Effect of Rifampin on the Development of Ribonucleic Acid Bacteriophage Qβ

(1)

Effect of

Rifampin

on the

Development

of

Ribonucleic Acid Bacteriophage

Qf

JADWIGA PASSENTI AND PAUL KAESBERG

Biophysics Laboratory and BiochemistryDepartment, University of Wisconsin,

Madison, Wisconsin 53706 Received for publication 29 April 1971

Thedrug rifampin, when added atthe time of infection, inhibits synthesis of the phage

QW.

Both viral ribonucleic acids and viral proteins are made in nearly the sameamount asin theabsence ofrifampin, butthe rate of assembly into phage par-ticles islow.

Rifampin (formerly known as rifampicin) is 3-(4-methyl-1-piperazinyliminnmethyl) rifamycin SV(14).It is known to inhibit specificallythe de-oxyribonucleic acid (DNA)-dependent ribonu-cleic acid (RNA) synthesis inEscherichia cilo (5) by forming a stablecomplex with RNA polym-erase (19). Rifampin acts also as an antiviral agent.Itinhibitsvaccinia virus

replication

(6, 17)

by preventing the formation of the core poly-peptide fromalarger precursor (10).

According to Fromageot and Zinder (2), rifampin at a concentration of 45

Ag/ml,

when added atthetime ofinfection, severely limits f2 phage growth in E. coli K225. However, when added 5 min after

infection, growth proceeds

in nearly normal fashion. Friesen(1) notedasimilar inhibitory effect although he observed that addition of the antibiotic 15 min after infection caused a threefold reduction of f2 plaque-forming units (PFU). The extent and cause of inhibition resulting from

rifampin

addition at

time of infectionare not known. We have found thatrifampin inhibits the synthesis ofphage

Q0a

toanevengreaterextentthanit doesf2.Wehave studied thenature of this

early

inhibitioninsome detailandconclude thatearlyevents,

phage

RNA andprotein synthesis, proceed normally butthat a late event, phage assembly, is

markedly

in-hibited. Thus, both PFU and physical particles aremade in onlysmallamounts.

MATERIALS AND METHODS

Virus and bacteria. Phage

Qj3

was originally ob-tainedfrom C. Weissmann and phage R17 fromA.

Graham. E. coli Q13 strain, obtained from T.

Sugiyama, wasused throughout ourwork.

Growth and assay conditions.Cellsweregrown

over-1Present address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences,Warsaw, Poland.

night at 37 C in tryptone broth. Portions of such cultures were diluted into tryptone broth containing 1mM CaCl2 to optical density at 590 nm (0D590) between 0.05 and 0.07, and growth was allowed to continue. Cultures were infected with phage Q,3 at OD59o = 0.25, except as noted, and at multiplicity ofinfection (MOI) = 5. Rifampin (Schwarz/Mann, Orangeburg, N.Y.) was used at a concentration of 100.ug perml of culture. (i) To measure the number of infectious phage particles, 0.1-ml samples were withdrawnfrom theculturesatappropriate timesand combinedwith 10jMitersof 0.1 M tris(hydroxymethyl)-aminomethane (Tris) buffer, pH 8.0,containing5 ,g oflysozyme and 0.005 M ethyenediaminetetraacetic acid (EDTA). After 15 min ofshaking at 37C, cell debriswasremovedbycentrifugationandphagetiter

was determined by the standard agar overlay tech-nique. (ii) To measureuracil incorporation, 1 uCiof 4C-labeled uracil (specific activity, 52 Ci/mole; Schwarz/Mann) wasadded per1ml ofcultureatthe time of infection. Duplicate

50-,gliter

samples were transferred at appropriate times to glass-fiber filters treatedpreviously with0.1 ml of1% sodiumdodecyl sulfate (SDS),and coldtrichloroacetic acid-insoluble radioactivity was assayed. (iii) To measure lysine incorporation, 10

ICi

of 3H-lysine (specific activity,

15 Ci/mmole; Schwarz/Mann) was added per1 ml ofcultureattime ofinfection. Duplicate samplesof 50literswerehandledasin(ii)andweresubsequently assayed for hot trichloroacetic acid-insoluble radio-activitybythe method ofMansandNovelli(15).(iv)

To determine the proportion ofuracil incorporated into viral and cellular RNA, 10-mlportionsof E. coli Q13 were infected with and without addition of rifampin. A20-MCi amount of'4C-uracil was added

toeachportionattime ofinfection.After 40min,the cultures werecooled in a dry ice-ethanol bath, and cells were collected by centrifugation (5 min at 5,000 X g). To each sample, 0.2 ml ofa bentonite suspension (8% bentonite, 0.001 M EDTA, pH 7.5) and 0.1 ml of 10% SDS were added. Nucleic acids

wereextractedbythephenolmethod andprecipitated from theaqueouslayer with 2.5 volumes of absolute 286

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fluid [ethoxyethanol-toluene = 1:1, containing 5 gof1-2,5-diphenyloxazole (PPO) perliter and 0.1 g of 1 4-bis-2-4-methyl-5-phenyloxazolyl)-benzene (Me2POPOP) perliter]wasadded, andradioactivity

wasassayed. (v) Tomeasuresynthesis ofphage pro-teins,rifampinwas addedtoonecultureattimeof in-fectionandto a secondculture 30 minafter infection. Reconstituted 14C-algal protein hydrolysate

(10jMCi)

and 14C-lysine (10 g&Ci, 231 Ci/mole, both from Schwarz/Mann) were added toeach culture40 min afterinfection.Fifteen minuteslater,thecultureswere

cooled inadry ice-ethanolbath andcells wereharvested bycentrifugation.Cellsweresolubilizedas describedby Jockusch etal. (8). Sampleswereelectrophoresed on

gels containing SDS, as described by Glowacki-Strauss and Kaesberg (3) and fractionated with an

automatic gel crusher. Gel fractions were left

over-nightwith 10mlofscintillationfluidconsistingofone

part of Beckman BBS-3 solubilizer and 10 parts of toluenecontainingS gof PPO and 0.1 gofM2POPOP perliter andwereassayed forradioactivity.

All radioactivity measurements above were made with a Packard Tri-Carb liquid scintillation counter

at an efficiency of about 80% for solubilized 14(j samples, about 60% for 14C-samples on filters, and about 25% for 3H-samples onfilters.

Theratio ofviable particles to physical particles in phage lysates was obtained as follows. One-liter portions ofE. coli Q13 were grown andinfected in the presenceorabsence ofrifampin. After 40 min of infection,thecultureswerecooledinadryice-ethanol bath andcellswerecollectedbycentrifugationfor 10 minat6,000 X g.Theyweresuspended in 1.5ml of 0.1 MTrisbuffer (pH 8.0) containing0.005 M EDTA and300 ,ug oflysozymeperml. Thesuspensionswere

frozen andthawed threetimes. One-tenthvolume of 0.1 MgSO4 and 10 ug of deoxyribonuclease per ml

wereadded, and thesuspensionwasincubated for 10 min at 37 C. It was then centrifuged for 30 minat

30,000 X g. The pellet wasextracted with 0.5 ml of Tris-EDTA buffercontaining lysozyme, andthe

ex-tracting liquid was added to the supernatant fluid. Plaque-forming titer was determined. Antigen-antibody precipitation was performed with bovine anti-Qj serum.Toaportionof supernatant fluid

con-taining 3 X 1010 PFU (a quantity far below that requiredtosaturatetheantiserum) wereaddedlysing mediumto a volume of 350 litersr, 2 M NaCl to a

final concentration of 0.15 M, and 0.2 ml of four-fold-diluted anti-Q4, serum. The diluted antiserum had beenpreadsorbed previouslywithanE. coliQ13 cell extract. The reaction was allowed to proceed overnight at4 C. Precipitates were collected by

cen-trifugationfor10minat5,000 X gandwashedtwice

DNA-dependent RNA polymerasewasassayed in crude extracts of E. coli Q13 and RQ13 as follows. Portions (50-ml) of RQ13 cells and Q13 cells were grown toODm = 0.55.Cells werecollectedand then disrupted by sonic oscillation in 0.01 M Tris buffer (pH 7.5) containing 0.01 M Mg'+ and 25%glycerol. The assay medium contained in 0.2 ml: uridine triphosphate, guanosine triphosphate, cytidine tri-phosphate,andadenosinetriphosphate,50mmoles of each; phosphenolpyruvic acid, 1 molel; pyruvate kinase, 2.5 ,ug; Tris buffer (pH 8.0), 20molese; g-mercaptoethanol, 2.5 Mmoles; MgCl2, 2

Mmoles;

3H-uridinetriphosphate, 5 ,uCi (specific activity, 17.1 Ci/mmole); and 20 Mliters of the crude extract. Rifampin (1 Mug) was added as indicated. Incubation

wascarriedoutfor 10 min at 37 C and was stopped by addition of 3 ml ofice-cold 10% trichloroacetic acid. After15min,thecold precipitateswerecollected

on glass-fiber filters and washed with 3X 5-ml por-tions of 10% trichloroacetic acid, absolute alcohol, and ether. The filters were dried, and radioactivity wasassayed.

RESULTS

Preliminary experiments showed that for E. coli Q13 asomewhathigher rifampin concentra-tion than used by Fromageot and Zinder (2) for E. coli K225 was required to inhibit cell growth. Therefore, in our experiments, we used 100 ,ug of rifampin per 1 ml of culture at cell concentrations of 2 X 108 to 3.5 x 108 cells/ml. Figure 1 shows that such rifampin concentration stopped E. coli Q13 cell growth within 5 min. Protein and RNA syntheses in uninfected cells werealsorapidly suppressed.

Production of viable phage. Addition of rifampin at the time of QB infection markedly affected development ofprogeny. Forty minutes afterinfection,the yieldof

Q3

PFU grown in the presence of rifampin was 18% of that in the absence ofrifampin. Afterthattime phage yield with rifampin leveled off, whereas yield in its absencesteadily increased. After 2

hr,

the phage yield in the presence of rifampin was only

5%7o

ofthenormalphageyield (Fig. 2).

Rifampin exerted a similar but quantitatively lessereffect on themultiplicationofR17,aphage serologically related to f2. The yield of R17 PFU wasabout20%ofnormal after2hr.

The absorbency of cell cultures infected with

Q$

increased for about50minand then declined rapidly,signaling the onsetof celllysis (Fig. 1).

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PASSENT AND KAESBERG

0.2

0.1

u

TI

40 80 120 160

IME AFTER INFECTION(min)

FIG. 1.Growthcurves. (A)Inifectedcells,norifampii

(B) Infected cells, rifampin added 5 mini after infection. (C) Infectedcells, rifampinaddedat timeof infection. (D) Infected cells, rifampin added5 mili before infec-tion. (E) Uninfected cells. (F) Cells with rifampini added.

Lysiswascomplete within120min. Infected cells treated with rifampin showed no substantial increase of absorbency, and there was only a small declineindicative of lysis. Nevertheless, in the absence of artificial lysis, 90%" of the phage had been released into the medium within 120min.

The yieldofQ3PFU varied with the time of additionofrifampin.It approachednormalyield whenthe drugwas added 30min afterinfection. Upon earlier addition, the yield varied approxi-mately exponentially with the time of addition (Fig. 3).Thiswouldsuggestthatthe "safe" time for addition ofrifampinis substantiallylater for Qu3 infection thanreportedforf2. Theinhibition

of phage development by rifampin was irre-versible. Itmay be seen (Fig. 4) that, when por-tionsof cells infected inthe presence ofrifampin

were transferred at various times to a medium

without rifampin, essentially complete inhibition ofphageyieldwasfound iftheremoval occurred later than 20min.

RNAsynthesis. Despite thelow yield of PFU uponearly rifampin addition, there was substan-tial production ofboth RNA and viral proteins prior to the time of lysis and, as we will show below, the RNA and proteins were functional.

Figure 5 shows kinetics of 14C-uracil

incorpora-TIME AFTER INFECTION (MIN) FIG. 2. Effect ofrifampin (rijampicin) addition oil, phage yield. RiJinmpiniwasaddedat the timeof

inJ&c-tiOI.

0100_

U-0L

o 80_

F

I

0 60_

CL40_- S

20_

dr y

-5 0 5 10 20 30 40 50

TIME(min)

FIG. 3.Effect oftime rifampiii (rifampicin)addition

onphage yield plottedas a percentof PFU obtained

in theabsence ofrifampiui.

tioninto RNA. Itmaybeseenthatincorporation is nil for uninfected cells treated with rifampin. Infected cells treated with rifampin 5 min after

infection, at infection, and 5 min prior to infec-tion synthesize RNA beginning about tO min

1.0-Z0.5.

H

0 -J L)0.3 0

aL

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TIME

(min)

FIG. 4. Effect ofthe time of rifampin (rifampicin) removal on phage yield. Rifampin was added at the timeof infection. Portions were removed after 10 and 20 min;cellswerecollected and transferred to medium withoutrifampin. After 90min of incubation, cells were lysed and phage titer was assayed. PFU in culture infected without rifampin wastakenas 100%. afterinfection and continueat anincreasingrate until the onset of lysis. Comparison of these kinetic curves with those of untreated, infected anduninfected (notshown) cells suggests that the synthesis of virus-induced RNA in rifampin-treated cells is comparable to that in untreated cells. Toverifythatitis,in fact,viral RNA that issynthesized, radioactive RNA wasisolated 40 min afterinfection from cells treated with rifam-pinin themanner of Fig. 5andwas analyzed by

polyacrylamide gel electrophoresis,

(Fig. 6).

The RNAfrominfected, untreated cells migrated as four well defined bands; the slowest moving corresponded toQ3 RNA (sedimentation coeffi-cient 30S) and the others corresponded to 23,

16, and 4 to 5S E. coli RNA. The

infected,

rifampin-treated

cells produced predominantly

viral-induced 30S RNA. The relative incorpora-tion into the several bands is given in Table 1. Combiningthesedatawith thoseofFig.5shows that inexperiments correspondingto curvesB,C, and D, 1.4,1.7, and 1.8 timesasmuch viral RNA was produced in rifampin-treated cells as in untreated cells. We may conclude that rifampin does not adversely affect the synthesis of

Qf3-inducedRNA.

Protein synthesis. Apart from the evidence of theproduction of Q3RNAsynthetaserequiredto

Po

I0

xI

>

0-C) 0

In:

TIME

(min)

FIG. 5. Kinetics of'4C-uracilincorporation into

in-fected and uninfected cells. (A) Infected cells, no

rifampin. (B) Infectedcells,rifampinadded5 minafter infection. (C) Infectedcells, rifampinaddedattimeof infection. (D) Infected cells, rifampinz added S mill before infection. (E)

Uninfected

cells with rifampin. Cellswereinfectedat OD59o = 0.4.

achievetheRNAsynthesis (see above), itcanbe showndirectly fromkineticsofradioactiveamino acidincorporationthat thereis substantial virus-induced protein synthesis in rifampin-treated cells. Figure7shows thetimecourseof3H-lysine incorporation. It may be seen that in rifampin-treated cells

incorporation

is

underway

within10 minafter infection and continues

throughout

the entire

period

ofvirus

production.

Jockuschetal. (8) have shown that

polypeptide

I, theviral spe-cific subunit of

Q3

synthetase, as wellas matura-tion protein and coat

protein

are synthesized in infected cells treated with rifampin. We have measured the synthesis of these

Q13-specific

pro-teins in the presence and absence of

rifampin

under a variety of conditions, but

quantitative

comparisonsaredifficulttoobtain becauseofthe large contribution ofcellularprotein

synthesis

in the absence of the drug.

Figure

8 shows

gel

patterns of '4C-amino acid

incorporation

into proteinsin the

period

40 to 55minafter infection.

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4000

3000

a-

I-> 2000

CM)

0

1000

FRACTION NUMBER

FIG. 6. Gel electrophoresis ofRNA synthesized in

injected cells. (A) No rifAmpin. (B) RifAmpinadded5

minafter infection. (C) Rifampin addedat time of

[image:5.480.229.434.62.368.2]

in-fection. (D) Rifampinadded5 miil beforeinfection.

TABLE 1. Distribution of radioactivity among the

fourRNA species

Rifampin addition RNA

Noe 5min With 5min

afterPhage phage beforephage

30S 13.5b 47.6 56.3 59.1

23S 38.3 20.6 18.5 12.9

16S 25.1 13.4 8.9 9.6

4-5S 23.1 18.4 16.3 18.4

aAs calculated from Fig. 6.

b Values expressed as percentages.

Without rifampin, only coat protein is clearly evident above the large background of cellular protein synthesis (Fig. 8). We calculate that about26% of the total amino acid incorporation is into coat protein. With rifampin, the

matura-tionprotein band is evidentas well andcontains about 12% of the total gel radioactivity; the remaining 88%hois inthecoatproteinband. From these data and a knowledge oftheratio oftotal

Il4C-amino acid incorporation corresponding to

profilesAandB,wecalculate thatincorporation

intocoatprotein inthepresence of rifampinwas

60% of that in its absence. From reasonable estimates of the amount of synthesis of cellular

0 50 100

TIME (MIN)

FIG. 7. Kinetics of 3H-lysine incorporation into

in-fected and uninfected cells. (A) Infected cells, no rifampin. (B) Infectedcells, rifampinaddedattime of infection.

proteins,togetherwiththe dataofFig. 7,wecan estimate the amount ofcoat protein synthesized during other time periods. With rifampin addi-tion, we estimate a minimum of50% of normal coat protein production during these times. Similarly we can compare theeffect of rifampin addition atthetime of infection with its addition 30min afterinfection under which condition we obtain a nearly normal yield of PFU (Fig. 3). When the drug was added at infection, protein synthesis was approximately 60% of that when the drug was added 30 min after infection, and

the ratio of maturation protein to coat protein

was nearly identicalinthetwocases.From these data we conclude thatcoat protein and

matura-tionproteinaremade innearlynormalamount in

the presence of rifampin. No polypeptide I

synthesis was detected during the 40- to 50-min incorporation period, presumably because of the repressive effect of coat protein. However, we

know from gel patterns of proteins labeled at

earliertimes,aswellasfrom the

uracil-incorpora-290 J. VIROL.

I / LYSINE

A INCORPORATION

0

B

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1-U 4

0 0

FS

25 50

FRACTION NUMBER

75

FIG. 8. Gel electrophoresis of proteins made upon infection. (A) No rifampin added. (B) Rifampin added at timeof infection.

tion experiments, that polypeptide I had been synthesized.

Yield of physical particles. The results of in-corporation of RNA and protein precursors together withphage

yield

couldsuggestanormal yield ofpoorly viable particles. Thus we assayed the amount of

Q0J

antigen

in cell

lysates

after infection in the presence and absenceofrifampin. Table 2 shows that the same quantity of viable particles resulted in formation of the same amount of

antigen-antibody

complex, regardless of the addition of

rifampin

and the consequent phage titer.We knowfromthework of Hunget al. (7) that unassembled components of Qua do not contribute significantly to the complex. It may be concluded then that decrease in the quantity of PFU upon addition of

rifampin

indicatedacorresponding decreaseinthe number

of

physical phage

particles.

Phage synthesis ina rifampin-resistant mutant. To exclude the

possibility

of a direct effect of rifampin on the assembly of

Q3

phage, we allowed phage to grow on a rifampin-resistant mutant, RQ13, derived from E. coli Q13. The resistanceofRQ13 stemsfromtheresistanceof its RNA polymerase, as judged by in vitro assays (Table 3), rather than a

permeability

barrier to

rifampin. After 2 hr of growth and

overnight

lysis, the phage were plated on both Q13 and RQ13.Table4shows that in the

rifampin-resist-ant mutrifampin-resist-ant

Q3

phagegrew

nearly

as well in the presenceofrifampinasinitsabsence.

Cell viability in the presence of

rifampin.

The

phage.... ... 8.6 X 1010 3 X 1010 109

Withphage. 7.1 X 1011 3 X 100 99 5minafter

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phage. 1.2 X 1012 3 X 1010 99

TABLE 3. DNA-dependent RNA polymerizing activity

Bacteria Rifampin Enzyme activity (counts/min)

Q13 33,100

+ 11,300

RQ13 - 35,000

+ 36,300

TABLE4. Comparison ofphage yield in rifampin-sensitive and rifampin-resistant strains of

Escherichia coli

Phagegrown on Plating_(PF_U/ml)onQ13 Plating on RQ13_(PFU/ml)

RQ13. 3.6 X 1011 3.4 X 1011

RQ13 + rif 2.7 X 1011 2.3 X 1011

Q13 .3.8 X 1011 3.6 X 1011

lowyield ofPFU isnot an obviousconsequence of cell death induced by rifampin. To explain Fig. 3 and the

incorporation

data, there would havetobealag period ofabout 30 min and thena rapid

exponential

cell demise aftertheadditionof rifampin. We measured colony-forming ability of cultures growninthe presenceand in the ab-senceof

rifampin. Figure

9showsthatcellsin the presence of rifampin lose

viability

slowly and withnodetectable

lag period.

DISCUSSION

Rifampin, added at the time of

infection,

inhibits the production of

Q(3

phage. Although theyield ofPFUis

only

about

5%7

ofnormal,at

least a normal

quantity

of phage RNA and a

nearly normal quantity of phage

proteins

are

made. The phage-induced RNA is indistinguish-able from its

rifampin-less

counterpart. Almost all of it migrates in

electrophoresis

as a

single

band characteristic of

undegraded

RNA. Its increasing rate of synthesis indicates that it

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E

U)

w

:

0

z

0

30 60 90 120

TIME (MIN)

FIG. 9. Colony.forming ability ofE. coli Q13cells

in thepresence andabsence ofrifampin (rifampicin). serves as a template for viral RNA synthesis. The vigorous synthesis of phage proteins

indi-catesthat it functionsasamessenger. The phage-induced proteins also show no aberrations. The induced RNA synthesis itself signifies the pres-ence of Q3 RNA synthetase, so we know that functional polypeptide I has been synthesized and that there exists an adequate pool of the cellular components of

Q0

RNA synthetase (9,11). The smalldepression inyield ofcoatand maturation protein can be accounted for bythe gradual loss ofcells in the presence ofrifampin (Fig. 9). From the cessation of polypeptide I

synthesis late ininfection, it is evident that coat

protein plays its normal role of repressor (16). We have no assay for the biological activity of maturationprotein, but byanalogywith

matura-tionprotein ambermutantsweknow thatevenin

the complete absence ofmaturationprotein

pro-duction ofphysicalparticles is not hindered.

Thus we judge that Qf phage precursors are made but they are not assembled into physical particles.

Early addition of rifampin is essential for inhibition of phage production, but the early

events in phage protein and RNA synthesis, including the assembly of RNA synthetasefrom polypeptide I and cellular proteins, appear

normal. It isalatefunction, phage assembly,that

fails. This suggests that the effect of rifampin is

indirect and probably is mediated through a cellular function. In cells carrying a rifampin-resistant E. coli RNA polymerase, Q3 synthesis

proceeds normally in the presence of rifampin.

Thusit is theinactivityof DNA-dependent RNA polymerase that results in the failure of phage assembly. We postulate that there must be a labile cellular site or factor essential for phage assembly, and in the presence of rifampin it is

depleted.

ACKNOWLEDGMENTS

This researchwassupported by Public Health Servicegrants

AI-1466 and AI-21-942 from the National Institute of Allergy and InfectiousDiseases andbygrantAEC 1633 from theDivision ofBiologyandMedicine,AtomicEnergyCommission.

WeareindebtedtoAndy Brugger for capableandconscientious technicalassistance.

LITERATURE CITED

1.Friesen, J. D. 1969. Dependence of f2 bacteriophage RNA replicationonamino acid. J. Mol. Biol.46:349-353. 2.Fromageot, H. P. M., and N. D. Zinder. 1968. Growthof

bacteriophagef2 inE. coli treated with rifampicin. Proc. Nat.Acad. Sci. U.S.A. 61:184-191.

3. Glowacki-Strauss, E., and P. Kaesberg. 1970. Acrylamide gel electrophoresis of bacteriophage Q$; electrophoresis of the intact virions and of the viral proteins. Virology42:437-452. 4. Godson, G. N., and R. L. Sinsheimer. 1967. The replication of

bacteriophageMS2. J.Mol. Biol. 23:495-521.

5.Hartmann, G., K. Hbnikel, F. KnUsel, and J. Nuesch. 1967. The specific inhibition of theDNA-directed RNA synthesis by rifampicin. Biochim.Biophys. Acta 145:843-844. 6. Heller, E., M. Argaman, H. Levy, and N. Goldblum. 1969.

Selective inhibition of vaccinia virus by the antibiotic rifampicin. Nature (London) 222:273-274.

7.Hung, P. P., E. M. Hale, and L. R. Overby. 1970. Compar-ative antigenicities of QOphage, its structuralcomponents

and reconstituted particles.Virology 38:703-706. 8. Jockusch, H., L. A. Ball, and P. Kaesberg. 1970. Synthesis of

polypeptides directed by the RNA of phage QB. Virology 42:401-414.

9. Kamen, R. 1970. Characterization of the subunits of Q0

replicas. Nature (London) 228:527-533.

10. Katz,E., and B. Moss. 1970. Formation ofvaccinia virus

structural polypeptide from a higher molecular weight

precursors: inhibition by rifampicin. Proc. Nat. Acad.Sci. U.S.A.66:677-684.

11. Kondo, M.R.Gallerani, and C. Weissmann. 1970. Subunit

structureofQ3 replicase. Nature (London) 228:525-527. 12. Lane, L., and P. Kaesberg. 1971. Multiple geneticcomponents

inbromegrassmosaic virus. Nature (London) 232:40-43.

13. Lowry, 0. H., N. J. Rosebrough, A. R. Farr, and R. J.

Randall. 1951. Proteinmeasurementswith theFolin phenol

reagent.J.Biol.Chem. 193:265-275.

14.Maggi, N.,C. R.Pasqualucci,R.Ballotta,and P.Sensi.1966. Rifampicin:a neworallyactiverifamycin. Chemotherapia 11:285-292.

15. Mans,R.J.,andG. D. Novelli. 1961.Measurementof the in-corporationofradioactive aminoacids intoprotein bya

filter-paper discmethod.Arch.Biochem.94:48-53. 16. Skogerson, L.,D.Roufa, andP.Leder.1971.

Characteriza-tion ofthe initialpeptideofQ0RNApolymerase and

con-trolofitssynthesis. Proc. Nat. Acad. Sci. U.S.A.

68:276-279.

17. Subak-Sharpe, J. H., M. C. Timbury, and J. F. Williams. 1969. Rifampicin inhibitsthegrowthofsomemammalian viruses. Nature (London) 222:341-345.

18. Sugiyama, T. 1969. Translationalcontrol ofMS2RNA

cis-trons.ColdSpringHarborSymp. Quant.Biol.34:687-694. 19. Wehrli, W., R. Knusel,K.Schmid,and M.Staehelin. 1968.

Interactionofrifamycin with bacterial RNA polymerase.

Proc. Acad. Nat.Sci.U.S.A. 61:668-673.

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