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.

Ultrastructural changes and antigen localization in tissues from foot-and-mouth disease virus-infected guinea-pigs.

Foot-and-mouth disease virus (FMDV)-induced ultrastructural changes in guinea-pig tongue, heelpad, mammary and liver tissues were examined using scanning and transmission electron microscopy. FMDV infection caused cell rounding and the release of virus in membrane limited vesicles in the animal tissues similar to that seen in other work in cell cultures. Microfilaments were present which may be responsible for cell rounding. Immunoperoxidase labeling revealed the attachment of the virus-infection associated (VIA) antigen to the smooth vacuoles of mammary and liver tissues, and to milk fat globules. The electron microscope immunoperoxidase procedure increased the sensitivity of detection sufficiently to allow the visualization of VIA antigen in tissues not previously shown to have the antigen. It is postulated that the release of the smooth vacuoles from the liver cells stimulates the animal's immune response to the VIA antigen.

Association of foot-and-mouth disease virus induced RNA polymerase with host cell organelles.

The localization of foot-and-mouth disease viral-induced RNA polymerase has been determined in situ and in partially fractionated cell components by using polymerase antisera tagged with either peroxidase or ferritin. Electron microscopic examination revealed the polymerase to be heavily concentrated on membranes of the smooth membranous vacuoles (SMV) which are newly formed during infection and which were previously shown to be the site where newly synthesized viral RNA appeared. Polymerase antigen was also seen to be associated with the endoplasmic reticulum (ER), the assumed site of original synthesis, and to a lesser extent with mitochondria and the Golgi apparatus. There was no significant polymerase attachment to nuclear and plasma membranes.

Effect of salts and other agents on foot-and-mouth disease virus poly (U) polymerase activity.

The activity of the purified poly(U) polymerase replication complex of foot-and-mouth disease virus was optimized when 100 mM NH4+ and either 0.75 mM Al3+ or 1.0 mM Fe3+ was added to the standard assay reaction mixture. Zn2+ at concentrations of 10(-5) mM to 5 mM inhibited enzyme activity although all polymerases examined to date have contained zinc. Mercaptoethanol and dithiothreitol inhibited polymerase activity despite the presence of cysteine residues in the viral induced polypeptide of the replication complex, possibly because of their action as metal chelators rather than as reducing agents.

Isolation of a foot-and-mouth disease polyuridylic acid polymerase and its inhibition by antibody.

A template-dependent polyuridylic acid [poly(U)] polymerase has been isolated from BHK cells infected with foot-and-mouth disease virus (FMDV). Enzyme activity in a 20,000 x g supernatant of a cytoplasmic extract was concentrated by precipitation with 30 to 50% saturated ammonium sulfate. The poly(U) polymerase was freed of membranes by sodium dodecyl sulfate and 1,1,2-trichlorotrifluoroethane extraction, and RNA was removed by precipitation with 2 M LiCl. The solubilized poly(U) polymerase required polyadenylic acid as template complexed to an oligouridylic acid primer and Mg2+ for activity, but was inhibited by Mn2+. Antisera from animals infected with FMDV had previously been shown to inhibit the activity of FMDV RNA replicase complexed to the endogenous RNA template. The same antisera also inhibited the activity of poly(U) polymerase. Antisera depleted of antibody by absorption with the virus infection-associated antigen of FMDV no longer inhibited replicase and polymerase activities. The evidence suggests that FMDV RNA replicase, poly(U) polymerase, and the virus infection-associated antigen share a common protein.

Chemically characterized media for study of foot-and-mouth disease virus in baby hamster kidney cells.

Foot-and-mouth disease virus can be grown in baby hamster kidney cells with a chemically characterized medium containing only tris(hydroxymethyl)-amino-methane (Tris) buffer, glucose, glutamine, and salts. Virus infectivity was only 0.5 log unit less than in a complex cell growth medium containing serum, tryptose phosphate, and lactalbumin hydrolysate. At high multiplicity of infection, production was maximal in 5 hr, with the virus remaining largely intracellular. Glucose and glutamine appeared to act independently of each other although both were required at about the same time during the virus production cycle. Glutamine had the greater effect and could not be replaced by amino acids, purines, and pyrimidines. Glutamine also stimulated cellular oxygen uptake in both normal and infected cells. Serum and other organic components added singly to the defined medium did not increase the virus yield. Studies on uninfected cells over a 5-hr incubation period showed that the defined medium maintained protein and ribonucleic acid synthesis at rates similar to the complex cell growth medium. These rates were much lower in media containing only inorganic salts and Tris buffer. Glucose, however, was more important to uninfected cellular metabolism than was glutamine. Defined medium containing dialyzed calf serum produced the highest rate of protein synthesis.

Serological differentiation of foot-and-mouth disease virus on electron microscope grids coated with protein A and antibody.

A serological technique using electron microscope grids coated with protein A and antiserum was able to detect foot-and- mouth disease virus particles in oesophageal-pharyngeal fluids from infected cattle without the need for prior concentration of the sample. The technique was adapted to differentiate serologically among foot-and-mouth disease virus types A, O and C with antigen-adsorbed sera. When grids were coated with heterotypic antigenadsorbed antisera, the homotypic antigen could be observed in viral specimens containing 10(5) PFU/mL, but the heterotypic antigen was not visualized until its concentration was about tenfold higher. Grids coated with the appropriate antigen-adsorbed antiserum can thus be used to indicate foot-and-mouth disease viral serotypes in specimens containing less than 10(6) PFU/mL.

Characterization of a 70S polyuridylic acid polymerase isolated from foot-and-mouth disease virus-infected cells.

A polyuridylic acid polymerase complex isolated from foot-and-mouth disease virus-infected cells sedimented at 70S in a sucrose gradient and appeared in the exclusion volume of an agarose column whose molecular weight cutoff was 5 x 10(6). Phenol extraction of the complex yielded a heterogeneous band of virus-specific RNA and an apparently host cell-derived 4.5 to 5S RNA, both of which are essentially single stranded. Neither RNA served as a template in the cell-free enzyme reaction. Polyacrylamide gel analysis revealed five polypeptides with molecular weights of 50,000, 56,000, 60,000, 70,000, and 74,000 and with molar ratios of 1:2:2:1:1, respectively. Autoradiography showed P56 to be the only major virus-induced polypeptide; the other proteins are apparently of host cell origin. Electron microscopic examination suggested a cartwheel shape for the polymerase complex which was seen to dissociate as polyadenylic acid was added. Antibody previously shown to inhibit enzyme activity aggregated the 70S units.

Viral interference phenomena induced by foot-and-mouth disease temperature-sensitive mutants in bovine kidney cells.

Cultures of bovine kidney (BK) cells infected with temperature-sensitive (ts) mutants of foot-and-mouth disease virus (FMDV) were incubated at 38.5 degrees C, a temperature nonpermissive for mutant virus growth and RNA synthesis. The cells were subsequently resistant to viral growth and RNA synthesis when superinfected with wild-type FMDV and with heterologous fowl plague virus. The extent of interference was proportional to the multiplicity of infection of the ts mutant. It increased with time elapsed between infection with mutant and challenge infection, becoming greater than 99 percent after 24 hours. Interference was not proportional to decreased levels of cellular protein synthesis. The interference could be produced in the presence of actinomycin D, and thus was apparently mostly caused by the ts mutant itself rather than by interferon. The interference could not be produced in other less susceptible cell lines. Supernatant fluids from the BK cells infected with ts mutant virus interfered with wild-type FMD viral growth and RNA synthesis in fresh BK cells, and also showed low levels of activity in a vesicular stomatitis virus-plaque reduction assay. The properties of the supernatant fluid-interfering agent resembled to some extent those of an interferon. The ts mutant-mediated interference factor was apparently not able to diffuse into the supernatant fluid.

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 Zn(2+), aurintricarboxylic acid, polyribocytidylic acid, polyriboinosinic acid, phosphonoacetic acid, and the viral contact inactivator N-methyl isatin beta-thiosemicarbazone alone and with CuSO(4). The most effective agent, Zn(2+), inhibited foot-and-mouth disease virus production in primary calf kidney cells by 1 log unit at 0.05 mM Zn(2+) 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 [(35)S]methionine-labeled, virus-specific proteins showed increasing amounts of higher-molecular-weight material, in accord with reports that Zn(2+) inhibits post-translational cleavages of other picornavirus precursor polypeptides.

Further studies of the physical and metabolic properties of foot-and-mouth disease virus temperature-sensitive mutants.

Three temperature-sensitive (ts) mutants of foot-and-mouth disease virus were classified as ribonucleic acid negative and as belonging to the same complementation group when measured by virus yields and [3H] uridine incorporation in paired, mixed infections at the nonpermissive temperature (38.5C). Mutant ts-22, the only mutant able to produce plaques at 38.5 C, was more sensitive to acid than were the parental wild-type or other mutant viruses. Diethylaminoethyl-dextran did not enhance the plaque-forming ability of the mutant viruses at 38.5C. All of the viruses inhibited host cell protein syntehsis at both permissive (33C) and nonpermissive (38.5C) temperatures.

Foot-and-mouth disease virus-induced RNA polymerase is associated with Golgi apparatus.

Electrophoretic analysis of the Golgi apparatus isolated by differential centrifugation from radiolabeled cells infected with foot-and-mouth disease virus showed about 10 protein bands. The virus-induced RNA polymerase was identified by immunoprecipitation and electron microscope staining procedures. Pulse-chase experiments indicated that the polymerase passed through the Golgi apparatus in less than 1 h.

Correlation of surface and internal ultrastructural changes in cells infected with foot-and-mouth disease virus.

The surfaces of primary and continuous line cell cultures displayed the same sequence of morphological changes during the course of infection with foot-and-mouth disease virus. These changes could be classified into four broad stages: I) cells were flattened, closely attached to one another and microvilli appeared, II) cells rounded, microvilli began to disappear and the cells started to separate from one another by cytoplasmic strands, III) cells were discrete, rounded structures and IV) cells were rounded and had numerous attached buds, some of which contained virus. The internal changes included the appearance of increasing amounts of smooth membranous vacuoles lined with the viral induced RNA polymerase and the presence of buds, some with viral particles inside. While the different cell cultures showed similar internal and external changes as a result of infection, they responded to infection at different rates and contained subpopulations of resistant cells.

The isolation of two enzyme-ribonucleic acid complexes involved in the synthesis of foot-and-mouth disease virus ribonucleic acid.

The foot-and-mouth disease virus-RNA polymerase complex was released from membrane particulates present in the cytoplasm of infected baby hamster kidney cells. The soluble polymerase complex was fractionated by zonal centrifugation in sucrose gradients. Two polymerase complexes (RNA and protein complex) active in the cell-free system were isolated and had S-rate ranges of 20-70S and 100-300S, respectively. The light polymerase complex contained 20S double-stranded RNA; and the heavy polymerase complex contained a polydisperse, partially RNase-resistant RNA. The cell-free product of these two polymerase complexes was analyzed by zonal centrifugation in sucrose gradients. The light polymerase complex synthesized only 20S double-stranded RNA. The product of the heavy polymerase complex contained no detectable 20S double-stranded RNA and only a peak of single-stranded RNA with S-rate corresponding to 37S viral RNA. A third polymerase complex was isolated with S-rate greater than 300S, and it contained a polydisperse, partially RNase-resistant RNA. This third polymerase complex synthesized both 37S viral RNA and 20S double-stranded RNA in the cell-free system, and it is probably the native polymerase complex still bound to cellular particulates.

Localization of foot-and-mouth disease--RNA synthesis on newly formed cellular smooth membranous vacuoles.

Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [3H] uridine. Both membrane formation and RNA synthesis became significant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the sites of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity.

Characterization of a foot-and-mouth disease mutant temperature-sensitive for viral RNA synthesis.

A temperature-sensitive (ts) mutant of foot-and-mouth disease virus (FMDV) did not produce RNA polymerase activity nor synthesize viral RNA when incubated in cells solely at the nonpermissive temperature (38.5 degrees C). Infected cells initially incubated at 38.5 degrees C and then shifted down to 33 degrees C synthesized increased amounts of viral RNA at earlier times compared to infected cells kept at 33 degrees C throughout, indicating that RNA polymerase precursors were synthesized at 38.5 degrees C. In cells shifted up to 38.5 degrees C from 33 degrees C, the total amount of viral RNA synthesized after infection increased sharply for about 15 minutes and then rapidly increased over the next 2 hours. RNA polymerase activity presented a similar pattern in its initial twofold increase and subsequent rapid decrease. Pulse labeling experiments showed that mutant viral RNA synthesis continued at a diminishing rate for 2 hours in cells shifted up to 38.5 degrees C. The data from temperature after shift-up was degraded. The FMDV ts mutant is apparently additionally defective in being unable to protect viral RNA synthesized after shift-up to 38.5 degrees C.

Effect of actinomycin D on virus-induced ribonucleic acid polymerase formation in foot-and-mouth disease virus-infected baby hamster kidney cells.

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.

Electrophoretic characterization of foot-and-mouth disease virus-specific ribonucleic acid.

Foot-and-mouth disease virus (FMDV)-specific ribonucleic acid (RNA) was analyzed by electrophoresis on 0.5% agarose gels. Four classes of RNA were resolved as a function of mobility in agarose: two classes of slowly migrating multistranded RNA, the infectious viral RNA with intermediate mobility, and a minor fast-moving class of lower-molecular-weight single-stranded RNA. The major RNA species were infectious viral RNA and the slowest migrating class of multistranded RNA. The latter RNA was polydisperse when analyzed by sucrose gradient centrifugation, it was partially ribonuclease resistant, and it was the predominant RNA species labeled during the initial period of (3)H-uridine triphosphate incorporation in the cell-free system. Heat treatment studies indicated that part of the slowest-moving RNA was degraded at 60 C and almost complete degradation was detected at 100 C. It was concluded that this RNA is the replicative intermediate in viral RNA synthesis. The second class of multistranded RNA contained both a ribonuclease-resistant RNA and a second RNA peak which was detected only after heat treatment at temperatures above 75 C. Fractions of FMDV-specific RNA isolated by sucrose gradient centrifugation were analyzed by agarose-gel electrophoresis. Infectious viral RNA was detected only in the 37S zone and was the major species of RNA in this part of the gradient. The ribonuclease-resistant RNA (the 20S zone) contained about equal amounts of multistranded RNA (both classes) and the low-molecular-weight single-stranded RNA. All sucrose gradient fractions between 20 and 40S were found to contain the replicative intermediate, although the major portion was detected in the 20 to 25S region.