Proc. Nat. Acad. Sci. USAVol. 71, No. 12, pp. 4892-4896, December 1974

Reverse Transcriptase: Correlation of Zinc Content with Activity(avian myeloblastosis virus/RNA-directed DNA polymerase/RNA virus)

BERNARD J. POIESZ, GITA SEAL, AND LAWRENCE A. LOEB

The Institute for Cancer Research, Fox Chase Center for Medical Sciences, 7701 Burholme Ave., Philadelphia, Pennsylvania 19111

Communicated by Thomas F. Anderson, August 14, 1974

ABSTRACT Evidence is presented that DNA poly-merase of avian myeloblastosis virus has an obligatoryzinc requirement for activity. Previous studies indicatethat the purified polymerase contains zinc in a stoichi-ometry of about 1 g-atom/mole. We now find that theenzyme-bound zinc is exchangeable with radioactive65Zn; after isoelectric focusing, the radioactive 65Zn iscoincident with polymerase activity. Dialysis of the 65Zn-labeled polymerase against the chelator, 1,10-phen-anthroline, results in a progressive loss of radioactive 65Znand polymerase activity. Thereupon, incubation of theinactivated enzyme with Zn2+ fully restores activity.Thus, the DNA polymerase present in an oncogenic RNAvirus, like animal DNA polymerases, can be rigorouslyclassified as a zinc metalloenzyme. DNA polymerase ofavian myeloblastosis virus is inactivated by 1,10-phen-anthroline at a much faster rate than the bacterial andanimal DNA polymerases that have been tested. It may,therefore, be possible to inactivate selectively DNA poly-merases from animal tumor viruses by brief exposure toappropriate metal chelators.

Recent metal analysis and inhibition by chelating agentssuggest that DNA polymerases are zinc metalloenzymes.Homogeneous DNA polymerase I of Escherichia coli hasbeen shown to contain 1.0 + 0.15 g-atom of zinc per mole ofenzyme (1, 2). Removal of zinc is accompanied by a pro-portional loss of activity; addition of zinc to the apoenzymeresults in full restoration of activity (2). DNA polymerasesfrom sea urchin nuclei (1) and T4 bacteriophage (2) have alsobeen shown to contain zinc in stoichiometric amounts. Inaddition, partially purified DNA polymerases from phylo-genetically divergent sources are inhibited by metal chelators(1-5). These correlated findings suggest that DNA poly-merases generically are zinc metalloenzymes.

Since the DNA polymerases present in RNA tumor virusescan copy RNA templates, it was initially thought that themechanism for catalysis of viral polymerases was differentfrom that of cellular polymerases. For this reason they weretermed "reverse transcriptases." However, the ability to copyRNA does not appear to be a unique property of viralenzymes; recently E. coli DNA polymerase I has been shownto copy faithfully a variety of natural RNA templates (6-8).Also, metal and substrate requirements for catalysis by viraland cellular polymerases appear to be similar, if not identical.It is therefore crucial to determine if viral polymerases arealso zinc metalloenzymes. Zinc has been found in the purified"reverse transcriptase" from avian myeloblastosis virus byus, using atomic absorption spectroscopy (9), and indepen-dently by Auld et al., using microwave induced emissionspectrometry (10, 11). However, in order to establish thisenzyme as a zinc metalloenzyme, it is necessary to show an

obligatory requirement of zinc for enzymatic activity (12).We now report evidence for such a requirement. The removalof zinc from avian myeloblastosis DNA polymerase resultsin loss of activity. The readdition of zinc entirely restoresactivity.

MATERIALS AND METHODSPurification of DNA Polymerases. Avian myeloblastosis

virus (AMV) was initially isolated from the plasma of in-fected chickens by velocity and equilibrium centrifugation asdescribed (13). The holoenzyme was purified from the isolatedvirus by chromatography on DEAE-cellulose and phospho-cellulose and by sedimentation in glycerol gradients asdescribed by Kacian and Spiegelman (14). Both the holo-enzyme and the isolated a-subunit were also obtained by theprocedure of Hurwitz and Leis (15). In both purifications,EDTA was omitted from all solutions. The purified poly-merases were stored under liquid nitrogen in buffer A: [20%glycerol, 50 mM Tris -HCl (pH 8.0), 0.6 mM dithiothreitol].DNA polymerase from Rauscher murine leukemia virus waspurified by chromatography on Sephadex and phospho-cellulose (15). The large DNA polymerase from phytohemag-glutinin-stimulated human lymphocytes (16) was purified asoutlined by Lewis et al. (17). DNA polymerase of sea urchinnuclei (18) and E. coli DNA polymerase I (2, 19) werepurified as described.

Contamination. Solutions were made "metal-free" by eitherpassing them over columns packed with the chelating resin,Chelex-100, (2), or by repetitively mixing them directly withone-tenth volume of Chelex beads and removing the Chelex bycentrifugation (9). Each of the reagents used in the assay orin the final step of the purification contained less than 0.1 ,uMzinc, as determined by atomic absorption spectroscopy. Alllaboratory vessels were washed with 0.1 mM o-phenanthrolineand distilled water to render them "zinc-free," and all solu-tions were stored in polyethylene bottles.

Electrofocusing and Atomic Absorption Spectroscopy. Elec-trofocusing was carried out in plastic minicolumns by amodification of the technique outlined by Cohen et al. (20).The columns consisted of two plastic pipettes (Falcon no. 7529)connected at the tips by Tygon tubing. The dense solution[0.2 M phosphoric acid in 80% (w/v) sucrose] surroundedthe anode and filled the pipette, the connecting tubing, andthe tip of the adjoining pipette. A 5-ml 10-40% glycerolgradient, containing 5 mM 2-mercaptoethanol and 0.8%carrier ampholytes (pH 3-10), was layered upon the top of thedense solution in the tip of the second pipette. The protein tobe analyzed [1-100 ,ug in 0.2 ml of 20% glycerol, 20 mMdithiothreitol, 20 mM Tris HCl (pH 7.8)] was introducedinto the middle of the gradient from the top of the pipette by

4892

Abbreviation: AMV, avian myeloblastosis virus.

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Zinc and Reverse Transcriptase 4893

means of thin Tygon tubing. Finally, 2 ml of 2% (v/v)ethylenediamine was placed at the cathode end of thegradient. Platinum electrodes were inserted in the top ofeach pipette, and electrofocusing was carried.out for 2 hr at600 V at 4°. Fractions (0.15 ml) were collected into wellsin plastic plates from the anode end of the gradient. Greaterresolution can be obtained by electrofocusing for a longer time,4-6 hr; however, zinc is progressively leached out of theapparatus and increases the background. Zinc content wasdetermined within 2 hr on l-Ml samples of each fraction with aVarian Techtron atomic absorption spectrometer equippedwith a carbon rod attachment. All analyses were carried outwith a noise level equivalent to less than 0.03,M zinc. Fe,Cu, and Ni were analyzed by the same procedure.

DNA Polymerase Activity. The reaction mixture of 0.05ml contained 50 mM Tris-HCl (pH 8.0); 0.6 mM dithio-threitol; 0.1 mM KCl; 5 mM MgCl2; 1 Asg of poly(rA) oligo-(dT)12_18 (ratio 1:1.5); 10 Ag of bovine serum albumin; 50,uM[3H]dTTP or [a-32P]dTTP; and 0.1-20 nM AMV DNApolymerase. Incubation was for 30 min at 370, and incorpora-tion into an acid-insoluble and alkali-resistant product wasdetermined (13). When o-phenanthroline was present in thereaction mixture, the enzyme was added immediately beforeincubation. Protein was determined by the fluorescaminemethod of Bohlen et al. (21), using homogeneous E. coli DNApolymerase I (2) as a standard.Plasma of infected chickens containing avian myeloblastosis

virus was a generous gift of Dr. J. W. Beard. Radioactivenucleotides and 65Zn were obtained from New EnglandNuclear Corp. Poly(rA) and oligo(dT)12_18 were productsof Collaborative Research, Waltham, Mass. Glycerol (spectro-quality grade) was obtained from Matheson Coleman andBell, and ampholines were from LKB.

RESULTS

Inhibition of AMV DNA Polymerases by a Metal Chelator.The inhibition of DNA polymerases by chelating agents sug-gests that these enzymes contain divalent cations essentialfor their activity. The chelating agent 0.8 mM 1,10-phen-anthroline (o-phenanthroline) has been shown to cause a 90%reduction in AMV DNA polymerase activity (9). This inhibi-tion was not due to chelation of the added divalent cationsince in these assays Mg2+ was present in excess (10 mM)and o-phenanthroline has a 105 less affinity for Mg2+ thanZn2+ (22). Figure 1 shows that the inhibition of AMV DNApolymerase by o-phenanthroline is time-dependent. Theinitial inhibition can be lowered by diluting the o-phen-anthroline from 0.8 mM to a final concentration of 0.16 mM.With time, however, simple dilution is insufficient to reversethe inhibition of polymerase activity.The nature of the immediate inhibition ofAMV DNA poly-

merase by o-phenanthroline was analyzed by observing theeffects of varying separately each of the components in theassay. Fig. 2A shows that the initial effect of o-phenanthrolineon AMV DNA polymerase is noncompetitive with the sub-strate, dTTP. The reciprocal activity plotted against thereciprocal concentration of the poly(rA) oligo(dT) complexin the absence and the presence of different amounts of o-phenanthroline is shown in Fig. 2B. Since the inhibition isnot competitive, the results suggest that o-phenanthrolineand the template-initiator complex, poly(rA) -oligo(dT), donot interact at the same site on the enzyme. This is in con-trast to studies on terminal transferase (23) and sea urchin

< 60

C

0 40 -20 ,~~Activity in 0.16mM

,I

30 60 90 120 150 180

Preincubation with 0.8 mM o-phenanthroline (min)

FIG. 1. Time-dependent inhibition of DNA polymeraseactivity by 1, 10-phenanthroline. AMV DNA polymerase (2,ug/ml) in buffer A was preincubated with 0.8 mM o-phen-anthroline at 150 for the times indicated. Then its activity wasdetermined using a standard assay mixture containing o-phen-anthroline at a final concentration of 0.16 mM or 0.8 mM. Onehundred percent activity corresponds to 200 pmol of ["2P]dTMPincorporated for 15 min at 37°.

nuclear DNA polymerase (1) in which the initiator or thetemplate-initiator complex, respectively, appears to becompetitive with o-phenanthroline.

Presence of Zinc in AMV DNA Polymerase. Purified AMVDNA polymerase consists of two polypeptide chains in equi-molar amounts: an active a-subunit (molecular weight of60,000-70,000) and a a-subunit (molecular weight of 105,000-115,000) of unknown function (13, 14, 24). Precise chemicaldetermination of zinc is difficult because the enzyme is obtain-able only in relatively small amounts, and zinc, being a ratherubiquitous cation, is a contaminant of most reagents andlaboratory vessels. For removal of any contaminating un-bound cations, purified AMV DNA polymerase was subjectedto isoelectric focusing, before the content of zinc and othermetals was measured by atomic absorption spectroscopy.As previously reported (9), zinc is coincident with poly-

merase activity after the holoenzyme, containing both thea- and ,B-subunits, is subjected to isoelectric focusing (Fig. 3).No detectable amounts of Fe, Cu, or Ni were found in theholoenzyme by atomic absorption spectroscopy. The sen-sitivity of the method is such that if these metals are present,the stoichiometry is less than 0.2 g-atom/mole. It is unlikelythat during electrofocusing the enzyme sequestered zinc bynonspecific metal-protein binding, because electrofocusing ofbovine serum albumin (pI 5.5) at a concentration 50 timesthat of the enzyme produced no zinc peak. Moreover, thezinc peak cannot be explained by coincidental binding ofzinc to particular carrier ampholytes because no zinc peakappears in the same fractions from a column to which noprotein was added. From the specific activity of a homo-geneous sample of polymerase assayed at the same time, weestimate the polymerase concentration of the peak fractionto be 6.5 MM. The zinc concentration is about 8 ,uM, giving.a stoichiometry of 1.3 g-atoms of Zn per mole of enzyme.A similar analysis of the active a-subunit yielded a value of1.2 g-atom/mole (9).

Exchangeability of Enzyme-Bound Zinc. Even though theenzyme-bound zinc is not removable by electrofocusing, itappears to be exchangeable with inorganic Zn2+. Exchangewas accomplished by incubating the purified polymerase with

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4894 Biochemistry: Poiesz et al.

0'cvEaT0

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0-

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o

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100

80 .

60 "

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Ao-phen.2 0.2mM

.,hn- .IM

o-ph.o0h1n.m 0A IA o-oe.0

0 20 40 60 0 o10 200 300

1mM dTTP

1poly(rA),oligo(dT) (mg/mi)

FIG. 2. Noncompetitive inhibition by 1,10-phenanthroline (o-phen.) with respect to the template-initiator complex and substrate.Polymerase activity was determined as given in Materials and Methods. (A) The concentration of poly(rA)-oligo(dT) was 20 Mg/ml.(B) The molecular ratio of poly(rA) to oligo(dT) was 1 to 1, and the concentration of [c-"2P]dTTP and MgC12 was 0.1 mM and 7 mM,respectively. Incubation was for 15 min and was initiated upon the addition of the enzyme.

excess OZn2+, extensively dialyzing the mixture against"zinc-free" reagents, and reisolating the enzyme by isoelectricfocusing. As seeen in Fig. 4, the radioactive 65Zn profilecoincides with polymerase activity and there is no accumula-tion of 65Zn at the cathode, indicating that the unbound 65Znwas removed by dialysis and the remaining OZn is tightlybound to the enzyme. In contrast, when similar experimentsare carried out with a zinc-free protein, bovine serum albumin

< Inorganic2 Zn/

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Cu Fe~~~z^Iinner~~~~~ I *

f I

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2

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FIG. 3. Isoelectric focusing of AMV DNA polymerase.AMV DNA polymerase (50 ,g) was subjected to electrofocusingin "mini-columns," as detailed in Materials and Methods. DNApolymerase activity was measured on 5SMl aliquots of eachfraction, and metal was analyzed. The position of bovine serum

albumin (BSA) and inorganic Zn was determined in separateexperiments. The pH distribution was essentially the same in allthree gradients.

(1), a small amount of uZn is not removed by dialysis. Uponelectrofocusing the remaining 65Zn is not tightly bound sinceit migrates to the cathode and is not coincident with thealbumin, pI 5.5 (Fig. 4, lower part).

ECCM

00o.< CDw,,AdO)i }

10

5

A-x

0.

FRACION

FIG. 4. Isoelectric focusing of "'Zn-labeled AMV DNA

polymerase. AMV DNA polymerase (5 gg) or bovine serum

albumin (50 Msg) in 20% glycerol, 0.5 M Tris HC1 (pH 8.0), and0.6 mM dithiothreitol was incubated with 50 uCi of 16Zn2+ for

12 hr at 00. After dialysis against buffer A for 24 hr, each samplewas subjected to electrofocusing separately. 65Zn concentrationwas determined on 25 ;l of each fraction on a gamma spectrometerat 00, and the same sample was then used to measure polymeraseactivity with [a-32P]dTTP as the radioactive substrate. Theinitial amount of "Zn corresponds to 600 dpm/25 M1.

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Zinc and Reverse Transcriptase 4895

Against 0.8 mM o-phen..~100

C

510vv5Zn ~ Against Against

W 50 C^10lmM Bfe

0 1 2 3 4 1 4

Dialysis (hr)

FIG. 5. Dialysis of 65Zn-labeled AMV DNA polymeraseagainst o-phenanthroline. One milliliter of buffer A containing 3jg of 65Zn-labeled AMV DNA polymerase (Fig. 4) was first sub-jected to open dialysis against "zinc-free" buffer A. Then it wasdialyzed against a similar buffer, which contained 0.8 mM o-phenanthroline for 4 hr at 100. At the time indicated, the amountof 65Zn remaining in the dialysis tubing was determined by gammaspectroscopy, and DNA polymerase activity was determined on10-Mul aliquots. At 4 hr, the retentate was divided into equal por-tions and dialyzed either against buffer A (0.2MAM Zn) or againstthe same buffer with added 0.4 mM ZnCl2. One hundred percentcorresponds to 200 pmol of dTTP incorporated in 15 min at 37°.o-phen., o-phenanthroline.

Correlation of Zinc with Polymerase Activity. In order todemonstrate unambiguously that an enzyme is a zinc metallo-enzyme, it is necessary to show that the bound zinc is requiredfor activity. Since the viral polymerase is only available inminute amounts, it was necessary to quantitate zinc contentby measuring radioactivity using the 65Zn-labeled polymerase.In the absence of o-phenanthroline, dialysis against "zinc-free" buffer was without effect for as long as 8 hr. As shown inFig. 5, upon addition of. o-phenanthroline to the buffer, theactivity of the 65Zn-labeled polymerase decreases at a ratesimilar to that observed with the nonlabeled enzyme (Fig. 1).The loss of activity is not reversible by simple dilution, sincein this experiment (Fig. 5), the o-phenanthroline concentra-tion in the dialyzed enzyme was decreased 10-fold in theassay. The initial loss of 65Zn from the enzyme during dialysisappears slower than the decrease in polymerase activity. Thelag in the loss of 65Zn can be accounted for by time requiredfor 65Zn and o-phenanthroline to diffuse across the dialysismembrane. By 2 hr, both the polymerase activity and the zinccontent decreased 75%. Most importantly, dialysis against0.4 mM ZnCl2 results in the full restoration of polymeraseactivity within 1 hr, while dialysis against a buffer containingonly contaminating amounts of Zn (0.1 MM) results in a slightincrease in polymerase activity. In other experiments it wasfound that if the polymerase was inactivated by more than80% or maintained inactive for more than 2 hr, only partialrestoration of activity was achieved by dialysis against zinc.It is possible that the zinc-free apoenzyme is unstable andeasily inactivated irreversibly. After total inactivation byo-phenanthroline, complete restoration of polymerase activitycan be achieved by dialysis against zinc in a nitrogen atmo-sphere. So far, we have not been able to achieve significantrestoration of polymerase by dialysis even under nitrogenof the "zinc-free" enzyme against other divalent cations(Co2+, Mn2+, Mg2+, Ni2+, Hg2+, Cd2+, or Fe2+).

Human Lymphocyte

E 9 C

E 0: Sea Urchin >

\ RLV

AMV-I0 10 20 30 40

Dialysis Against 0.8 mM o-phenanthroline (hr)

FIG. 6. Time-dependent inactivation of different DNApolymerases by 1,10-phenanthroline. Preparations of DNApolymerase from AMV (10 Mug), Rauscher murine leukemia virus(RLV) (3Mug), sea urchin nuclei (0.1 ,ug), E. coli DNA polymerase I(0.02 ug), and phytohemagglutinin-stimulated human lympho-cytes (10,ug) each in 0.3 ml of buffer A were dialyzed in separatebags against a common reservoir of 500 ml of buffer A containing0.8 mM o-phenanthroline at 5°. When indicated, DNA polymer-ase activity was determined with lO-,l aliquots in a reactionmixture of 100 Ml. AMV DNA polymerase and RLV DNApolymerase activity was determined with poly(rA)- oligo(dT)as given in Materials and Methods. E. coli DNA polymerase I (2),sea urchin nuclear DNA polymerase (18), and lymphocyte DNApolymerase (6-8S) (16) were assayed as described with maxi-mally activated DNA as a template. In control experiments, therate of inactivation of AMV DNA polymerase with o-phen-anthroline was the same when the assays were carried out withpoly(rA)-oligo(dT) or with activated DNA as templates.

Rate of Inactivation of Different DNA Polymerases. A com-parison of the rate of inactivation of different DNA poly-merases by o-phenanthroline is shown in Fig. 6. Each enzymewas dialyzed against 0.8 mM o-phenanthroline at 50, andactivity was determined after the enzymes were diluted sothat the maximal concentration of o-phenanthroline in thefinal assay was noninhibitory (less than 80,MM). The DNApolymerases from Rauscher murine leukemia virus and avianmyeloblastosis virus were inactivated within 90 min. Onthe other hand, the time required for inactivation of thecellular enzymes was an order of magnitude greater.

DISCUSSION

The obligatory requirement for enzyme-bound zinc for theactivity of DNA polymerase from AMV establishes thisenzyme as a zinc metalloenzyme. It strengthens the hypothesisthat all DNA polymerases are zinc metalloenzymes (1) andsuggests that the fundamental mechanism of catalysis byDNA polymerases from oncogenic RNA viruses is the sameas that ofDNA polymerases from animal cells.The stoichiometry between zinc and AMV DNA poly-

merase deserves some comment. After the holoenzyme issubjected to isoelectric focusing, analysis for zinc by atomicabsorption spectroscopy yields about 1.3 g-atom of zinc permole of enzyme. A similar analysis of the isolated active a-subunits yields a value of 1.2. Auld et al. (10, 11) reported astoichiometry-of 1.8 to 2.0 upon analysis of the holoenzyme bymicrowave induced emission spectroscopy. Both methodsoffer equal sensitivity. It can be argued that the lower zinc

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4896 Biochemistry: Poiesz et al.

content in our studies results from contamination of thepolymerase with other proteins or from removal of the zincfrom the polymerase during electrofocusing. Alternatively,one of the zinc atoms might not be required for activity. Thehomogeneous AMV DNA polymerase used in our studies(13) had a specific activity of about 5000 nmoles of dTTPincorporated per 30 min/mg at 370, which is equal to orgreater than that reported by Auld et al. (11). In initialstudies, E. coli DNA polymerase I was reported to have 1.7-2.5 g-atom of zinc per mole of enzyme (1). However, afterprolonged dialysis against metal-free buffers, the zinc contentof the active enzyme was consistently found to be 1.0 i 0.15g-atom/mole (2). There was a proportional reduction ofpolymerase activity and zinc content only after the remainingzinc was removed by dialysis against o-phenanthroline.The obligatory requirement for zinc of E. coli DNA poly-

merase I and of AMV DNA polymerase suggests either thatthe metal participates in catalysis or that the metal is astructural component of the enzyme necessary for activity.Kinetic and binding studies on related enzymes suggest thatenzyme-bound zinc coordinates with the 3'-OH terminus ofthe initiator. With sea urchin DNA polymerase, inhibition byo-phenanthroline is competitive with DNA in the reactionmixture (1). A similar competition was initially observed byChang and Bollum (23) with calf thymus terminal transferase,an enzyme that polymerizes nucleotides on to the 3'-OHterminus of a DNA initiator but does not require a DNAtemplate. E. coli RNA polymerase has also been reported tocontain zinc (25), and binding studies using equilibrium dialy-sis suggest that o-phenanthroline interferes with coordinationof the enzyme to the initiating purine nucleotide triphosphate.Furthermore, studies on E. coli DNA polymerase I usingnuclear quadrupolar relaxation indicate that oligonucleotidesbut not deoxynucleotide triphosphates can displace Br- ionsfrom the enzyme-bound zinc (2). In contrast to these studies,we find that with AMV DNA polymerase the inhibition byo-phenanthroline is not competitive with the template-initiator, poly(rA) - oligo(dT), suggesting a possible differencein the mechanism of inactivation.

Metabolic and nutritional studies (26, 27) as well as theresponse of cells to chelators (28-30) have long suggested arole of zinc ions in DNA replication. The presence of zinc inDNA polymerases could explain this cellular requirement.Differences in zinc metabolism between normal and leukemiclymphocytes led to a hypothesis on the role of zinc unique tothe pathophysiology of leukemia (10). However, the presenceof zinc in both cellular and viral DNA polymerases arguesagainst a simple relationship between altered zinc metabolismand leukemia. An understanding of the quantitative differ-ences in the rate of removal of zinc from viral and cellularpolymerases may allow one to use enzyme-bound zinc as achemotherapeutic target. Thus, the preferentially rapidinactivation of the DNA polymerases from two RNA tumorviruses by o-phenanthroline as compared with cellular poly-merases suggests the possibility of interfering selectively withviral replication by briefly exposing infected cells to metalchelators.

We thank Dr. A. S. Mildvan for generous counsel. This in-vestigation was supported by grants from the National Institutesof Health (CA-12818, CA-11524) and The National ScienceFoundation (GB-36812), by grants to this Institute from theNational Institutes of Health (CA-06927), (RR-05539), andby an appropriation from the Commonwealth of Pennsylvania.We thank Dr. J. W. Beard and Dr. M. A. Chirigos for generouslysupplying avian myeloblastosis virus. B.P. is a medical studentat the University of Pennsylvania. L.A.L. is also a member of theDepartment of Pathology, University of Pennsylvania.

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