Chem.-Biol. Interactions, 16 (1977) 347--357 347 © Elsevier/North-Holland Scientific Publishers, Ltd.

EFFECT OF STREPTOVARICIN ON THE INCORPORATION OF URIDINE INTO CELLULAR AND VIRAL RNA

K.B. TAN * and B.R. McAUSLAN *

Roche Institute of Molecular Biology, Nutley, New Jersey 07110 (U.S.A.)

(Received August 12th, 1976) (Revision received November 22nd, 1976) (Accepted December 7th, 1976)

SUMMARY

Poxvirus replication is inhibited by streptovaricin. The most readily ob- served effect is the inhibition of incorporation of [3H]uridine into viral mRNA, suggesting an inhibition of RNA synthesis. Streptovaricin also inhibits the incorporation of [SH]uridine into cellular RNA but not as severely as viral RNA. On the other hand, [3H]uridine incorporation into the RNA of Semliki Forest virus (SFV), which contains a positive strand RNA genome, does no t seem to be inhibited by streptovaricin. The inhibitory effect of streptovaricin is completely reversible after removal of the inhibi- tor. In addition to inhibiting RNA synthesis, streptovaricin also may inhibit the methylat ion of cellular RNA. Viral RNA is stable in the presence of streptovaricin.

INTRODUCTION

Streptovaricin complex, consisting of a mixture of streptovaricins A through G, was found to inhibit the replication of poxvirus in primary chick embryo fibroblasts at levels of the inhibitor that had little effect on cellular macromolecule synthesis [1]. The primary target of the inhibitor in pox- virus-infected HeLa cells appears to be viral messenger RNA.

We present here data from a more extensive investigation of this phe- nomenon. In particular, we want to know whether or no t poxvirus mRNA

* Present addresses: (K.B.T.) The Wistar Institute, 36th Street at Spruce, Philadelphia, PA 19104 (U.S.A.) and (B.R.M.) Division of Animal Genetics, C.S.I.R.O., P.O. Box 90, Epping, N.S.W. (Australia). Abbreviations: CP, cowpox virus; pfu, plaque forming units; SDS, sodium dodecyl sulfate; SFV, Semliki Forest Virus; TCA, trichloroacetic acid.

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is indeed inhibited by the streptovaricin complex and which streptovaricin is active. Some unexpected properties of the streptovaricins, which made an unequivocal interpretation of our results difficult, must be considered when studying the antiviral or antibiotic effect of novel ansamycins.

MATERIALS AND METHODS

Cells HeLa-S3 cells were maintained as suspension cultures. Procedures for

maintenance and infection with virus have been described [2,3].

Virus SFV was grown in BHK/21 cells from a plaque-cloned stock prepared in

chick embryo fibroblasts [4]. CP was grown in embryonated eggs and purified before use as described previously [3].

Streptovaricin Streptovaricin complex (referred to throughout this paper as streptovari-

cin M) was a mixture of streptovaricins A through G [1]. Streptovaricins M, A and C were all obtained from the Upjohn Company, Michigan. Strepto- varicins B, D, E, F and G were a gift from K. Rinehart. The streptovaricins were dissolved in a few drops of dimethylsulphoxide, diluted to a final con- centration of 1 mg/ml in water and stored at 0 ° C.

Labeling of cells with [3H] uridine A 10 ml sample (5 • 106 cells) was incubated for 10 min with 10 pCi of

[3H]uridine (specific activity > 2 Ci/mmole, New England Nuclear, Mass.). After adding cold uridine (0.1 mg/ml), the cells were sedimented by centrifugation (500 g, 3 min), washed once with 0.1 M Tris--HC1, pH 7.4, and again sedimented. Af te r swelling in 1 ml RSB (0.01 M Tris--HC1, pH 7.4, 0.01 M NaC1, 0.0015 M MgC12), for 10 min at 0°C the cells were dis- rupted with a Dounce homogenizer to break open more than 90% of the cells wi thout disrupting the nuclei. The sample was centrifuged (500g , 3 min) and the cytoplasmic fraction was treated with cold 5% TCA at 0°C. Acid-insoluble material was collected on glass fibre filter papers (GF/C What- man), washed 4 times with 5% TCA, and the radioactivity determined by liquid scintillation spectrometry in a Beckman LS 250 counter.

Procedure for labeling early mRNA The method described by Kates and McAuslan [5] and McAuslan [6]

was used. Briefly, cells were pretreated with cycloheximide (200 pg/ml), infected with 50 pfu of CP per cell for 30 min and maintained in cyclo- heximide-containing medium at 37°C. At intervals, the cells were incubated for 10 min with [3H]uridine. They were then fractionated into nuclear and cytoplasmic fractions and the incorporation of label into cytoplasmic RNA was determined.

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Methylation o f HeLa RNA After incubation at 37°C for 1 h in amino acid-free medium, 150 ml of

cells (5 .10S/ml) were sedimented and resuspended in 30 ml of fresh medium containing 10 /ICi/ml [3H]methyl methionine (specific activity 11 Ci/mmole; Amersham/Searle, Ill.), adenosine (2 • 10 -s M) and guanosine (2 • 10 -s M). After 30 min at 37°C, nuclear RNA was prepared and analyzed on sucrose gradients.

Acrylamide gel analysis o f radioactive RNA The cytoplasmic fraction of cells prepared as described above was treated

as follows: Disodium EDTA was added to 0.01 M, SDS to 1% and the mixture was incubated at 37°C for 15 min. Sodium percholate was added to 0.5 M. The mixture was then shaken with an equal volume of chloroform (24 parts)-isoamyl alcohol (1 part) mixture for 2 min, chilled in ice for 5 min and centrifuged at 1000 g for 10 min. The aqueous phase was reextracted with chloroform-isoamyl alcohol. RNA was precipitated (at --20°C for 16 h) from the final aqueous phase with 2.5 volumes of ethanol after the addition of NaC1 to 0.2 M. The precipitate was then processed for electrophoresis in a 2.6% polyacrylamide gel (8 mm diameter × 8 cm long) by the method of Loening [7]. Electrophoresis was performed at 5 mA per gel for 3 h. The gels were then frozen and sliced into 2 mm slices. Each slice was incubated with 0.5 ml NCS solubilizer (Amersham/Searle, Ill.) at 50°C for 15 h and mixed with 10 ml of spectrafluor scintillation fluid (Amersham/Searle) for measurement of radioactivity.

Sucrose gradient analysis o f radioactive nuclear RNA Nuclear RNA was extracted by the method of Penman [8] with slight

modifications. A 1 ml sample (representing RNA from 5-106 cells) was layered over a 30 ml gradient of 15--30% (w/v) sucrose (in 0.1 M NaC1, 0.05 M Tris, pH 7.2, 0.001 M EDTA and 0.5% SDS) and centrifuged (Spinco SW 27 rotor; 15 000 rpm, 20°C, 15 h). 1 ml fractions were collected from the bot tom of the tubes and acid-insoluble radioactivity was determined as described above.

Acid soluble pool in [3H]uridine labeled cells [3H]Uridine in the acid soluble pool was measured by the method

described by Tan and McAuslan [9].

RESULTS

Effect o f streptovaricin on [3H]uridine incorporation into viral early mRNA CP virus is a DNA-containing virus and directs the synthesis of an early

mRNA (prior to DNA replication) and a late mRNA (subsequent to DNA replication). When poxvirus-infected cells are treated with cycloheximide to inhibit viral DNA synthesis and then labeled with [3H]uridine, the labeled RNAs detectable in the cytoplasm are cellular 4 S RNA and early viral

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mRNA [5,10,11]. Early viral mRNA has a sedimentation coefficient of 10--16 S in sucrose gradients [5,11]. In our experiments, the cytoplasmic RNA from CP-infected cells was analyzed by polyacrylamide gel electro- phoresis because this technique gives a better separation of viral mRNA from host 4 S RNA than by sedimentation techniques.

Cells were treated with cycloheximide and infected with CP (see MATE- RIALS AND METHODS). Streptovaricins were added to the cultures 30 min prior to infection and were maintained at a concentrat ion of 10 pg/ml throughout the experiment. At 1 h after infection, cultures were incubated for 10 min with [3H]uridine. Cytoplasmic extracts were prepared and the RNA was fractionated on polyacrylamide gels (Fig. 1). Apart from the complex, streptovaricins, B, D, and F inhibited [3H]uridine incorpora- tion (more than 10% inhibition) into early viral mRNA and cellular 4 S RNA

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Fig. 1. Streptovaricin inhibition of [3H]uridine incorporation into CP early mRNA. Infected ceils treated with cycloheximide and streptovaricin were labeled with [3H]- uridine and cytoplasmic RNA was extracted and analyzed in 2.6% gels. The top left panel shows the superimposed profiles of RNA extracted from uninfected and CP- infected cells incubated in the absence of inhibitor. Uninfected cell extract contains only labeled 4 S RNA whereas the infected cell extract contains in addition to the 4 S RNA a heterogeneous RNA (fractions 5--17) which is CP early mRNA. For comparison, the profile o f CP early mRNA from nontreated cells, shown in the top left panel, is included as a broken line in the profiles of RNA extracted from infected cells treated with dif- ferent streptovaricins. The arrows indicate, from left to right, the positions of cellular 28 S, 18 S and 4 S RNA.

351

(Table I). Streptovaricins A, E and G were wi thout any marked effect as was the structurally related compound rifampicin [16] (and Tan and McAuslan, unpublished data). Under similar conditions the only RNA detected in un- infected cells was 4 S RNA.

Effect of streptovaricin on incorporation of [3H]uridine into late CP mRNA Viral late m R N A is synthesized after the onset of viral DNA synthesis.

The rate of late m R N A synthesis was determined by briefly labeling infected cells and measuring the incorporation of [3H]uridine into the acid-insoluble cytoplasmic fraction of the cells. This gives a measure of late viral m R N A synthesis since host RNA synthesis is no t only depressed at late times after infection but the labeling time is too short to detect cellular RNA (except 4 S RNA) in the cytoplasm. Streptovaricins M and D inhibited the incorpora- tion of [3H]uridine into late viral m R N A whereas streptovaricin A did no t (Fig. 2).

Effect o f streptovaricin on incorporation of [3H]uridine into cellular RNA It was of interest to investigate whether HeLa cell RNA metabolism was

affected by doses of streptovaricin that inhibit the replication of CP in this system. When HeLa cells are incubated briefly (less than 15 min) with [3H]uridine, the RNA species labeled are the nuclear 45 S (ribosomal pre- cursor RNA), rmclear heterogeneous RNA and cytoplasmic 5 S, 4 S and 4 S-precursor RNA [10,12]. The low molecular weight RNA, referred to collectively as "4 S" RNA, migrated as a single peak in 2.6% polyacrylamide gels (see Fig. 1).

At the doses used in this series of experiments, only streptovaricins D and M inhibited the incorporation of [3H]uridine into cellular RNA (Fig. 3). The inhibition of labeling of viral early mRNA and cellular RNA by the different

TABLE I

STREPTOVARICIN INHIBITION OF [3 H]URIDINE INCORPORATION INTO CELLU- LAR RNA AND VIRAL mRNA

Streptovaricin Percent inhibition of [ 3 H]uridine incorporation into RNA a

HeLa 45 S HeLa "4 S" b CP early mRNA

None 0 0 0 A 5 9 7 B 3 12 28 F 0 36 49 D 39 44 75 M 75 69 97

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n o Fig. 2. Inhibition of incorporat ion of [3H]uridine into late CP mRNA. At various times after infection, aliquots of uninfected or infected cells were each incubated for 10 rain with [3H]uridine (1 pCi/ml) and acid-insoluble cytoplasmic radioactivity was determined as described in MATERIALS AND METHODS. Each sample contained 5 • 106 cells in 10 ml of medium. Streptovaricin (10 pg/ml) was added at 1 h after infection. (a) CP- infected cells in the absence (o t ) or presence (o ~) of streptovaricin M. (4 i), uninfected cells treated with streptovaricin M., (b) CP-infected cells in the absence ( . - ) or presence of streptovaricin A (L~ ~) or streptovaricin D (D ~ ); (A A) uninfected cells not treated with streptovaricin.

Fig. 3. Inhibition of uridine incorporation into HeLa nuclear 45 S RNA. Cells treated with streptovaricin (10 pg/ml) for 60 rain were labeled for 10 rain with 2.5 pCi of [3H]- uridine/ml. Nuclear RNA was extracted and 1 ml samples (representing RNA from 5 . 1 0 6 nuclei) were centrifuged on 30 ml 15--30% (w/v) sucrose gradients (15 000 rpm; 15 h, 20°C, Spinco SW 27 rotor). Cellular ribosomal RNAs were included as markers.

s t r e p t o v a r i c i n s is c o m p a r e d in T a b l e I. In al l i n s t a n c e s t h e i n c o r p o r a t i o n o f [ 3 H ] u r i d i n e i n t o v i ra l m R N A was m o r e s e v e r e l y i n h i b i t e d t h a n t h a t i n t o c e l l u l a r R N A .

Is RNA synthesis inhibited by streptovaricin M or D ? We h a v e s h o w n p r e v i o u s l y [ 9 ] t h a t s t r e p t o v a r i c i n D i n h i b i t s n u c l e o s i d e

u p t a k e b y ce l l s ( a b o u t 50% i n h i b i t i o n o f u r i d i n e u p t a k e ) . T h u s t h e o b s e r v e d i n h i b i t i o n o f [ 3 H ] u r i d i n e i n c o r p o r a t i o n i n t o b o t h c e l l u l a r a n d CP R N A m i g h t r e f l e c t n o t a n i n h i b i t i o n o f D N A - d i r e c t e d R N A s y n t h e s i s p e r se, b u t a r e d u c e d i n c o r p o r a t i o n o f [ 3 H ] u r i d i n e i n t o R N A r e s u l t i n g f r o m a r e d u c e d

353

[3H]uridine pool. The following experiment was conducted to test this hypothesis.

Streptovaricin M causes a 50% inhibition of uridine uptake by HeLa cells [9]. If, in addition, streptovaricin M inhibits RNA synthesis, then the incorporation of [3H]uridine into SFV RNA should be inhibited to about the same extent as poxvirus mRNA. SFV contains a positive strand RNA genome and can replicate in the absence of host cell RNA synthesis. Ac- cordingly, HeLa cells were pretreated with actinomycin D, so that only SFV RNA would subsequently be detected [4] and infected with SFV. The effect of streptovaricin M added at various times after SFV infection on the incor- poration of [3H]uridine into viral RNA is shown in Fig. 4. Streptovaricin M caused a 50% inhibition, up to 6 h after infection, of the rate of [3H]uridine incorporation into actinomycin D-resistant RNA (SFV RNA) in the cell cytoplasm.

We investigated the possibility that the uptake of [3H]uridine into strep- tovaricin M-treated cells could be reduced further after infection with CP virus. This could then account for the 97% or more inhibition of [3 H]- uridine incorporation into CP mRNA. However, the soluble radioactive pools in infected or uninfected cells were the same after streptovaricin treat- ment.

Messenger stability in streptovaricin-treated cells The recovery of reduced amounts of labeled CP mRNA could have been

due to a streptovaricin-mediated breakdown of the mRNA into acid soluble material. This was tested by prelabeling early viral mRNA, which normally has a comparatively long half life [11] and determining its stability in the presence of streptovaricin.

Cycloheximide-treated cells were infected with CP and early viral mRNA was labeled with [3H]uridine. m R N A synthesis was then arrested with act inomycin D. The culture was resuspended in fresh medium containing cycloheximide, act inomycin D and non-radioactive uridine. To equal aliquots were added streptovaricin M or streptovaricin A and one aliquot, not treated with streptovaricin served as a control. No significant decay of early v~al mRNA was detected during the 60 min incubation of infected cells inJ~reptovaricins M or A (Fig. 5).

We are unable to determine if early viral mRNA made in the presence of streptovaricin is inherently less stable than the mRNA made in the absence of the inhibitor.

Reversibility of streptovaricin inhibition The inhibition of [3H]uridine incorporation into CP early mRNA by

streptovaricins M or D was completely and rapidly reversible as shown in the following experiment. Cultures of cycloheximide-treated cells were infected with CP in the absence or presence of streptovaricin M. 60 min after infec- tion, aliquots of cells were labeled with [3H]uridine and early m R N A was isolated for analysis. A duplicate streptovaricin-treated culture was washed

354

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Fig. 4. Synthesis of Semliki Forest Virus RNA in HeLa cells. Cells infected (50 pfu/cell) in the presence of 5/~g actinomycin D/ml were labeled 20 rain with 1 pCi [3H]nridine/ml at various times postinfection. Acid insoluble radioactivity in the cytoplasmic fraction of disrupted cells was determined as described in MATERIALS AND METHODS. Infected (e 8 ) and uninfected ( , , ) actinomycin-treated cells in the absence of strepto- varicin; infected cells with streptovaricin A (10 /~g/ml) added at 1 h after infection (o o) and streptovaricin M (10/~g/ml)-added at 1 h (A A) and 3 h ([] o) after infection.

Fig. 5. Stability of CP early mRNA in the presence of streptovaricin. Cells infected with CP in the presence of cycloheximide were incubated for 10 min with 5/~Ci [3H]uridine/ ml at 1 h after infection. Actinomycin D (10 pg/ml) and unlabeled uridine (50 pg/ml) were added and the cells chilled, centrifuged and resuspended in the same volume of warm growth medium containing the same concentrations of cycloheximide, actinomycin D and unlabeled uridine as before. The cells were divided into 3 aliquots. Streptovaricin M (10 pg/ml) was added to one aliquot, streptovaricin A (10 pg/ml) to another and the third served as a control. At time 0 (time streptovaricin added), 20 and 60 rain after streptovaricin addition, cytoplasmic RNA was prepared and analyzed on 2.6% gels.

free of streptovaricin with warm cycloheximide-containing medium and resuspended in this medium. After 60 min the washed cells were incubated for 10 min with [3H]uridine and labeled early mRNA was analyzed by electrophoresis. We found that the inhibition of [3H]uridine incorporation into viral early mRNA was readily and fully reversible (Fig. 6). Similarly, the

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f r a c t i o n n o Fig. 6. Reversal of streptovaricin inhibition of [3H]uridine incorporation into CP early mRNA. Streptovaricin M (10 pg/ml) was added to cells infected with CP in the presence of cycloheximide. After 1 h at 37°C, aliquots of nontreated and streptovaricin°treated (e i ) cells were each incubated for 10 rain with 5 ]~Ci [3H]uridine/ml and cyto- plasmic RNA prepared. Another aliquot of streptovaricin-treated cells (o o ) was washed twice with medium lacking streptovaricin but containing cycloheximide and resuspended in medium containing cycloheximide. 1 h after streptovaricin removal, the cells were labeled for 10 rain with 5 pCi [3H]uridine/ml and cytoplasmic RNA was prepared and analyzed in 2.6% gels.

Fig. 7. Methylation of HeLa RNA. Nuclear RNA extracted from cells not treated (e e) or treated (o o) with 10 pg streptovaricin M/ml and labeled with [3H]methyl methionine were centrifuged on 15--30% (w/v) sucrose gradients (60 000 rpm; 60 rain, 25°C, Spinco SW 65 rotor). The positions of ribosomal RNAs are indicated.

streptovaricin inhibition of [3H]uridine incorporation into viral late mRNA and HeLa RNA was also completely reversible (data not shown).

Methylation of RNA HeLa cell 45 S RNA is methylated [10] but methylated CP mRNA has

not been detected (K.B. Tan, unpublished data). The effect of streptovaricin on RNA methylat ion was investigated. HeLa cells were resuspended in medium containing [3H]methyl methionine. One culture was incubated with streptovaricin M for 1 h and another was incubated wi thout the inhibitor. Cytoplasmic RNA was extracted and analyzed by sucrose gradient velocity sedimentation. The results show that less methylated RNA was recovered from streptovaricin-treated cells (Fig. 7). Whether this is due to an inhibition of methylat ion or synthesis of RNA remains to be determined. Strepto- varicin M had no effect on the uptake of either [3H]methyl methionine or [~H]leucine into the amino acid pool of HeLa cells (K.B. Tan, unpublished data).

356

DISCUSSION

Previous studies by Quintrell and McAuslan [1] showed that low doses of streptovaricin M (2 pg/ml) had little effect on chick embryo fibroblast RNA synthesis but inhibited CP replication. Streptovaricins M and D also inhibit the replication of CP in HeLa cells ([1] and McAuslan and Tan, un- published data). At tempts to show the primary target of inhibition have been rendered difficult by the multiplicity of effects of the inhibitor. Diffi- culty in obtaining practical amounts of streptovaricin E, F and G has obliged us to work mainly with the complex mixture streptovaricin M and with streptovaricin D which showed similarities in action with streptovaricin M.

The arguments that streptovaricin M (and D) inhibits early and late CP mRNA and HeLa RNA synthesis are as follows:

(i) [3H]uridine incorporation into CP mRNA is abolished at concentra- tions of the inhibitor that cause only 50% inhibition of [aH]uridine uptake into the cell.

(ii) In contrast, [aH]uridine incorporation into ribovirus RNA (SFV) is not inhibited beyond that expected from the depression of the rate of up- take of [aH]uridine into the cells produced by streptovaricin (in both cases an inhibition of 50%). If this is taken into consideration, then SFV RNA synthesis per se is not inhibited by streptovaricin M, but the 97% or more inhibition of uridine incorporation into CP mRNA indicates a selective inhibition of CP mRNA synthesis.

(iii) Streptovaricin F appears to inhibit selectively the incorporation of [3H]uridine in early CP mRNA (see Table I). We have not been able to obtain sufficient amounts of this inhibitor to conduct infectivity inhibition tests.

The inhibition of CP replication in chick fibroblasts at very low strepto- varicin M concentrations indicates the potential value of some component of streptovaricin M as a potent antiviral agent. The present study gives an indication of what the mechanism of action might be and demonstrates the difficulties in determining the primary action of streptovaricin as an anti- viral agent. Streptovaricins M and D do cause a significant inhibition of [3H]uridine uptake into cells [9] which makes accurate measurement of RNA synthesis difficult. Other compounds that do this are substituted benzimidazoles [13,14] and some acridines [15]. Presumably streptovaricins can reversibly affect transport across membranes and might inhibit mRNA synthesis as a secondary effect.

Streptovaricin M at high concentrations does not inhibit early mRNA synthesis by poxvirus in vitro [1]. The ease and rapidity with which the in vivo streptovaricin inhibition can be reversed suggest that the inhibitor does not bind tightly to the viral RNA polymerase. It is conceivable that streptovaricin is rapidly converted in vivo to an active form that inhibits virus transcription and replication.

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ACKNOWLEDGEMENTS

We are University ponents.

most grateful to Dr. K.L. Rinehart, Department of Chemistry, of Illinois, Urbana, for a generous supply of streptovaricin c o r n -

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2 B.R. McAuslan, Control of induced thymidine kinase activity in the poxvirus-infected cell, Virol., 20 (1963) 162.

3 K.B. Tan and B.R. McAuslan, Effect of rifampicin on poxvirus protein synthesis, J. Virol., 6 (1970) 326.

4 K.B. Tan, J.F. Sambrook and A.J.D. Bellett, Semliki Forest virus temperature- sensitive mutants: isolation and characterization, Virology, 38 (1969) 427.

5 J.R. Kates and B.R. McAuslan, Messenger RNA synthesis by a "coated" viral genome, Proc. Natl. Acad. Sci. USA, 57 (1967) 314.

6 B.R. McAuslan, Rifampicin inhibition of vaccinia replication, Biochem. Biophys. Res. Commun., 37 (1969) 289.

7 U.E. Loening, The fractionation of high molecular weight ribonucleic acid by poly- acrylamide gel electrophoresis, Bioehem. J., 102 (1967) 251.

8 S. Penman, RNA metabolism in the HeLa cell nucleus, J. Mol. Biol., 17 (1966) 117. 9 K.B. Tan and B.R. McAuslan, Inhibition of nucleoside incorporation into HeLa cells

by streptovaricin, Biochem. Biophys. Res. Commun., 42 (1971) 230. 10 J.E. Darnell, Ribonucleic acids from animal cells, Bacteriol. Rev., 32 (1968) 262. 11 K. Oda and W.K. Joklik, Hybridization and sedimentation studies on "Early" and

"Late" vaccinia messenger RNA, J. Mol. Biol., 27 (1967) 395. 12 D.B. Mowshowitz, Transfer RNA synthesis in HeLa cells, II. Formation of tRNA

from a precursor in vitro and formation of pseudouridine, J. Mol. Biol., 50 (1970) 143.

13 R.A. Bucknall, The effects of substituted benzimidazoles on the growth of viruses and the nucleic acid metabolism of host cells, J. Gen. Virol., 1 (1967) 89.

14 J.J. Skehel, A.J. Hag, D.C. Burke and L.N. Cartwright, Effects of actinomycin D and 2 mercapto-l(~-4-pyridethyl) benzimidazole on the incorporation of [3H]uridine by chick embryo cells, Biochim. Biophys. Acta, 142 (1967) 430.

15 C. Scholtissek and H. Becht, Action of acridines on RNA and protein synthesis and on active transport in chick embryo cells, Biochim. Biophys. Acta, 123 (1966) 585.