Jerome Polatnick

Foot-and-mouth disease virus-induced ribonucleic acid polymerase in baby hamster kidney cells

Abstract Baby hamster kidney cells infected with foot-and-mouth disease virus contain a newly synthesized RNA-dependent RNA polymerase not found in uninfected cells. This polymerase catalyzes the incorporation of nucleoside triphosphates in the presence of actinomycin D into acid-insoluble RNA composed of 37 S viral RNA, 20 S RNase-resistant RNA, and heterogeneous 26–28 S components. These products correspond to components derived from infected cells. Enzyme activity, dependent upon Mg++, the 4 ribonucleoside triphosphates, and an adenosine triphosphate generating system, is strongly inhibited by Mn++. Activity reaches a maximum at 3.5 hours after infection and decreases to half this value by 5 hours, the time of maximal virus production. Infected cells incubated with cell growth medium yield 3- to 3.5-fold greater amounts of both virus and enzymatic activity than do cells incubated with a minimal medium. Polymerase activities have ranged up to 900 μμ/Amoles of labeled uridine incorporated into polyribonucleotides per milligram of protein in 60 minutes. Enzyme preparations from infected cells pretreated with a concentration of guanidine previously found to inhibit virus production have strongly decreased viral RNA polymerase activity. Sodium dodecyl sulfate (0.5%) almost completely inhibits cell-free enzyme activity. Sodium deoxycholate at the same concentration did not decrease the incorporation of labeled precursors into acid-insoluble products, although the viral RNA profile of products from such reaction mixtures was changed from the untreated. Deoxycholate rendered the incorporation system more sensitive to RNase.

Studies on foot-and-mouth disease virus ribonucleic acid synthesis

Abstract Ribonucleic acid (RNA) was extracted from baby hamster kidney cells infected with type A119 foot-and-mouth disease virus (FMDV) in the presence of actinomycin-D. Virus-specific RNA components, resolved by ultracentrifugation in sucrose gradients, were first detected between 90 and 180 minutes after infection; the maximum rate of synthesis was found 5 hours after infection. The virus-specific RNA components which have been identified are viral RNA and a complex having a sedimentation coefficient of about 20 S which is partially ribonuclease (RNase) resistant. The RNase-resistant RNA is synthesized in amounts similar to viral RNA, and “pulse-labeling” experiments indicate that it is formed before virus RNA.

Inhibition of cell-free foot-and-mouth disease virus-ribonucleic acid synthesis by antibody

Abstract The cell-free synthesis of virus specific RNA by foot-and-mouth disease virus (FMDV) RNA polymerase was inhibited by antisera from infected guinea pigs and cattle, by γ-globulin fractions from sera of infected guinea pigs, and by antibody isolated from antibody-antigen precipitates. Deoxycholate, at a concentration of 0.5%, increased the inhibitory effect from 20% to 90%. Ribonucleic acid profiles of antibody-inhibited enzyme products showed a small peak corresponding to 20 S RNase-resistant material and no 37 S viral RNA peak as usually seen in the in vitro deoxycholate pattern. Antisera from animals vaccinated with inactivated virus, which differed from that obtained by infection in not possessing antibody to a third antigenic component, did not inhibit polymerase activity. This third antigenic component was previously found to be a nonviral constituent associated with infection. Preparations of antisera from infected animals from which antibody to this antigen had been removed in stepwise amounts, showed corresponding decreases in inhibition of polymerase activity. Polymerases from FMDV types A and O appeared to be immunologically related, whereas that from type SAT 2 did not. The evidence indicates that FMDV-RNA polymerase may be the third antigenic component.

Foot-and-mouth disease virus RNA. Presence of 3'-terminal polyriboadenylic acid and absence of amino acid binding ability.

Foot-and-mouth disease virus RNA contains a segment of polyriboadenylic acid (poly(A)) that is somewhat heterogenous in nature in polyacrylamide gels containing 8 M urea; however, a prominent peak of radioactive poly(A) migrates to the position of the 4 S marker RNA. The evidence derived from a number of experiments indicates that the poly(A) segment contains 60–80 nucleotides or less and is covalently attached to the 3′-terminus of the RNA. The RNA of the virus could not be aminoacylated with valine, histidine, tyrosine, or several other amino acids.

Detergent-Solubilized RNA Polymerase from Cells Infected with Foot-and-Mouth Disease Virus

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.

Protein kinase activity in African swine fever virus

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Increased deoxyribonucleic acid polymerase activity in African swine fever virus-infected culture cells

I t was reported previously that baby hamster kidney (BHK) cells infected with the deoxyribonucleic acid (DNA) virus causing African swine fever (ASF) contain increased amounts of thymidine kinase (1). Also, the thymidine kinases from normal and infected cells responded differently to changes in substrate concentration and pH, indicating possible differences between them. The present report concerns the increase of DNA polymerase activity in BI-tK cells infected with ASF virus. BHK-21, clone 13 cells were grown in t{oux flasks to the stationary phase using a modified Eagles medium containing 10% tryptose phosphate and 10% bovine serum (2). The Tengani isolate of ASF virus was concentrated by foeelectric precipitation and assayed by hemadsorption in swine leukocyte cultures (3, 4). The supernatant fractions from sonicated infected B H K cell suspensions were used for enzyme studies (1). The enzyme assay measured the incorporation of tritiated dATP into DNA as follows: To 0.1 ml of sample was added 0.1 ml of reaction mixture containing 2_5 ~zmoles MgCI~, 1.25 ~xmoles ATP, 12.5 ~moles Tris(hydroxymethyl aminomethane), pH 7.3, 2.5 mBmoles each of dGTP, dCTP, TTP and tritiated d-ATP at a specific activity of 0.4 ~Ci per m~mole and 125 Bg heat-denatured calf thymus DNA (5). After incubation at 37 ~ C for the desired time, the reaction was stopped by adding 5% trichloroaeetic acid (TCA). The precipitate was washed with TCA and counted for radioactivity in the Packard Tri-Carb Liquid Scintillator.

Metabolic studies of bovine kidney cultures infected with foot-and-mouth disease virus

Abstract Virus-host cell interrelationships have been studied in primary bovine kidney cultures infected with foot-and-mouth disease virus (FMDV). Multiplicities of infection of 4–10 plaque-forming units per culture cell were used; virus progeny appeared at 100–110 minutes; and virus production reached its maximum 270–300 minutes after inoculation. Virus activity was distributed about equally between cells and supernatant fluid at this time. The respiration rate of infected cells was unaffected during the period of virus production up to 300 minutes post inoculation, but it began to decline thereafter. No differences were detected in the total synthesis of ribonucleic acid, deoxyribonucleic acid, and protein. Glucose utilization and lactic acid production were increased as a result of infection, indicating an essential requirement for glycolysis rather than oxidative processes during virus production.

Effect of Zinc and Other Chemical Agents on Foot-and-Mouth Disease Virus Replication

Chemical agents reported to inhibit the growth of various ribonucleic acid and deoxyribonucleic acid viruses were tested against foot-and-mouth disease virus in cell culture. These included Zn2+, aurintricarboxylic acid, polyribocytidylic acid, polyriboinosinic acid, phosphonoacetic acid, and the viral contact inactivator N-methyl isatin β-thiosemicarbazone alone and with CuSO4. The most effective agent, Zn2+, inhibited foot-and-mouth disease virus production in primary calf kidney cells by 1 log unit at 0.05 mM Zn2+ and completely at 0.50 mM. Zinc was inhibitory even when added late in infection and was nontoxic to uninfected cells as measured by protein and nucleic acid syntheses. Polyacrylamide gel patterns of [35S]methionine-labeled, virus-specific proteins showed increasing amounts of higher-molecular-weight material, in accord with reports that Zn2+ inhibits post-translational cleavages of other picornavirus precursor polypeptides.

Changes in protein and nucleic acid metabolism in baby hamster kidney cells infected with foot-and-mouth disease virus

At a multiplicity of infection by foot-and-mouth disease virus sufficient to induce maximum changes in host cell metabolism, baby hamster kidney cells showed a decreased turnover of protein, RNA and DNA. Up to 50% inhibition of protein synthesis occurred by 90 minutes postinfection, when viral-induced synthesis of RNA polymerase had not yet started. Viral inhibition of protein synthesis was not decreased by guanidine at a concentration which inhibited production of viral-specific RNA polymerase. At 300 minutes postinfection, when virus production had reached its maximum, 70 to 90% of cellular protein synthesis had stopped. The relative ease of obtaining this value and its reproducibility at various time intervals make it a useful experimental reference parameter. Deoxyribonucleic acid synthesis was unchanged and RNA synthesis was only decreased 10 to 20% at 90 minutes. At 300 minutes, the respective maximum decreases were 90 and 55%. Analyses of RNA profiles from pulse labeled cells indicated, however, that ribosomal RNA synthesis was more strongly reduced at 300 minutes in infected cells than the 55% average value would indicate. The 45 S peak, present in uninfected cells and postulated to be a precursor of ribosomal RNA, was missing. Cells infected 300 minutes possessed a new 37 S RNA peak which probably represented viral RNA.