Proc. Natl. Acad. Sci. USAVol. 75, No. 12, pp. 6263-6267, December 1978Microbiology

Normal human placentas contain RNA-directed DNA polymeraseactivity like that in viruses

(retrovirus/virus cores/isopycnic centrifugation/reverse transcriptase/DNA nucleotidyltransferase)

JAY NELSON*, Jo-ANN LEONG*t, AND JAY A. LEVYt*Department of Microbiology, Oregon State University, Corvallis, Oregon 97331; and the *Cancer Research Institute and Department of Medicine,University of California, San Francisco, California 94143

Communicated by Charlotte Friend, September 18,1978

ABSTRACT Extracts from over 100 normal human pla-centas have been examined for RNA-directed DNA polymerase(DNA nucleotidyltransferase, EC 2.7.7.7) activity. More than80% of these placentas contained this enzyme activity, whichbanded at a density of 1.15-1.17 g/ml in sucrose. After heattreatment, this enzyme activity was shifted in density to1.22-1.24 g/ml. The enzymatic activity was greater with(rq 0(dGh) j8 than with (dC).i(dG)18 and was not stimulatedby (dG)12 18 alone. The product of the endogenous reaction,which was sensitive to RNase, had the characteristics of a smallDNA associated with a large RNA by hydrogen bonding. Elec-tron microscopic inspction of the material with a density of1.15-1.17 g/ml revealed numerous retrovirus-like particles withcentral electron-dense cores and double-membraned envelopes.The enzyme may be associated with the retroviruslike particlesnoted in the trophoblast layer of some human placentas.

Endogenous B, C, and D type viruses have been identified intissues of many vertebrates, including humans (cf. refs. 1 and2). The viruses were first detected in solid tumors and leukemias(cf. ref. 3) but have since been observed in normal tissues ofseveral different animals as well (cf. ref. 4). They have beendescribed in syncytiotrophoblast cells in normal rhesus monkey,marmoset, and baboon placentas (5-8). In humans, particlesresembling endogenous RNA viruses have been detected byelectron microscopy in embryos (9) and placentas (5, 10-13).

Spiegelman and associates (14) noted the association ofRNA-directed DNA polymerase (RDDP; DNA nucleotidyl-transferase, EC 2.7.7.7) with virus-like particles in humantissues. In their studies, the enzyme was detected only in humanmalignancies. We report here that normal human placentascontain this RDDP activity associated with particles possessingthe density characteristics of endogenous RNA viruses. Webelieve this activity resides in the virus-like particles detectedin the trophoblast layer of human placentas (5, 10, 13).

MATERIALS AND METHODSExtraction of Human Placental Tissues. Human placentas

were obtained within 1 hr after delivery from Good SamaritanHospital, Corvallis, OR. Tissues were used immediately orstored at -700C. The trophoblast layer facing the maternal sideof the placenta was excised and approximately 100 g of thistissue was rinsed in ice-cold STE buffer (0.01 M Tris-HCl, pH7.8/0.1 M NaCl/0.001 M EDTA). This tissue was then mincedand the fragments were suspended in two volumes of the STEbuffer and homogenized on ice in a VirTis homogenizer(maximum speed) in 10-sec episodes for 10-20 times. Thesuspension was centrifuged at 2000 X g for 10 min at 40C, andthe resulting supernatant was centrifuged at 10,000 X g for 10min at 4°C. The supernatant was collected and then layered

onto a 20-ml column of 20% sucrose in STE and centrifuged at95,000 X g for 60 min at 4°C in a Beckman type 35 rotor. Theresulting pellet was resuspended in STE and homogenized byforcing the suspension through an 18-gauge needle repeatedlyand then a 23-gauge needle twice. The suspension was thenlayered onto a linear gradient of 20-60% sucrose (wt/vol) inSTE with a 65% sucrose cushion and centrifuged for 13 hr at90,000 X g, 4°C, in a BeckmanSW 41 rotor. The gradient wascollected from below into 25 equal fractions. Unless indicatedotherwise, each fraction was assayed for (rC)n - (dG)1218 tem-plate-primer-directed DNA polymerase activity. PNE buffer(0.1 M NaH2PO4, pH 7.8/0.1 M NaCl/0.001 M EDTA) wasused in some experiments because it appeared to stabilize theenzyme activity.

Assays for DNA Synthesis with Homopolymerie-Oligo-meric Template-Primers. A 20- to 30-Al aliquot was taken fromeach fraction collected from a sucrose gradient after isopycniccentrifugation of the placenta extract. Each sample was dis-rupted by the addition of 10 pgl of buffer E [0.267 M Tris-HCI,8.3/41.7% (vol/vol) glycerol/0.55 M KCI/1.67% NonidetP40/0.03 M dithiothreitol/0.4 M NaCl) for 15 min at 0°C. A75-,Ml reaction mixture was then established that contained 50,ug of the appropriate template-primer (P-L Biochemicals) 6mM Mg(CH3COO)2, and 0.18 gmol of [3H]dGTP (3000-4000cpm/pmol) (ICN Pharmaceuticals, Irvine, CA). After 30 minat 37°C, the reaction was stopped by the addition of 10% tri-chloroacetic acid/0.01 M sodium pyrophosphate. The precip-itate was collected on Millipore cellulose nitrate filters and itsradioactivity was measured in a Beckman LS-8000 liquidscintillation counter. The enzyme activity was generally stableat 4°C in sucrose for 4-6 days. We were unable to preserveactivity by storage at -20°C in either 50% glycerol/i mg bo-vine serum albumin per ml or 10% fetal calf serum.

Effect of RNase on the Endogenous DNA PolymeraseActivity. Fractions obtained after equilibrium density gradientcentrifugation of placental extracts were assayed for endoge-nous DNA polymerase activity. A 30-,l aliquot of the appro-priate fraction was diluted to 78.5 ,l with H20 and pretreatedwith 10 Ml of lysing buffer (0.5 M Tris-HCI, pH 7.8/0.1%Nonidet P40/0.2 M dithiothreitol/0.6 M NaCl/33 mMMg(CH3COO)2 and 10 /l of [3H]dTTP (New England Nuclear)(0.02 mM, 3200 cpm/pmol) for 5 min at room temperature.The reaction was started by the addition of 0.5 Mg each of un-labeled dATP, dGTP, and dCTP (1.5 Mul). Duplicate sampleswere incubated with boiled pancreatic RNase A (320 ,g/ml,Sigma) and Ti RNase (60 Mg/ml, Sigma) for 15-30 minm 37°C.All reactions were stopped by the addition of 1 ml of 0.1 M so-dium pyrophosphate and 1 ml of 10% trichloroacetic acid. After15 min at 0°C, the acid-insoluble radioactivity was collected

Abbreviation: RDDP, RNA-dependent DNA polymerase; STE,saline/Tris/EDTA buffer.t To whom all correspondence should be addressed.

6263

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Proc. Natl. Acad. Sci. USA 75 (1978)

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FIG. 1. RDDP activity in the microsomal pellet from the extractsof a human placenta. The microsomal pellet was fractionated bysedimentation to equilibrium in a 20-65% sucrose gradient. B, bottomof gradient. A 30-,ul aliquot of each fraction was assayed for RDDPactivity (0) with (rC)Q-(dG)12 18. Density (-) was determined byrefractometry.

on Millipore filters and measured in a liquid scintillationcounter.

Electron Microscopy. Fractions corresponding to peaks ofRDDP activity at 1.15-1.17 g/ml in sucrose were pooled, di-luted with phosphate-buffered saline, and centrifuged at100,000 X g for 60 min. The supernatant fluid was removedand the pellet was fixed with glutaraldehyde. Subsequently,the pellet was postfixed with osmium tetroxide, dehydrated,and embedded in Epon/araldite (Electron Microscopy Sciences,Fort Washington, PA). After thin sectioning, the material wasstained with lead citrate and uranyl acetate and examined ina Phillips EM300 electron microscope by A. Soeldner.

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Table 1. Summary of template-primer requirements of particlesobtained from human placental extracts*

LabeledTemplate-primer precursor Activityt

(rC)n-(dG)12 18 dCTP 1.83(rCm)n-(dG)12 18 dGTP 1.17(rA)n-(dT)12_18 dTTP 2.56(dG)12 18 dGTP 0.05(dC)n (dG)12-18 dGTP 0.23Endogenous dTTP 1.8770S RNA (avian myeloblastosis virus) dGTP 3.29

* Reactions were performed under standard reaction conditions.t Enzyme activity is expressed as pmol of 3H-labeled deoxyribonu-cleoside triphosphate incorporated per 100 ,ul of reaction mixturein a 30-min reaction.

Analysis of Endogenous Reaction Product. An endogenousDNA polymerase reaction mixture was incubated with [3H]-dTTP (0.02 mM, 41,000 cpm/pmol) and [3H]dGTP (0.001 mM,20,000 cpm/pmol) at 37°C for 30 min and the reaction wasterminated by the addition of sodium dodecyl sulfate and NaClto final concentrations of 1% and 0.4 M, respectively. After theaddition of an equal volume of phenol/cresol/chloroformmixture (7:1:7, vol/vol), containing hydroxyquinoline (0.185g/100 ml), the mixture was shaken at room temperature for 5min and centrifuged at 2000X g for 5 min. The aqueous phasewas reextracted once more and then analyzed by cesium sulfateequilibrium centrifugation in a Beckman SW 50.1 rotor,110,000 X g for 66 hr, 15°C.

RESULTSDetection of RDDP Activity in Placental Extracts. The

gradient fractions obtained from extracts of the trophoblastlayer of human placentas, banded to equilibrium in sucrose,were assayed for RDDP activity with (rC)n-(dG)12-18. Most ofthe RDDP activity banded at 1.15-1.17 g/ml (Fig. 1). An ad-ditional peak of enzymatic activity at 1.22-1.25 g/ml was ob-served in some cases, and in certain placental extracts a distinct

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FIG. 2. RDDP activity in human placentas and number of pregnancies. Each point represents the (rC)n-(dG)12 18-directed activity for a

different placenta. Activity is measured by [3H]dGTP incorporation into acid-precipitable material under standard assay conditions for fractions

at 1.21-1.24 g/ml (A), 1.15-1.17 g/ml (B), and 1.12-1.13 g/ml (C).

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6264 Microbiology: Nelson et al.

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Proc. Natl. Acad. Sci. USA 75 (1978)

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MI/tubeFIG. 3. RDDP activity in placental extract banding at 1.15-1.17

g/ml in the presence of (rCm)n-(dG) 121s1 (A) Enzyme dependencyof the reaction. The reaction mixture with various amounts of pla-cental extract was incubated at 370C for 30 min. (B) Kinetics of theDNA synthesis. DNA polymerase activity was assayed with 30 j1 ofthe pooled fractions at 1.15-1.17 g/ml as enzyme for the indicatedlengths of time.

peak of enzymatic activity was detected at 1.12 g/ml. Thedistribution of RDDP activity in over 100 placentas is shownin Fig. 2. The amount of enzyme activity detected did notcorrelate with the number of pregnancies or the age of thewoman. The absence of activity in some placentas appears tobe related to the presence of an enzyme inhibitor. This inhibitoralso prevents the RNA-dependent DNA synthesis by Rauschermurine leukemia virus (unpublished data).The gradient fractions were also analyzed with the tem-

plate-primers (rA) -(dT)12s18, (dC)n-(dG)12 18, and (dG)12 18alone (Table 1). The (rCm)n (dG)12 18 template-primer isspecific for viral RDDP (15), and enzymatic activity detectedwith this template-primer coincided with activity assayed with(rC)n (dG)i2 18. Assays done with (dC)n-(dG)1218 'or (dG)12 18alone did not detect any enzymatic activity at 1.15 to 1.17 g/ml.Divalent cation was required for the reaction with (rC)n-(dG)12i18; Mg2+ was preferred over Mn2+ under the reactionconditions employed.

Incorporation of labeled dGTP into acid-insoluble material

5

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FIG 4. Effect of RNase treatment on the endogenous polymerasereaction. Material obtained at 1.15 g/ml was used to catalyze an en-dogenous DNA polymerase reaction. The kinetics ofDNA synthesiswith (0) and without (0) pretreatment with RNase A and T1 areshown.

in the exogenous reaction depended on concentration of theenzyme preparation (Fig. 3A); the kinetics of incorporationwere linear for at least 30 min (Fig. 3B).RNase Sensitivity of the Endogenous DNA Polymerase

Activity. The pooled fractions of the 1.15-1.17 g/ml densityregion of the sucrose gradient had endogenous activity in thepresence of [3H]dTTP and three other nonradioactive deoxy-ribonucleotide triphosphates. The peak of endogenous activityoccurred with fractions from the density region 1.15-1.17 g/ml.The rate of incorporation was linear for the first 30 min andincorporation of radioactivity into acid-insoluble material wasRNase sensitive (Fig. 4).Conversion of Activity to Core Density. Upon heating at

370C, the structures banding in sucrose at 1.15-1.17 g/ml were

-0 4 8 12 16 20 24

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Fraction FractionFIG. 5. Production of "cores" from human placenta extracts. The microsomal pellet was obtained from 140 g of placental tissue from a woman

with 11 previous pregnancies. The preparation was layered on a 20-65% linear sucrose gradient and centrifuged at 150,000 X g for 12 hr, 40C(Spinco SW 50.1 rotor). (A) Each fraction was assayed for exogenous DNA polymerase activity with (rC)n. (dG)12_18. Material banding at 1.15g/ml was pooled, pelleted at 190,000 X g, and resuspended in STE buffer. (B) After heating for 15 min at 370C, the material was layered on a

20-65% linear sucrose gradient and centrifuged for 3 hr, 190,000 X g, 40C (Spinco SW 50.1 rotor). The gradient was fractionated and each fractionwas then analyzed for exogenous DNA polymerase activity with (rC)n- (dG)1218. Density-was measured by refractometry (0).

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Microbiology: Nelson et al. 6265

Proc. Natl. Acad. Sci. USA 75 (1978)

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4 81I216 20 2428 32 4 812 1620 2428 32Fraction Fraction

FIG. 6. Cesium sulfate buoyant density analysis of the endogenous, reaction product of placenta extract banding at 1.15 g/ml in sucrosedensity gradients. (A) [3HJDNA product without treatment. (B) [3H]DNA product denatured at 1001C for 2 min. DNA and RNA markers bandedat 1.44 g/ml and 1.64 g/ml, respectively.

found to undergo a shift in density to 1.22-1.24 g/ml (Fig. 5).After heating, no loss in activity was observed, although 50%of the activity originally present at 1.15 g/ml shifted in densityto 1.24 g/ml. RDDP activity that banded primarily at 1.24 g/ml

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was also observed in extracts from placentas that had beenfrozen at -850C for more than 24 hr and in placentas frozenand thawed more than once. The freezing procedure appearedto increase the activity banding at 1.24 g/ml at the expense ofthe activity present at 1.15 g/ml.

Characterization of Endogenous Reaction Product. Thematerial banding at 1.15 g/ml was used to catalyze an endog-enous reaction in which radioactively labeled DNA was syn-thesized. The [3H]dTTP and [3H]dGTP-labeled DNA productwas subjected to Cs2SO4 gradient centrifugation. Most of thelabeled DNA banded in the RNA density region, 1.63 g/ml, andsome labeled DNA was bound in the equimolar DNA-RNAhybrid region of 1.54 g/ml. Labeled DNA was also detectedin the DNA region (1.44 g/ml) (Fig. 6A). After heat treatmentat 100'C for 2 min, the majority of labeled material shifted indensity to the DNA region (Fig. 6B).

Electron Microscopic Examination of Fractions Con-taining Polymerase Activity. The material found at 1.15 g/mlwas fixed, sectioned, and stained with uranyl acetate and leadcitrate. Numerous virus-like particles approximately 100 nmin diameter were observed. These structures contained centralelectron-dense cores and double-unit membrane envelopes (Fig.7).

DISCUSSIONRetrovirus-like particles have been detected by electron mi-croscopy in nonhuman and human primate placentas. RDDPactivity has been described in normal placental tissue of rhesusmonkeys (16) and rabbits (17) and in human amniotic fluid (18).The experiments described in this paper demonstrate thatnormal human placental tissue also possesses RDDP activityassociated with structures banding at densities characteristicof complete retroviruses and viral cores. The results indicatethat RDDP like that in viruses, previously recognized only inhuman tumors, can be detected in normal human tissues aswell.

This RDDP activity in human placentas was demonstratedby assays using the exogenous template-primer (rC)n-(dG)1-18and not (dC)Q-(dG)12 18. To date, only the methylated tem-

6266 Microbiology: Nelson et al.

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Proc. Natl. Acad. Sci. USA 75 (1978) 6267

plate-primer has been reported as specific for viral RDDP (15).No terminal nucleotidyltransferase activity was found in thoseplacental fractions that had high RDDP activity, althoughfractions at different density regions did show low levels of thisenzymatic activity for some placentas. Although templatespecificities cannot be used to prove unambiguously the pres-ence of viral enzyme, the results suggest that the enzyme is ofviral rather than cellular origin.The endogenous activity found at density 1.15-1.17 g/ml was

sensitive to treatment with RNase, and preliminary evidencesuggests that the endogenous DNA product synthesized byplacenta particles of density 1.15-1.17 g/ml sediments in amolecular weight region corresponding to DNA associated witha 35S template RNA. We have been unable to obtain a labeled70S RNA from these particles. Either nucleases are preventingits detection or the placental RDDP activity is associated withimmature viral particles that contain 35S instead of 70S RNA.The shift of the material containing RDDP activity from 1.15g/ml to the density characteristic of viral cores would be ex-pected of a viral enzyme. Further data in support of a viralenzyme in human placentas is the fact that the DNA synthe-sized in an endogenous reaction banded in cesium sulfate gra-dients either as RNA (presumably because the RNA is highmolecular weight) or as an RNA-DNA hybrid (1:1). Moreover,after boiling this labeled DNA banded as DNA. This latter resultindicates that the labeled DNA that banded as RNA was indeedhybridized to a high molecular weight RNA.

As seen in the electron microscope, material banding at 1.15g/ml had numerous particles of 100-150 nm. These structureshave several common structural features, including a two-unitmembrane envelope and a centrally located electron-densecore. In some cases, surface subunits were visible in the core andresembled the surfaces of murine leukemia virus cores (19).The significance of these placental virus-like particles is not

known. More than 80% of all placentas examined containedsome RDDP activity. Whether these structures represent con-genital transplacental infection by a virus or whether they areendogenous viruses involved in developmental processes in thehost is not known.The enzyme activity in crude preparations from isopycnic

gradients was not inhibited by antisera prepared against RDDPof the primate-derived viruses, baboon endogenous virus,RD-1 14, and Rauscher murine leukemia virus. The immuno-

logical relationship of purified human placenta RDDP to otherprimate, cellular and viral, polymerases will be the subject ofa future report.

We thank M. Johnson for her capable technical assistance. We areindebted to G. Beaudreau, A. 0. Deeney, and J. M. Bishop for helpfulsuggestions and to the Obstetrics staff of Good Samaritan Hospital,Corvallis, OR, for their assistance. The work was supported by ContractN01 CP 4338 of the National Cancer Institute. J.A.L. is a recipient ofResearch Career Development Award 70990 from the National CancerInstitute.

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14. Schlom, J. & Spiegelman, S. (1971) Science 174, 840-843.15. Gerard, G. (1975) Biochem. Biophys. Res. Commun. 63, 706-

711.16. Mayer, R., Smith, R. G. & Gallo, R. (1974) Science 185, 864.17. Bedigian, H. G., Fox, R. R. & Meier, H. (1976) Cancer Res. 36,

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Microbiology: Nelson et al.