Abstract
The RNA-dependent RNA polymerase induced in baby-hamster kidney cells by infection with foot-and-mouth-disease virus can be detected as early as 60min. after infection, which is 60min. before viral RNA synthesis commences. The time at which the polymerase can first be detected coincides with the latest time at which actinomycin D (50,ug./107 cells) or guanidine (lmg./107 cells) inhibits virus replication. However, by increasing the concentration of guanidine, viral replication can be inhibited later in the growth cycle, casting doubt on the validity ofthe hypothesis that guanidine acts specifically on the formation of the viral RNA polymerase. (Dulbecco & Vogt, 1954) and the cells harvested with a rubber 'policeman'. The cells were homogenized in water (108 cells/ml.) in a Teflon-glass homogenizer (ten strokes). Inspection at this stage showed the presence of cell debris and clean intact nuclei only. The cell homogenate was then made 10mM with respect to tris-HCl buffer, pH7-6, and 5mM with respect to MgC92.

We found in preliminary experiments that an induced RNA-dependent RNA polymerase was present in BHK cells infected with FMDV, at least 60min. before the commnencement of virus-induced RNA synthesis. As this delay in viral RNA synthesis after formation of the RNA polymerase was not observed in the other virus systems so far examined, the kinetics of formation of the enzyme were investigated in some detail.
The time at which viral RNA polymerase activity could first be detected corresponded to the latest time at which guanidine (Brown, Martin & Underwood, 1966) or actinomycin D (Black & Brown, 1968) inhibited virus replication. Guanidine is generally regarded as inhibiting viral replication by preventing the formation of the viral RNA polymerase (Caliguiri, Eggers, Ikegami & Tamm, 1965) . We show, however, that increasing concentrations of guanidine cause inhibition of virus * Abbreviations: BH[K cells, baby-hamster kidney cells; FMDV, foot-and-mouth-disease virus. replication at later stages of the growth cycle. Reasons for this later inhibitory action and for the inhibitory action of actinomycin D are suggested.
MATERIALS AND METHODS
Virus 8trains. FMDV (type 0 and type SAT 1) was grown in monolayers of BHK cells (BHK21, clone 13; Macpherson & Stoker, 1962) and assayed by the plaque method (Mowat & Chapman, 1962) .
Chemicals. Labelled ribonucleoside triphosphates and uridine were obtained from The Radiochemical Centre, Amersham, Bucks. Phosphoenolpyruvate (sodium salt), unlabelled ribonucleoside triphosphates and pyruvate kinase were obtained from Sigma (London) Chemical Co., London S.W.6, and guanidine hydrochloride was from British Drug Houses Ltd., Poole, Dorset. Actinomycin D was a gift from Merck, Sharp and Dohme Inc., Rahway, N.J., U.S.A.
I8olation of BNA polymerase. Monolayers of 108 BHK cells were infected with FMDV (50 plaque-forming units/ cell) and incubated for 30min. at 37°. The unadsorbed virus was then poured off, Eagle's medium (30ml.) added and incubation continued to 150min. after infection. (In the experiment on the kinetics of induced polymerase forma. tion, incubation was terminated at the specific times shown in the Results section.) The cell sheets were washed twice with ice-cold phosphate-buffered saline (Dulbecco & Vogt, 1954) and the cells harvested with a rubber 'policeman'. The cells were homogenized in water (108 cells/ml.) in a Teflon-glass homogenizer (ten strokes). Inspection at this stage showed the presence of cell debris and clean intact nuclei only. The cell homogenate was then made 10mM with respect to tris-HCl buffer, pH7-6, and 5mM with respect to MgC92.
The method of isolation ofthe enzyme from this stage was varied, depending on whether the total-particle fraction or the large-particle fraction was to be used.
(a) Total-particle fraction. The cell homogenate was rapidly mixed with 5M-NaCl (01 vol./vol. of homogenate) and kept at 00 for 10 min. An equal volume of water was added and the mixture was centrifuged at 600g for 10min. at 0°in an MSE Mistral centrifuge. The supernatant was centrifuged at 80000g for 1-5hr . in a Spinco model L ultracentrifuge. After removal of the 80OOOg supernatant, the pellet and centrifuge tube were rinsed with 0-25M-sucrose-i mM-MgCl2 and the pellet was suspended in sucrose-MgCl2 (0.2ml./108 original cells). This suspension was used as the total-particle fraction.
(b) Large-particle fraction. The nuclei and the cell debris were removed by centrifugation of the cell homogenate at 600g for 10min. at 00 in the MSE Mistral centrifuge. The supernatant was mixed with 5m-NaCl (0.1 vol./vol. of supernatant) and an equal volume of water was added. The mixture was kept at 00 for 10min. and then centrifuged at 40000g for 15min. in the Spinco model L ultracentrifuge. After removal of the 40000g supernatant, the pellet and centrifuge tube were rinsed with sucrose-MgCl2 and the pellet was suspended in sucrose-MgCl2 (0.2ml./108 cells originally). This suspension was used as the large-particle fraction.
Assay systeM. The assay system contained (,umoles):
ATP, GTP, CTP and UTP, 0-1 each; tris-HCl buffer, pH8.1, 25; MgCl2, 5; phosphoenolpyruvate, 5; pyruvate kinase, 2 pg.; enzyme preparation, 0.1 ml. (400-600,ug. of protein); total volume, 0-4ml. The mixture was incubated for 15min. at 37°. This reaction mixture (with minor modifications as indicated in the Results section) was used throughout the work. In each incubation one of the ribonucleoside triphosphates was labelled with 14C (specific radioactivity 5mcfm-mole). After incubation the reaction mixture was rapidly chilled and 10% (w/v) trichloroacetic acid (5 ml.) was added. The reaction mixture was kept at 00 for 60min. and the precipitate was washed by centrifugation with 3 x 5ml. of 10% trichloroacetic acid and 5ml. of ethanol-ether (1:1, v/v (Brown & Cartwright, 1964; Black & Brown, 1968) , although host-cell RNA synthesis is inhibited more than 99%. The cells were infected with virus (100 plaqueforming units/cell) for 10 min. at 370, and the unadsorbed virus was poured off and replaced by Earle's saline (10ml.).
[3H]Uridine (29c/im-mole) (10,ec/bottle) was added to a series of bottles for successive 15min. periods throughout the growth cycle. The labelling period was terminated by removing the medium and adding 10% trichloroacetic acid (4ml.) at 00. After standing for 1 hr. at 00 the tissue was removed from the glass and washed by centrifugation with 10% trichloroacetic acid (3 x 4 ml.) and methanol (2 x 4 ml.). The residual precipitate was dissolved in 0 5 ml. of Hyamine lOX hydroxide (Packard Instrument Co. Inc., La Grange, Ill., U.S.A.) at 370 and counted in a Packard scintillation counter with a toluene-2,5-diphenyloxazole-1,4-bis-(4-methyl-5-phenyloxazol-2-yl)benzene (1000:5 :0-3, v/w/w) system at 20-30% efficiency. In experiments with guanidine, the Earle's saline was replaced by Earle's saline containing guanidine at the specific time, and the labelling procedure was continued.
RESULTS
Polymerase activity in the total-particle and largeparticlefraction8. Table 1 shows that the microsomal fraction from infected cells incorporated ribonucleoside triphosphate into an acid-insoluble product when incubated with the complete assay system. The omission of one ribonucleoside triphosphate precursor caused a decrease in the quantity of labelled ATP or UTP incorporated into acid-insoluble material. This indicated a dependence of the polymerase activity on the presence of all four ribonucleoside triphosphates. A difference was apparent between the incorporation with labelled ATP and that with labelled UTP. With ATP as the labelled precursor the decrease in the incorporation when only three ribonucleoside triphosphates were present was 74%. When UTP was used as the labelled precursor the corresponding decrease was greater than 90%. This suggested the presence of a contaminating polyadenylate homopolymerase. This was in fact confirmed in the experiments with the large-particle fraction. Table 2 shows that the large-particle fraction had more polymerase activity/mg. of protein than the total-particle fraction and that a partial separation of the heteropolymerase and the homopolymerase had been achieved. Whereas in the large-particle fraction the homopolymerase/heteropolymerase ratio was 1:10, in the particle preparation isolated from the 40 OOOg supernatant it was 1: 3. The total protein in the large-particle preparation was twice that in the particle preparation from the 40000g supernatant. Thus from Table 2 it can be calculated that 92% of the heteropolymerase was in the largeparticle fraction.
Dependence on Mg2+ ion8, enzyme concentration and the effect of ribonuclease. The effect of varying the Mg2+ ion concentration in the assay system on the polymerase activity is shown in Fig. 1 . The optimum concentration was 5-7,umoles/0.4mL (15mi). Fig. 2 shows the effect of varying the amount of enzyme (large-particle fraction) on the incorporation of [14C]GTP into acid-insoluble material. Over the range of enzyme concentrations studied, the incorporation of labelled precursor into acid-insoluble product was directly proportional to the amount of enzyme used. Ribonuclease (50,g.) decreased the incorporation of [14C]ATP into acidinsoluble product by 42% in a 10min. incubation period and by 50% (compared with the assay system plus bentonite) in a 30min. incubation period (Table 3) .
Kinetics ofincorporation. A large-particle fraction preparation from cells elliottkimberly@example.org. after infection was used. Fig. 3 Kinetics of polymerase formation. Infected BIlK cells were harvested at different times after infection and the total-particle fraction or the large-particle fraction was prepared and assayed as described in the Materials and Methods section. Fig. 4 shows that polymerase activity was detectable in the large-particle elliottkimberly@example.org. after infection and in the total-particle elliottkimberly@example.org.; Fig. 4 also shows that viral RNA synthesis was not detectable until 60min. after the appearance of RNA polymerase activity. Brown et al. (1966) showed that, under similar conditions of growth, viral RNA synthesis was not detectable until 120miin. after infection.
Effect of actinomycin D on virus replication and viral RNA polymerase formation. Actinomycin D inhibits DNA-dependent RNA polymerase (Reich, Franklin, Shatkin & Tatum, 1962) , but is without effect on RNA-dependent RNA polymerases. Table 3 shows that actinomycin D had no effect on the incorporation of [14C]ATP into acid-insoluble material in the assay system used here. The replication of FMDV is inhibited by actinomycin D (50,ug./107 cells), provided that the drug is added within 60min. of infection; the extent of inhibition is dependent on the time at which the drug is added. For example, there was only 10% of the normal yield of virus if the actinomycin D was added with the virus (Black & Brown, 1968) . Fig. 4 shows that polymerase activity was first elliottkimberly@example.org. after infection, suggesting that the . 9 o co xl4-Time after infection (hr.) Fig. 4 . Polymerase activity and viral RNA synthesis during viral replication. A, Polymerase activity in totalparticle fraction; *, polymerase activity in large-particle fraction; *, viral RNA synthesis. Experimental details are given in the Materials and Methods section. actinomycin D, added at different times after infection, on the appearance of polymerase is shown in Fig. 5 . When added later than 60min. after infection, actinomycin D had no effect on polymerase formation.
Effect of guanidine on virus multiplication, viral RNA replication and viral RNA polymerase formation. Guanidine has been shown to inhibit the growth of certain strains of FMDV, but not that of others (e.g. type 0 is guanidine-resistant and type SAT 1 is guanidine-sensitive; Pringle, 1964) . Table  4 shows that guanidine had no effect on the polymerase isolated from BHK cells infected with the two immunological types of virus used. (At the concentration of guanidine used, the replication of virus type SAT 1 is suppressed.) Table 5 shows that adding guanidine inhibits the formation of RNA polymerase in BHK cells infected with FMDV type SAT 1, but does not affect the formation of RNA polymerase in the cells infected with type 0.
Previous studies (Brown et al. 1966 ) on the effect of guanidine on the replication of FMDV type SAT 1 in BHK cells showed that complete inhibition of virus replication occurred only when the guanidine (lmg./107 cells) was added within 60min. of infection. If the guanidine was added later than 105 min. after infection it had no effect on virus replication. Fig. 6(a) shows the effect of different concentrations of guanidine (0.5-3.Omg./107 cells) on viral replication when added at different times during the growth cycle of the virus. It is possible to inhibit the replication of the virus by the addition of guanidine as late as 150min. after infection by using 3mg./107 cells. The relationship between log (guanidine concentration) and the latest time at which it has maximum effect is linear (Fig. 6b) . It has been shown (Lwoff, 1965; Brown et al. 1966) 
DISCUSSION
The properties of the enzyme preparation described in this paper are similar to those reported for RNA polymerases induced by other viruses (e.g. Horton et al. 1966 ). There was, however, an optimum Mg2+ ion concentration (15mM) for the large-particle preparation whereas, in the work of Polatnick & Arlinghaus (1967) with the same virus-cell system, the dependence on Mg2+ ions increased linearly up to 8mM and then slowly up to 18mM. The specific activity of the preparation used here was also considerably higher than that used by Polatnick & Arlinghaus (1967) .
RNA polymerase activity was detectable 60min. before the start of viral RNA synthesis (Fig. 4) However, the apparent dependence of actinomycin D inhibition on the time of its addition to the infected cells may be an artifact, because we have found that the sensitivity of virus replication to guanidine depends on the concentration of the guanidine as well as on the time of its addition.
It has been accepted that guanidine inhibits the replication of small RNA viruses by specific interference with the formation of the induced viral RNA polymerase (Caliguiri et al. 1965) . Lwoff (1965) suggested that the inhibition by guanidine is due to its preventing the aggregation of the monomer sub-units of the RNA polymerase into the active enzyme. By using a guanidine-requiring mutant of poliovirus, this guanidine-sensitive step was shown to occupy only 10min. of the growth cycle. Other workers have favoured the hypothesis that the action of the guanidine is on the translation of the viral RNA.
Earlier studies with the virus-cell system used here (Brown et al. 1966) showed that there was complete inhibition of virus replication when the guanidine (Img./107 cells) was added within 60min. ofinfection, which is the time at which virus-induced RNA polymerase is first detected. At later times of addition, the guanidine had less effect, and there was no inhibition with elliottkimberly@example.org. These results are in agreement with the hypothesis that the action of guanidine is due to its specific interference with the formation of the viral RNA polymerase.
By increasing the concentration of guanidine, however, we were able to inhibit virus replication at much later times (Fig. 6a) . It is impossible to correlate this result with Lwoff's (1965) hypothesis because (a) guanidine suppresses viral replication over a time range extending from the time of infection to 150min. after infection and (b) whenever the guanidine is added there must be sufficient to prevent the monomers already synthesized from forming the active enzyme, and also to prevent any monomers subsequently synthesized from forming active enzyme. Thus it would not be possible for guanidine of a certain concentration to be effective only during the early part of the growth cycle (this assumes that the polymerase has a short halflife). Even if the polymerase is stable, guanidine added late in the growth cycle would not affect the polymerase already formed, so that viral replication would not be inhibited.
From Fig. 6(b) we can deduce that, irrespective of the site with which the guanidine reacts, the number of such sites must increase exponentially. If the transcription of viral RNA at any time depends on the number of viral RNA strands available at that time (i.e. it follows first-order kinetics) we could suggest two possibilities for the action of the guanidine: (1) on the transcription process, or (2) on the translation of viral RNA, if translation can occur on progeny RNA as well as on the parental RNA. Fig. 7 shows that the increase in the rate of synthesis of viral RNA is immediately stopped by adding guanidine, but RNA synthesis still continues. If the site of guanidine action is the start of transcription, then the residual RNA synthesis could be due to the completion of strands already in the process of synthesis. If, however, the site of action is in the translation step, it is possible that RNA synthesis would be stopped by the blockage of RNA by incomplete polypeptide chains. It is thus impossible from the evidence in the present paper to decide which alternative is more likely to be correct. However, Jacobson & Baltimore (1968) showed that viral proteins continue to be synthesized in poliovirus-infected HeLa cells in the presence of guanidine. It is tempting to postulate, therefore, that the site of guanidine action is connected with transcription of the viral RNA. During the preparation of this manuscript Caliguiri & Tamm (1968) presented evidence suggesting that guanidine interferes with the initiation step of viral RNA replication.

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