hybridisation experiments indicate that the 'heavy' oligo(dT)-primed and oligo(dG)-primed DNA fractions hybridise to overlapping sequences of EMC virus RNA and place the priming regions of EMC virus RNA approximately 500 nucleotides apart during reverse transcription.
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We have adapted the chain-termination method for determining the nucleotide sequence of DNA of Sanger, Nicklen, and Coulson [(1977) Proc. Natl. Acad. Sci. USA 74, 5463--5467] for use with reverse transcriptase (RNA-directed DNA nucleotidyltransferase) on RNA templates. With this method and using a primer (the octanucleotide pdT7rC) directed at the 3'-terminal poly(A), we have determined a sequence of 166 residues in the genomic RNA of the picornavirus encephalomyocarditis virus.
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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.
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The polyadenylic acid contained in 35S mengovirus RNA produced in infected BHK-21 cells contained approximately 94% AMP and was estimated to contain an average of 50 to 55 nucleotides. The polyadenylic acid is placed at the 3'-end of the genomic RNA based on the presence of significant levels of [3H]adenosine in complete alkali or RNase T2 digests of polyadenylic acid from [3H]adenosine-labeled 35S viral RNA.
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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.
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Incubation of Mengo encephalomyelitis virus at slightly acidic pH in the presence of chloride or bromide ions results in the dissociation of the viral capsid into uniform protein subunits (13.4 S) with the release of the intact viral genome (Mak et al., 1970). Electron microscopic studies have shown that the 13.4 S subunit has a well defined, slightly ellipsoidal shape, with surface dimensions of 16.8 ± 0.3 × 14.2 ± 0.2 nm. It is concluded from these studies that the virus capsid is composed of 12 of these subunits, arranged in icosahedral symmetry. The 13.4 S subunits can be further dissociated into 4.7 S fragments by incubation in 2 M urea. The 4.7 S fragment has an approximately spherical shape, with a diameter of 6.8 ± 0.3 nm; and has the same polypeptide composition as does the 13.4 S subunit. These observations suggest that there are 5 such fragments in each 13.4 S subunit, for a total of 60 in the complete virus capsid.
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The secondary structure of encephalomyocarditis (EMC) virus RNA has been studied in situ and in free solution by absorbance-temperature relationships and by circular dichroism (CD). Extracted EMC virus RNA melts reversibly and has a hypochromicity of about 20%; analysis of CD spectra and formaldehyde treatment suggests that approx. 60% of the nucleotides are involved in base-pairing at 25 degrees C. It is shown that the RNA within the virus particle is less structured than when it exists in free solution, being partially stabilized by capsid protein against melting until the virion is disrupted to release the intact RNA. Upon clarification to remove denatured capsid protein, the released RNA gives a melting profile identical with that of phenol-extracted virus RNA. The results suggest that the intact structure of the virus is dependent upon intimate non-covalent bonds between RNA and protein together with hydrophobic bonds between the protein subunits.
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Poly(A) molecules containing on average 25, 45 and 90 nucleotide residues are all eluted from DEAE-Sephadex in the presence of 7 M urea by approximately the same NaCl concentration which is higher than that required to elute 4 S and 5 S RNA. The same poly(A) molecules have electrophoretic mobilities on 12% polyacrylamide gels which are proportional to the logarithm of the number of nucleotide residues they contain but not to the number found in 4 S and 5 S RNA, even after denaturation of the RNA and performing electrophoresis in the presence of 2.2 M formaldehyde. As a result, many reported estimates of poly(A) size derived from such techniques are probably too large and need re-evaluation. Corrections are suggested for the use of 4 S and 5 S RNA as molecular weight markers for electrophoresis on 12% polyacrylamide gels.
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A bovine enterovirus (serotype VG-5-27) was grown in BHK 21 cells and purified using gel filtration and sedimentation procedures. Infective particles sedimented at 165s and the empty capsids or procapsids at 75s. Proteins extracted from each type of component were separated by polyacrylamide-gel electrophoresis. The infective component yielded four polypeptides of molecular weight 34,000, 28,000, 26,000 and 9,000 and were present in molar ratios of 1:1:1:0.5, respectively. Three polypeptides were extracted from the procapsid. These have molecular weights 37,000, 34,000 and 26,000 and were present in molar ratios of 1:1:1, respectively. We interpret these results as indicating that the small polypeptide of the virus particle may have a specific location in the virus in relation to the three major structural proteins.
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Conditions for the propagation, plaque assay, and purification of radioactively labeled rhinovirus 1A are described. Purified virus was free of empty capsids. It sedimented as a single peak at a rate indistinguishable from that of type 1 poliovirus and ME virus on density gradients. Treatment at pH 4, which left ME virus intact, completely disrupted rhinovirus 1A to produce largely insoluble products. Buoyant density measurements in cesium chloride confirmed that rhinovirus 1A is denser (d = 1.386) than ME virus (d = 1.342).Analysis of purified rhinovirus 1A on SDS-polyacrylamide gels revealed five components; of these, four with apparent molecular weights of 33,800, 29,500, 26,000 and 7000, respectively, were present in roughly equimolar proportions. Roughly as much of the fifth component, weighing about 36,700 daltons, was observed. This distribution of polypeptides, which is similar to that of ME virus and of poliovirus, supports the notion that all three viruses represent a common structural archetype. Thus acid lability and high buoyant density which is characteristic of rhinoviruses cannot be attributed to any gross differences from other picornaviruses in size or number of polypeptide chains in the virion.It is proposed that the rhinoviral capsid is comprised of 60 identical four-chain subunits each weighing about 96,000 daltons. Assuming an RNA content of 30–32% this model predicts that the size of the virion is 8.4 × 106 daltons and the size of the RNA is 2.6 ± 0.1 × 106 daltons.
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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.
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This chapter discusses the unique potentialities of the ultracentrifuge that is an aid to the general investigation of viruses, particularly those of plants, as opposed to its use for the sole purpose of the determination of the physicochemical properties of virus preparations that have been purified to the extreme before they have been subjected to this type of examination. It is quite easy to detect small quantities of a homogeneous substance, such as a virus, in the presence of quite large amounts of other proteins provided that the virus sediments at a rate differing from that of the latter by 10% and that the absolute amount of the virus is greater than about 0.1 mg. per milliliter. The use of a double sector cell, although regarded with approval by the more theoretical authorities, is liable to give misleading results because it is only reliable if the cell walls are exactly parallel under the conditions of the ultracentrifuge run. The prismatic effects of the cell, which are usually evident, are modified by any refractive index gradient and result in a lateral or vertical displacement of the baseline.
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Methods are described for the isolation in pure form of three variants of Mengo virus and their constituent ribonucleates. The resulting preparations have been subjected to a detailed physico-chemical investigation using the techniques of sedimentation velocity, diffusion at low centrifugal fields (method of Möller, 1964), optical rotatory dispersion and electron microscopy. It was established that all three variants are identical with respect to the measured hydro dynamic parameters (S20,w0 and D20,w0), and a similar conclusion was drawn for their RNA constituents. The particle weight of the virus, as estimated from the Svedberg equation, is 8.32 ± 0.7 × 106, while that of the RNA is 1.74 ± 0.2 × 106. In the electron microscope the three Mengo variants are indistinguishable from each other and appear as spherical particles with diameters of 26.2 to 27.2 mμ. The hydrated diameter calculated from the experimental diffusion coefficient is 29.3 mμ, which is consistent with a hydrated sphere having 0.23 g water per gram dry virus. The frictional ratio of the virus particle, estimated from S20,w0 and D20,w0, is 1.10, which also suggests that it is essentially spherical in shape. A comparison of the optical rotatory dispersion of the three variants of Mengo virus and their constituent ribonucleates, shows that the curves are essentially identical to one another, with similar cross-over points, positions of troughs and peaks, and amplitudes. This would suggest that the manner in which the proteins are arranged around the RNA chain in the virus is precise and similar in all three cases. By subtracting the contribution of the RNA from that of the virus, the optical rotatory dispersion curve of the protein in situ was obtained. The small amplitude of [m′]234 suggests that the protein in its native environment possesses negligible α-helical content.
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IN their experiments with bacteriophages, Hershey and Chase1 have shown that only the nucleic acid component plays a part in the intracellular multiplication. There are also indications that in simple viruses containing ribonucleic acid the nucleic acid plays a dominant part in the infection. Thus, experiments with tobacco mosaic virus have shown that the protein can be changed chemically without affecting the activity and the genetic properties2; recently, it was even found3 that part of the protein can be removed from tobacco mosaic virus without destroying the activity.
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Methods are described for the concentration and purification of EMC virus grown in L cells in tissue culture. Virus concentrates obtained by treatment of lysates with protamine sulfate and methanol were purified with little or no loss of infectivity by a sequence of steps including digestion with trypsin, treatment with sodium pyrophosphate and ribonuclease, and column chromatography on hydroxylapatite.
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This chapter deals with the absorption spectra of proteins and amino acids. The colored proteins are conjugated proteins in which the protein carrier is colorless. This transparency of protein solutions extends into the ultraviolet region of the spectrum and many proteins do not absorb radiation of longer wavelength than 2500 Ǻ. The essential protein fabric, consisting of a peptide chain in various forms, is not responsible for absorption at longer wavelengths. In case of fibrous proteins, there is some evidence that the peptide fabric is responsible for absorption in this region. Many proteins absorb in this region. This absorption is due to the aromatic amino-acids present in the protein. The advent of quantitative methods of spectrophotometry is the basis of a method of determining tyrosine and tryptophan in proteins. The striking property of proteins is their transparency, indicating a high degree of electronic saturation. The configurational stability of the protein molecule depends entirely on extra-valence forces and not on unsaturation, which would result in high absorption in the ultraviolet. The absence of such rigidifying bonds endows the protein with its unique characters of plasticity, while the number-sequence of side chains gives its chemical constancy. These two properties allow these molecules to be arranged in large polymorphic masses to form a matrix fabric of recurrent pattern in media, which are essentially aqueous. The material reviewed is principally derived from the study of homogeneous absorbing systems, in which the inhomogeneity is finer in grade by several orders than the dimensions of the exploring light beam.