Retroviral “terminal deoxynucleotidyl transferase” activity is reverse transcription

VIROLOGY

84, 247-259

Retroviral

(1978)

“Terminal

STUART Memorial

Deoxynucleotidyl Transferase” Reverse Transcription L. MARCUS

Sloan-Kettering

AND

Cancer Accepted

Center,

September

NURUL New

Activity

Is

H. SARKAR York,

New

York

10021

7,1977

Detergent-disrupted preparations of murine mammary tumor virus (MuMTVl, Mason-Pfizer monkey virus (MP-MV), and Rauscher murine leukemia virus (RLV) were all found to be capable of catalyzing poly(dT) synthesis when only the exogenous primer oligo(dT) was supplied. Analysis of this apparent terminal deoxynucleotidyl transferase (TdTl-like DNA polymerase activity revealed that: (a) Optimal concentrations of divalent cations and the effect of KC1 addition were similar or identical to those observed for reverse transcriptase activity; (b) sensitivity to inhibition by inorganic phosphate for both TdT-like and reverse transcriptase activity were identical and virion specific; (cl sedimentation coefficients obtained for TdT-like and reverse transcriptase activity from each virion were identical, although almost all TdT-like activity was lost following velocity sedimentation of solubilized virions; the TdT-like activity could be restored through the addition of 705 RNA to reaction mixtures; (d) the products of TdT-like and reverse transcriptase reactions were found to be equally sensitive to S, nuclease treatment. Disruption of viral cores through the use of the ionic detergent sodium deoxycholate rendered TdT-like activity almost completely sensitive to RNase treatment. Additionally, antisera prepared against MP-MV DNA polymerase inhibited both TdT-like and reverse transcriptase activity to an identical degree. Our results therefore suggest that the “TdT” observed in MuMTV, MP-MV, and RLV is actually reverse transcriptase activity directed by endogenous virion poly(A1 annealed to the exogenously provided oligo(dT) primer. INTRODUCTION

and Gallo, 1973; Green and Gerard, 1974) found these enzymes to be devoid of detectable TdT-like activity. Due to the apparent clinical importance of TdT and to the absence of such activity in purified reverse transcriptase preparations, the recently reported TdT-like activity in MuMTV core preparations (Ashley et al., 1977) suggested a new enzyme activity within virions of possible significance. We have investigated purified preparations of RLV, MuMTV, and MP-MV, as representing three biochemically, immunologically, and morphologically distinct mammalian oncornavirus groups, respectively, for the presence of TdT-like activity similar to that reported by Ashley et al. (1977). Our results indicate the presence of a similar enzymatic activity in detergent-disrupted preparations from all three viruses. Subsequent critical biochemical

Terminal deoxyribonucleotidyl transferase (TdT) has been shown to be unique among DNA polymerases in its ability to catalyze deoxynucleotide polymerization from the 3’-OH of a DNA primer molecule without template direction (Bollum, 1974). Although TdT is normally found only in thymocytes and in bone marrow, its presence in peripheral leukocytes has been shown in certain cases of human acute leukemia (McCaffrey et al., 1975; Marcus et al., 1976). In studies on purified retroviral DNA polymerases (reverse transcriptases) from Rauscher murine leukemia virus (RLV), murine mammary tumor virus (MuMTV), and Mason-Pfizer virus (MP-MV), we (Modak and Marcus, 1977; Marcus et al., 1976a) and others (Abrell ’ To whom

reprint

requests

should

be addressed. 247

0042-6822/78/0842-0247$02.00/O

Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

248

MARCUS

AND

analysis indicated that such DNA polymerase activity, apparently directed only by exogenously added oligodeoxynucleotide primer molecules, is in fact dependent upon the presence of viral RNA. We report here that expression of apparent “TdT” activity is dependent upon the maintenance of virus “core” integrity, which protects the virion RNA from nuclease attack, but which allows the annealing of oligonucleotides serving as primer molecules. The genomic RNA thus appears to serve as a template to direct homopolymeric DNA synthesis, indicating that virion “TdT” activity is not true terminal addition, but rather RNA-directed DNA synthesis. MATERIALS

AND

METHODS

Viruses. Murine mammary tumor virus (MuMTV) was obtained from two sources. Virus was purified from RI11 mouse milk as previously described (Sarkar and Dion, 19751, and was also obtained from the MuMTV producer cell line MuMT-73 (Sarkar et al., 1977). MuMT-73 were grown in Eagle’s minimal essential medium (MEM) supplemented with 15% fetal serum. After clarification of medium following culture, virus was harvested by high-speed centrifugation and the pellet was resuspended and further purified by isopycnic banding through linear sucrose gradients (Sarkar et al., 1977). The RLV and MP-MV used in these studies were kindly provided by Dr. J. Gruber of the National Cancer Institute, through the Virus Cancer Program. The RLV used in this study was propagated on a chronically infected mouse f’ibroblast cell line JLSV-9, while MP-MV was propagated in the human lymphoid cell line NC-37. Both viruses were harvested and concentrated by double isopycnic banding on linear sucrose gradients. All viruses were stored frozen at -70” until used. Enzymes. RLV-DNA polymerase was purified from virions by poly(C)-agarose affinity chromatography as previously described (Modak and Marcus, 1977). MuMTV-DNA polymerase was purified using a two-step procedure involving poly(C)-agarose and phosphocellulose

SARKAR

chromatography (Marcus et al., 1976a1, and a similar procedure was used to obtained purified MP-MV DNA polymerase (unpublished results). Pancreatic RNase (fraction V) of bovine origin was purchased from Worthington Biochemicals. A purified preparation of RNase II from Escherichia coli, obtained as a by-product of poly(A) polymerase purification, was kindly provided by Dr. M. J. Modak of this Institute (Modak and Srinivasan, 1973). Reagents. r3H]dTTP, purchased from AmershamlSearle, had a specific activity of 40-60 Ci/mmol. Unlabeled deoxynucleoside triphosphates, poly(A) * (dT)lz-le, (dTjlz, and (dC)lz-l, were products of P-L Biochemicals. Avian myeloblastosis virus (AMV) 70 S RNA was obtained from purified virus supplied through the courtesy of Dr. M. A. Chirigos of the National Cancer Institute by Dr. J. Beard. The 70s RNA was extracted from virions and purified by velocity sedimentation through glycerol gradients as previously described (Modak et al., 1974). DNA polymerase assays. Reactions were carried out in a total volume of 0.1 ml and consisted of 50 mM Tris-HCl, pH 6.8 or 7.8 (unless otherwise noted), 1 mM dithiothreitol (DTT), and 10 pg of bovine serum albumin monomer (Miles Laboratories). Synthetic primers and/or poly(A) * (dT),,-,s were added to a final concentration of 5 pg/ml. Divalent cation requirements for optimal DNA polymerase activity were determined for each virus preparation, using MgCI, and MnC12, and are indicated in the legends to figures and the footnotes to tables. When virus was used as a source of enzyme activity, nonionic detergent NP-40 was added to reaction mixtures at a final concentration Of 0.25% (v/v). Inorganic phosphate was stored as a 1 M potassium phosphate buffer solution, pH 8. [3HldTTP was added together with unlabeled substrate to yield a final concentration of 5 PM with a final specific activity of approximately 5000 cpm/pmol. AMV 70 S RNA with or without exogenous primers was used at a concentration of 1 pg per assay. Reactions were generally initiated by the addition of reac-

VIRION

“TdT”

IS REVERSE

tion mixture to virus or enzyme fractions. Approximately 5 pg of virus proteins were used per assay unless otherwise indicated. Reactions were carried out at 37” for 30 min and were terminated by the addition of cold trichloroacetic acid solution containing sodium pyrophosphate. Acid-insoluble material was collected by vacuum filtration onto Whatman glass-fibers (GF/ B) (Marcus et al., 1974a). After drying, the filters were placed into toluene-based scintillation fluid and counted in a Packard liquid scintillation counter. For studies of the effect of nuclease on DNA polymerase activity, virions were incubated, in a 50-~1 vol, with nuclease and all reaction mixture components except for exogenous template and/or primer and substrate, at 0” for 30 min. The remaining reaction components were then added in 50 ~1, and the reaction was carried out as described above. Molecular weight estimation. Sedimentation coefficients were determined by centrifugation of solubilized virions or purified DNA polymerases through preformed lo-30% (v/v) linear glycerol gradients in 0.05 M Tris-HCl buffer, pH 7.8, containing 1 mM DTT and 0.4 M KC1 (Marcus et al., 1974). Approximately 75 ~1 of purified RLV, MuMTV, or MP-MV containing 50 to 75 pg of viral proteins were solubilized by mixing with an equal volume of disruption buffer to yield a mixture with the following composition: 0.05 M Tris, pH 7.8, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.4 M KCl, and 10 mM DTT. After incubating the mixture at 0” for approximately 10 min, 150 ~1 of 0.05 M Tris-HCl, pH 7.8 was added and 250 ~1 of the resultant solution was layered over 5ml gradients. Centrifugation was carried out in an SW 50.1 rotor at 48,000 rpm for 16 hr at 4”. Fractions were then collected from the bottom of the tube and 20 ~1 were assayed for reverse transcriptase activity with poly(A)* (dT),,-,*, and for TdTlike activity with (dT),,. Parallel gradients were run with purified reverse transcriptases from the same viral sources to serve as molecular weight markers. Inhibition of MP-MV DNA polymerase by antiserum. Rabbit antiserum prepared

249

TRANSCRIF’TASE

against homogeneous preparations of MPMV DNA polymerase (reverse transcriptase) was generously provided by Dr. M. Ahmed of Pfizer laboratories. This antisera is monospecific against the MP-MV enzyme (M. Ahmed, personal communication). Stock dilutions of antisera of l:lO, 1:20, 1:50, and 1:lOO (v/v), were prepared using 0.5 M Tris-HCl, pH 7.8 as diluent. For testing the ability of the antiserum to affect reverse transcriptase or TdT-like reactions, approximately 5 pg of RLV, MuMTV, or MP-MV, as viral protein, were mixed in a final volume of 50 ~1 with Tris-HCl, divalent cation, NP-40, DTT, and 10 ~1 of antiserum dilution or diluent alone and incubated at 0” for 15 min. The remaining components of the reaction mixture, as substrate, primer, or template-primer combination, and bovine albumin monomer, were then added in a 50-~1 vol and the reaction was carried out as described under DNA polymerase assays. RESULTS

Exogenous Primer and Substrate Utilization by MuMTV, RLV, and MP-MV Purified virus preparations were gently disrupted with NP-40 and tested for their ability to catalyze DNA synthesis on exogenously added primer molecules. In order to observe the substrate range available to this reaction, all four DNA precursors were tested. We found that such DNA synthesis was possible for all three viruses tested, and that for these viruses the combination of dTTP and (dT),, yielded optimal rates of synthesis (Table 1). The ability to use an exogenously provided primer molecule for DNA synthesis is suggestive of TdT-like activity, and confirms the observation previously reported for MuMTV (Ashley et al., 1977). It is interesting to note also that no significant levels of dATP or dCTP incorporation were observed with any of the viruses tested, and that very low levels of dGTP incorporation were only observed with MP-MV. Low levels of dTTP incorporation were, however, observed for all viruses and with all primers tested (Table 1). That one substrate should prove optimal for all primers tested has also

250

MARCUS

AND

utilization of other substrates and primers by the viruses is considerably different from the known properties of mammalian TdT. Since (dT),,-catalyzed dTMP incorporation was found to be optimal, we concentrated our efforts on the comparison of this activity with reverse transcriptase activity (Table 2). It is interesting to note that the divalent cation preference expressed for the apparent terminal addition reaction closely follows that of reverse transcriptase activity for each virus. The divalent cation preferences which we observed are identical to those reported for disrupted virion polymerase activity as well as for purified enzyme preparations (Modak and Marcus, 1977; Verma, 1975; Marcus et al., 1976; Dion et al., 1974; Abrell and Gallo, 1973). The ratio of TdTlike DNA polymerase activity to reverse transcriptase activity under these conditions varied from 0.05 to 0.25 depending upon the virus used and the specific viral preparation tested. These ratios did not change significantly when milk-derived MuMTV was used in place of tissue culture-derived virus (data not shown). Levels of dTMP incorporation seen with oligo(dC) as a representative alternate primer were generally not increased more than 50% above those observed using detergent-disrupted virus in the absence of exogenous template and/or primer (Table

been reported for the calf thymus TdT enzyme (Bollurn, 1974; McCabe-ey et al., 1975; Marcus et al., 1976), but the lack of TABLE

1

SUBSTRATE AND PRIMER PREFERENCE OF MuMTV, RLV, AND MP-MV DETERGENT-DISRUPTED VIRAL PREPARATIONS IN THE ABSENCE OF EXOGENOUS TEMPLATES L1 Primer

(dA),,-,,

(dG)‘12-18

(dC),,,,

(dT),,

Enzyme source

I-

3H-labeled

substrate

,dATP

,3CTP

dGTP

MuMTV MP-MV RLV

-3

Cl

MuMTV MP-MV RLV


4 <1

MuMTV MP-MV RLV




MuMTV MP-MV RLV

<1 <1


-

dTTP
3.7

6.8 10


4.4

2.6 9.6 4.8

5

7.8 4.6 8.0

4


100 100 100

3.5

-


a Assays were carried out as described in Materials and Methods, using the appropriate primer molecules at a tins1 concentration of 5 pg/ml. Reaction mixtures containing MuMTV or MP-MV virions (5 pg protein/assay) also contained Mg*+ (5 mh4) as divalent cation; RLV-containing assays contained 0.5 mM Mn2+ as divalent cation together with 50 n&f KCl. MuMTVand RLV-catalyzed reactions were carried out at pH 6.8, while MP-MVcatalyzed reactions were assayed at pH 7.8. The 100% levels of [3HldTMP counts per minute incorporated for MuMTV-, MP-MV-, and RLV-catalyzed reactions incubated at 37” for 30 min were 38,100, 12,800, and 66,700, respectively. Background 100 cpm was subtracted from all results.

2). Characterization Activity

TRANSCRIPTASE

AND TdT-LIKE

Template and/or primer PolyW

. (dT),,-,,

W’),, (dC),,-,, None added

DNA

of Retroviral

TdT-Like

In order to further analyze the nature of this apparently template-independent

TABLE REVERSE

SARKAR

2

POLYMERASE

MuMTV

ACTIVITIES

OF

MuMTV,

MP-MV,

MP-MV

AND RLVa

RLV

Mg*+

Mn2+

Mg*+

Mn2+

Mg*+

MnZ+

113,000” 15,000 800 310

8,240 1,200 100 60

102,000 23,000 2,100 1,400

9,500 500 400 300

6,200 2,000 720 690

824,000 28,000 1,400 925

a Assays were carried out using detergent-disrupted virions as described in Materials and Methods. Concentrations of Mg*+ (as MgC1.J and Mn*+ (as MnCl,) used were 10 m&f and 0.5 mM, respectively. Those reaction mixtures in which Mn*+ served as divalent cation also contained 50 mM KCl. Background levels of approximately loo-150 cpm were subtracted from all results. b f3HldTMP counts per minute incorporated per 30 min at 37”.

VIRION

“TdT”

IS REVERSE

virion DNA polymerase activity, we determined the optimal reaction conditions and compared them with those for reverse transcriptase expression. Although apparent divalent cation preferences for expression of both types of DNA polymerase activity were identical (Table 2), the optimal concentration of divalent cations were individually determined. For MuMTV (Fig. l), both DNA polymerase activities cation, alpreferred Mg2+ as divalent though the TdT-like activity could use a broader range of Mg2+ concentrations than could reverse transcriptase. The ratio of Mg*+ Mn2+ activity expressed as t3HldTMP counts per minute incorporated in the presence of Mg2+ to that incorporated in the presence of Mn2+ during the same incubation period was 2.7 for TdT-like activity, and 3.2 for reverse transcriptase. The value expressed for reverse transcriptase using the detergent-disrupted virions was considerably lower than those reported by investigators studying the purified MuMTV-DNA polymerase (Dion et al., 1973; Marcus et al., 1976a) or purified vii-ions under various conditions (Young et al., 1975; Sarkar et al., 1977). For both

I 0.2

0.4 Divalent

0.6

0.8 cabon

I 1.0 h4nc12

(mM)

FIG. 1. Determination of Mg*+ (solid symbols) and Mn2+ (open symbols) optima for MuMTV TdTlike (squares) and reverse transcriptase (circles) activity. Assays were performed as described in Materials and Methods with detergent-disrupted virions.

251

TRANSCRIPTASE

01 0

loo

200

300

mM KCI

FIG. 2. Effect of the addition of increasing concentrations of KC1 on MuMTV TdT-like (0) and reverse transcriptase (0) activities, (A) using Mn*+ (0.05 m&f) as divalent cation and (B) using Mg2+ (5 m&f) as divalent cation for both reactions. Assays were carried out as described in Materials and Methods, with pH 6.3 used in TdT-like reactions, and pH 7.8 used for reverse transcriptase assays. Values for 100% activity, as 13H]dTMP counts per minute incorporated per 30 min, for reverse transcriptase and TdT-like activity in (A) were 128,000 and 13,000, respectively, and in (B) were 260,000 and 31,000, respectively.

RLV and MP-MV, optimal divalent cation concentrations for expression of reverse transcriptase and TdT-like activity were similar (data not shown), suggesting that these two DNA polymerase activities did not differ significantly in this respect. The effect of increasing KC1 concentrations on the TdT-like and reverse transcriptase activities of detergent-disrupted MuMTV in the presence of Mn*+ and Mg2+ is shown in Fig. 2. In the presence of Mn2+, KC1 addition up to 60 n-J4 stimulated both DNA polymerase activities, while Mg2+-containing reactions were inhibited by the addition of increasing KC1 concentrations. Therefore, we could find no difference in the response of TdT-like and reverse transcriptase activities to KC1 addition. Similar results were obtained

252

MARCUS

AND

using detergent-disrupted preparations of MP-MV and RLV (data not shown). We were therefore unable to confirm the initial report that MuMTV reverse transcriptase and TdT-like activity differ in their response to increasing concentrations of KC1 (Ashley et al., 1977). Experiments carried out to determine the optimum pH for the two types of DNA polymerase activities indicated that the TdT-like activity of MuMTV has a pH optimum of 6.8 (Fig. 31, while the optimum pH for reverse transcriptase activity is approximately 7.8. RLV reverse transcriptase and TdT-like activities demonstrated

05--I PH FIG. 3. Determination of pH optima for MuMTV and MP-MV reverse transcriptase (0) and TdT-like (0) activity. Assays were carried out as described in Materials and Methods, using appropriately buffered Tris-HCl solutions at 50 mM concentration. Mg*+ (5 n&f) was present for both MuMTVand MP-MV-catalyzed reactions. The 100% values for F’HldTMP counts per minute incorporation in TdT-like and reverse transcriptaae assays by MuMTV are 105,000 and 26,000, respectively, and are 120,000 and 31,000, respectively, for the same reactions catalyzed by MP-MV.

SARKAR

pH optima of 7.2-7.8, and 6.8, respectively (data not shown). In contrast to these findings, the MP-MV TdT-like and reverse transcriptase activities responded identically to varying pH (Fig. 3). We have recently reported that it is possible to distinguish mammalian C-type DNA polymerase activity from those of other mammalian retroviruses by their relative sensitivity to inhibition by low concentrations (~2 m&f) of inorganic phosphate added to reaction mixtures (Modak and Marcus, 1977). We therefore attempted to determine whether the virion TdT-like activities could also be distinguished from each other by their response to inorganic phosphate. Additionally, it has been shown that thymus gland-derived TdT activity can be inhibited by ribonucleoside triphosphates (Kato et al., 1967), unlike other cellular DNA polymerases (Modak, personal communication). Therefore, as another means of comparison, the effect of exogenous ATP and dATP addition on virion TdT-like and reverse transcriptase activities was also determined. The results of these studies are shown in Table 3. Both RLV-DNA polymerase activities were susceptible to inhibition by inorganic phosphate, while the MP-MV and MuMTV reverse transcriptase and TdT-like activities were either unaffected or slightly stimulated. Neither dATP nor ATP, at a concentration 40-fold that of the available substrate, showed significant inhibition of either type of DNA polymerase activities with RLV, MuMTV, or MP-MV. These results indicate that virion TdT-like DNA polymerase activity differs significantly in the mechansim of catalysis from known mammalian cellular TdT, and appears similar to reverse transcriptase in degree of susceptibility to phosphate inhibition. Molecular DNA

Weight Estimation of Vi&n Polymerase Activities

The apparent molecular weights of the active forms of TdT-like DNA polymerase and reverse transcriptase activities were determined using velocity sedimentation through linear lo-30% glycerol gradients (see Materials and Methods). In order to

VIRION

“TdT”

IS REVERSE TABLE

EFFECTS Exogenous

OF EXOGENOUS

ADDITION

compounds

MuMTV 12 I8

(pH

8)

ATP,

AND dATP

ON RETROVIRUS

Cd%,

y&’

DNA

RLV

MP-MV CdT),,

I;;#A). None Potassium phosphate (2 mM) dATP (200 CJM) ATP (200 CJM)

3

OF INORGANIC PHOSPHATE, POLYMERASE ACTIVITIES”

-

253

TRANSCRIPTASE

(dT),,

yd$;A’.

12 18

12 18

120,000~ 130,500

24,300 28,300

110,000 140,600

24,200 30,200

750,000 228,000

22,000 8,200

112,000 132,000

26,000 28,000

105,000

100,800

23,600 25,200

680,000 625,000

23,000 21,000

(1 Assays were carried out using NP-40 disrupted virions as described in Materials and Methods. The pH of MuMTV and RLV (dT),,-containing reaction mixtures was 6.8, and the pH was 7.8 for those catalyzed by MP-MV. All poly(A) . (dT),,_,B-directed activity was assayed at pH 7.8. b t3H]TMP counts per minute incorporated.

carry out this study, virions were solubilized using sodium deoxycholate in addition to NP-40 prior to layering preparations onto gradients. The results of assays carried out on gradient fractions revealed (Fig. 4) that the TdT-like DNA polymerase activity from MuMTV, RLV, and MP-MV possessed sedimentation coefficients of 5.4, 4.2, and 5.4 S, respectively, all of which cosedimented with virion reverse transcriptase activity. Sedimentation coefficients obtained for reverse transcriptase from solubilized virions were identical to those previously reported for purified enzyme preparations (Modak and Marcus, 1977; Marcus et al., 1976a; Abrell and Gallo, 1973). Although TdT-like activity in NP-40-treated virions (Table 2) represented 5 to 25% of the reverse transcriptase activity, the ratio of similar activities in peak gradient fractions dropped to 0.20.9%. These extremely low levels of TdTlike activity were found not to be sensitive to RNase pretreatment. The apparent similarities in divalent cation requirement, susceptibility to inhibition by phosphate, and sedimentation coefficients between each virion reverse transcriptase and TdTlike activity suggested that both may be catalyzed by RNA-directed DNA polymerase. In order to test this hypothesis, attempts were made to restore TdT-like activity to peak glycerol gradient DNA polymerase fractions by the addition of AMV 70 S RNA to reaction mixtures. The results of this study, summarized in Table 4, indicate that addition of 70 S RNA to such reaction mixtures completely re-

B

Fraction

number

T

FIG. 4. Glycerol gradient centrifugation of solubilized MuMTV, MP-MV, and RLV, and detection of TdT-like (0) and reverse transcriptase (0) enzymatic activity. Virions were solubilized and centrifugation was carried out as described in Materials and Methods. TdT-like and reverse transcriptase activities were located by assaying with (dT),, and poly(A) (dT),,_,,, respectively. Assays were carried out in the presence of 5 nu%4 Mg’+ for MuMTV and MP-MV fractions, and 0.5 m&f Mn’+ for RLV, and using individually optimum pH (See Fig. 2 and text) at 37” for 1 hr.

stored and, in the case of RLV, greatly stimulated, (dT),,-primed [3H]dTMP incorporation into acid-insoluble material.

254

MARCUS

AND SARKAR

TABLE 4 “TdT” ACTIVITY BY ADDITION OF VIRAL 70s RNA TO GLYCEROL GRADIENT

RESTORATION FRACTIONS

OF

CONTAINING

Enzyme source

PEAK

ENZYME

ACTIVITY

D

Template and/or primer

n Reaction mixtures were prepared as described in Materials and Methods, except that NP-40 was omitted. Glycerol gradient fractions (Fig. 3) were stored overnight at o”, and 10 ~1 of peak DNA polymerase activity fractions from the appropriate gradients were assayed. Assay conditions were as described in the footnote to Table 2. Incubation at 37” was carried out for 1 hr. b 13HldTMP counts per minute incorporated.

Core Integrity Activity

and Expression of TdT-Like

The studies described above suggested that the observed TdT-like activity in NP40-generated virion cores might be RNAdirected. The report initially describing this activity in MuMTV as TdT (Ashley et al., 1977) stressed that the resistance of oligo(dT)-primed poly(dT)-synthesizing activity to preincubation in the presence of ribonuclease indicated lack of direction by an RNA template. Such a conclusion might not be valid if the eore structure could in some way protect virion 70s RNA from attack by ribonuclease, yet could allow the expression of homopolymeric DNA synthesis by permitting entrance of low-molecular-weight primers and substrates. We tested this possibility by preincubating MuMTV plus detergent in the presence and absence of RNase. Deoxycholate has been shown to be effective for solubilizing MuMTV (Marcus et al., 1976a; Teramoto et al., 1977). Preincubation of MuMTV in the presence of NP40 together with sodium deoxycholate lowered expression of TdT-like activity to a level approximately 40-50% that observed in the presence of NP-40 alone (Table 5). Under our conditions, TdT-like activity was observed to be wholly resistant to RNase A (Table 5). Some sensitivity to RNase II was observed, although the de-

gree of sensitivity observed (30-40%) in the presence of NP-40 alone would be sufficient to suggest that the majority of activity was independent of RNA direction. High concentrations of RNase A (~200 pug/ml) did cause some inhibition in the presence of NP-40 alone (data not shown). Preincubation of MuMTV with pancreatic RNase or RNase II in the presence of NP40 and deoxycholate caused >90% inhibition of TdT-like activity. These results suggest that maintenance of MuMTV core integrity was responsible for the lack of nuclease inhibition of TdT activity previously reported (Ashley et al., 19771, and that dissolution of the core structure revealed the RNA-dependent nature of the reaction. Similar results were obtained with RLV and MP-MV (data not shown). It is therefore apparent that the (dT),,primed DNA synthesis reported herein for all three viruses tested may be an expression of endogenous RNA directing homopolymeric synthesis of poly(dT). Single-Strandedness of “TdT-Like” and Reverse Transcriptase Reaction Products

The above results are suggestive TABLE

of the

5

EFFECT OF MuMTV ABILITY OF NUCLEASE

CORE INTEGRITY TO INHIBIT “TdT”

ON THE ACTIVITY

MuMTVprincubated

[!HldTMP mcorporated

Inhibition

(cpm)

a

6)

NP-40 5500 NP-40 + deoxycholate 2300 6570 0 NP-40 + pancreatic RNase NP-40 + deoxycholate + 200 92 pancreatic RNase NP-40 + RNase II 3500 36 NP-40 + deoxycholate + 200 92 RNase II a Assays were performed using detergent-disrupted virions as described in Materials and Methods and in the footnote to Table 2. Sodium deoxycholate, when added, was present in preincubation mixtures at 0.2% (w/v) concentration. Preincubation was carried out as described in Materials and Methods. RNase A was present at a concentration of 20 &g/ml during preincubation, while 50 units of E. coli RNase II were used in the preincubation mixture.

VIRION

“TdT”

IS REVERSE

possibility that reverse transcription was responsible for “TdT-like” DNA polymerase activity. In order to support the concept of terminal addition, the product of MuMTV-catalyzed oligo(dT)-primed synthesis should be sensitive to S, nuclease, suggesting that it is single stranded and therefore not template directed. The results of this study are shown in Table 6. Both the “TdT-like” polymerase activity product and the template-directed reverse transcriptase product were found to be equally sensitive (about 75%) to S, nuclease digestion when the assay was carried out at 50”, while only <1.7% of the same products were sensitive at an incubation temperature of 37”. Single-stranded poly(A), on the other hand, was equally well hydrolyzed at 37 or 50”. The incubation temperature which was used for the S, nuclease assay (50”) is close to the T, of poly(A)*poly(dT), 59” (Ts’O et al., 1966). Thus, it is likely from this study that the S, sensitivity of the “TdT-like” product at 50” is due to the “melting-out” of a poly(A) * oligo(dT) hybrid structure and not to the single strandedness of the initial product.

255

TRANSCRIPTASE

both TdT and reverse transcriptase activity from MuMTV, MP-MV, and RLV using NP40-disrupted virions is shown in Fig. 5. Although addition of increasing quantities of antiserum to detergent-disrupted preparations of RLV or MuMTV did not cause significant inhibition of either TdT-like or reverse transcriptase activity, both activities catalyzed by detergent-disrupted MP-MV were inhibited by the antiserum directed against MP-MV reverse transcriptase. Moreover, the degree of inhibition of each type of activity observed at each dilution of antiserum was identical. These results strongly suggest that, in the case of MP-MV, both reverse transcriptase and TdT-like DNA polymerase activities are catalyzed by the same enzyme. DISCUSSION

We have described the presence in detergent-disrupted MuMTV, RLV, and MPMV of a DNA polymerase activity capable of catalyzing poly(dT) synthesis on (dT),, primer molecules in the absence of exogenously added poly(A) template. Such activity may initially be termed TdT-like due to the apparent lack of dependence on template direction. Our observations have Effect of Specific Antisera on “TdT-Like” served to confirm, extend, and explain the and Reverse Transcriptase Activity recent report by Ashley et al, (1977) in While the above results are suggestive which a similar activity was reported in of the concept that observed TdT-like ac- MuMTV. The apparently unique nature tivity is a manifestation of endogenous of this kind of DNA polymerase activity reverse transcriptase activity, the possibilnecessitated its characterization. The TdTity still existed that they might be similar like activity from each virion was very yet discrete entities. The presence of RNA similar to that specific virion reverse tranmight stimulate, yet not be essential for, scriptase with respect to: (a) divalent catexpression of TdT-like activity at low lev- ion preference and optimal concentration els. The results observed from velocity of the preferred divalent cation; (b) resedimentation analysis could be due to sponse to addition of exogenous potassium severely different labilities between the phosphate or increasing concentrations of two putatively different enzymes. In order KCl; and (c) sedimentation velocity. In to provide a more resolute test of the our studies with RLV, MuMTV, and MPpossible identity between reverse tranMV, dTTP was the only substrate showing scriptase and TdT-like activity, an immusignificant incorporation in reactions catnological approach was initiated. Antisealyzed by all three viruses (Table 1). We rum prepared against near-homogeneous were also, in contrast to the previous repreparations of MP-MV reverse transcripport, unable to show significant levels of tase was used. The ability of the anti13HldTMP incorporation above endogenous levels using oligo(dC), (dA), or (dG) MP-MV reverse transcriptase antiserum at increasing concentrations to inhibit as primers, indicating that oligo(dT) was

MARCUS

256 S, nucmzAsx

SENSITIVITY

OF

MuMTV

Source of product

AND SARKAR

TABLE 6 “TdT-LIKE” AND REVERSE TBANSCRIPTASE REACTION PRODUCTS 37 AND 50”” Trichloroacetic acid-precipitable 3H counts per minute Control sample

After S1 treatment

AT

Percentage S1 sensitive

37” 50” 37 50” 37” 50” (dT),,-primed reaction without ad12,200 12,200 11,900 3,100 1.7 75 dition of exogenous template Poly(A). (dT),,,,- erected synthesis 14,200 14,700 14,100 3,600
I/Antiserum

dilution

FIG. 5. Inhibition of MP-MV (circles), MuMTV (squares), and RLV (triangles) TdT-like (open symbols) and reverse transcriptase (filled symbols) activity by antisera against purified MP-MV reverse transcriptase. Antiserum dilution and preincubation of detergent-disrupted virions was carried out as described in Materials and Methods. Conditions of assay for the respective virion DNA polymerase activities were as described in the legend to Fig. 4, except that assays were incubated for 30 min at 37”. Values for 100% of control DNA polymerase activities, as [3H1dTMP counts per minute incorporated, were equivalent to those seen in Table 2.

the preferred primer for TdT activity from all virions tested (Tables 1 and 2). The classification of the TdT-like activ-

ity we found in all three retroviruses as true TdT was dependent upon rigorous proof that such activity was independent of template direction. The previous study, using MuMTV alone (Ashley et al., 1977), stressed resistance to RNase A and the synthesis of single-stranded product (determined through S, nuclease sensitivity) as suggesting lack of template direction. Our studies, based upon preincubation with RNase A as well as with a specific poly(A)-degrading RNase activity from E. coli (Modak and Srinivasan, 1973), suggest that NP-40 treatment alone is not suffrcient to allow nuclease activity to reach virion RNA (Table 5). Upon incubation in the presence of NP-40 and sodium deoxycholate, the virion core structure was sufficiently disrupted to allow inhibition of TdT-like activity by RNase. Therefore, the (dT),,-primed synthetic activity was indeed dependent upon access to virion 70s RNA, or to other RNA packaged within the virion. Additional support for this concept was found in studies on velocity sedimentation gradient fractions from solubilized virions containing peak reverse transcriptase activity and low but detectable levels of TdT-like activity (Fig. 4). Restoration of original TdT-like activity levels could be accomplished through the addi-

VIRION

“TdT”

IS REVERSE

tion of AMV 70s RNA to reaction mixtures containing such fractions (Table 4). Furthermore, we have shown (Table 6) that the products of the “TdT-like” reaction and of poly(A) . (dT),,,,-directed reaction (reverse transcription) are equally sensitive to S, nuclease when the assay is carried out at 50”. Neither product showed significant degradation when the nuclease assay was carried out at 37”. This result suggests that the product of the “TdT-like” reaction is double stranded, and is only susceptible to S, nuclease when the assay temperature approaches the T, of the poly(A).poly(dT) hybrid (Ts’O et al., 1966). Therefore, true terminal addition to (dT),, in the absence of template direction does not occur. Such results are sufficient to provide a simple model by which the RNA-directed, (dT),,-primed poly(dT) synthesis might be mistaken for TdT activity. Treatment of virions with NP-40 or other nonionic detergents (Bader et al., 1970; Stromberg, 1972; Bolognesi et al., 1972; Sarkar et al., 1971; Teramoto et al., 1977) may produce structures in which the 70s RNA is at least partly protected from nuclease attack by the surrounding internal virion proteins. The core structures would, however, allow the entrance of oligodeoxynucleotides as well as DNA precursor molecules into the immediate microenvironment of the RNA. Incubation at 37” would allow the annealing of oligo(dT) to the poly(A) stretch found at the 3’end of retroviral RNA molecules (Schlom et at., 1973), thus allowing reverse transcriptase to carry out homopolymeric synthesis. Due to the “tightness” of the core molecule, this activity would be RNase resistant. The ability of pancreatic RNase A, at low ionic strength, to digest even poly(A) stretches has been documented (Beers, 1960). We have reported that partially purified MuMTV-DNA polymerase preparations catalyze a disproportionately high level of dTMP incorporation in the presence of 70s RNA and oligo(dT) primer, even in the absence of other DNA precursors (Marcus et al., 1976a). We have also used a similar property of E. coli DNA polymerase I as the basis for development of an enzymatic test for the rapid detection of such poly(A)

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257

stretches in RNA (Modak et al., 1974a). The studies reported in this paper suggest that similar catalysis might occur with RLV and MP-MV reverse transcriptase. The above findings, together with the apparent immunological identity between MP-MV reverse transcriptase and TdTlike activity (Fig. 5) suggest that the “TdTlike” DNA polymerase activity observed in detergent-disrupted virions is truly DNA synthesis dependent upon an endogenous template together with an exogenous primer catalyzed by virion reverse transcriptase. However, the observations of different pH optima for each type of activity in MuMTV (Ashley et al., 1977) and in RLV (Fig. 3), and the presence of low levels of “TdT-like” activity in peak velocity sedimentation gradient fractions from solubilized virions (Fig. 3) still require explanation. The very low (0.2 to 0.9% of reverse transcriptase activity) but detectable “TdT-like” activity in gradient fractions is probably due to the addition of a single [3HldTMP molecule to ~0.01% of the 125 pmol of available (dT),, 3’-OH ends provided in the reaction mixture. This would represent an inefficient mimicry of a phenomenon previously observed with cellular DNA polymerase /3 (Weissbath, 1976). At present we cannot readily explain the different pH optima for “TdTlike” and reverse transcriptase activity seen in detergent-disrupted MuMTV (Fig. 3) and RLV preparations. It is likely that, although the virion core structure produced by gentle detergent treatment is of a particular conformation, the availability of the poly(A) stretch of virion RNA to reverse transcriptase or (dT),, may be altered by changing pH. In order to clarify this possibility, the effect of varying pH on the affinity of retroviral RNA-binding proteins for RNA, and on the susceptibility of RNA in such RNA-protein complexes to nuclease digestion will have to be examined. It is interesting to note that, for MP-MV, in which “TdT-like” and reverse transcriptase activity have identical responses to varying pH, the core has been noted to have a more fragile, easily damaged or opened structure than those prepared from either C- or B-type murine retroviruses (Kramarsky et al., 1971).

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AND SARKAR

Therefore, although our results indicate that the previously observed “TdT” activity within retroviral cores is truly reverse transcriptase activity, the methods used to determine this may point the way toward the dissection of the organization of virion core structure. Future studies using this approach may be helpful to determine the possible interaction of retroviral RNAbinding, and possibly other, core proteins. ACKNOWLEDGMENTS The authors wish to express their sincere thanks to Dr. M. J. Modak of this Institute for helpful discussions and for his gift of RNase II, to Dr. J, Gruber for R-MuLV and MP-MV, to Dr. J. Beard for AMV, and to Dr. M. Ahmed for antiserum prepared against purified MP-MV reverse transcriptase. We are grateful to Mr. S. W. Smith and Mr. E. S. Whittington for excellent technical assistance. This study was supported by grants nos. CA-08748, CA-17129, and CA-18369 from the National Cancer Institute. REFERENCES J. W., and GALLO, R. C. (1973). Purification, characterization, and comparison of the DNA polymerases from two primate RNA tumor viruses. J. Viral. 12,431-439. ASHLEY, R. L., CARDIFF, R. D., and MANNING, J. S. (1977). Characterization of terminal deoxynucleotidy1 transferase activity in mouse mammary tumor virus. Virology 77, 367-375. BADER, J. P., BROWN, N. R., and BADER, A. V. (1970). Characteristics of cores of avian leukosarcoma viruses. Virology 41, 718-728. BEEREI, R., JR. (1960). Hydrolysis of polyadenylic acid by pancreatic ribonuclease. J. Biol. Chem. 235,2393-2398. BOLLUM, F. J. (1974). Terminal deoxynucleotidyl transferase. In “The Enzymes” (P. D. Boyer, ed.), Vol 10, pp. 145-171. Academic Press, New York. BOL~GNESI, D. P., GELDERBLOM, H., BAUER, H., MOELLING, K., and HUPER, G. (1972). Polypeptides of avian RNA tumor viruses. V. Analysis of the virus core. Virology 47, 567-578. DION, A. S., VAIDYA, A. B., Four, G. S., and MOORE, D. H. (1974). Isolation and characterization of RNA-directed DNA polymerase from a Btype RNA tumor virus. J. Viral. 14.40-45. GREEN, M., and GERARD, G. F. (1974). RNA-directed DNA polymerase -properties and functions in oncogenic RNA viruses and cells. Progr. Nucleic Acid Res. Mol. Biol. 14, 187-334. KATO, K., G~NCALVES, J. M., HOUTS, G. E., and BOLLUM, F. J. (1967). Deoxynucleotide-polymerizing enzymes of calf thymus gland. J. Biol.

ABRELL,

1780-1784. B., SARKAR, N. H., and MOORE, D. H. (1971). Ultrastructural comparison of a virus from a rhesus monkey mammary carcinoma with four oncogenic viruses. Proc. Nut. Acad. Sci. USA 68, 1603-1607. MARCUS, S., MODAK, M. J., and CAVALIERI, L. F. (1974). Purification of avian myeloblastosis virus DNA polymerase by affinity chromatography on polycytidylate-agarose. J. Viral. 14,853-859. MARCUS, S. L., MODAK, M. J., and CAVALIERI, L. F. (1974a). Evidence for template-specific sites on DNA polymerases. Biochem. Biophys. Res. Commum 56,516-621. MARCUS, S. L., SMITH, S. W., JAROWSKI, C. J., and MODAK, M. J. (1976). Terminal deoxyribonucleotidy1 transferase activity in acute undifferentiated leukemia. Biochem. Biophys. Res. ComChem. 242, KRAMARSKY,

mun. 7937-44. MARCUS, S. L., SARKAR,

N. H., and MODAK, M. J. (1976a). Purification and properties of murine mammary tumor virus DNA polymerase. Virology 71,242-254. MCCAFFREY, R., HARRISON, T. A., PARKMAN, R., and BALTIMORE, D. (1975). Terminal deoxynucleotidy1 transferase activity in human leukemic cells and in normal human thymocytes. N. Engl. J. Med. MODAK,

292, 775-780.

M. J., and SRINIVMAN, P. R. (1973). Purification and properties of a ribonucleic acid primer-independent polyriboadenylate polymerase from Escherichia coli. J. Biul. Chem. 248, 6094-6910. MODAK, M. J., and MARCUS, S. L. (1977). Purification and properties of Rauscher leukemia virus DNA polymerase and selective inhibition of mammalian viral reverse transcriptase by inorganic phosphate. J. Biol. Chem. 252, 11-19. MODAK, M. J., MARCUS, S. L., and CAVALIERI, L. F. (1974). Synthesis of DNA complementary to AMV 70s RNA by E. coli DNA polymerase I. Biochem. Biophys. Res. Commun. 56, 247-255. MODAK, M. J., MARCUS, S. L., and CAVALIERI, L. F. (1974a). A new sensitive method for detecting

polyadenylate in viral and other ribonucleic acids using Escherichia coli deoxyribonucleic acid polymerase I. J. Biol. Chem. 249, 7373-7376. SARKAR, N. H., and DION, A. S. (1975). Polypeptides of the mouse mammary tumor virus. I. Characterization of two group-specific antigens. Virology 64, 471-491. SARKAR, N. H., NOWINSKI, R. C., and MOORE, D. H. (1971). Characteristics of the structural components of the mouse mammary tumor virus. 1. Morphological and biochemical studies. Virology 46, l-20. SARKAR, N. H., POMENTI, A. A., and DION, A. S. (1977). Replication of mouse mammary tumor virus in tissue culture. I. Establishment of a

VIRION mouse ization, rology

“TdT”

IS REVERSE

mammary tumor cell line, virus characterand quantitation of virus production. Vi77, 12-30. SCHLOM, J., COLCHER, D., SPIEGELMAN, S., GILLESPIE, S., and GILLESPIE, D. (1973). Quantitation of RNA tumor viruses and virus-like particles in human milk by hybridization to poly A sequences. Science 1’79, 696-698. STROMBERG, K. (1972). Surface-active agents for isolation of the core component of avian myeloblastosis virus. J. Viral. 9, 684-707. TERAMOTO, Y. A., CARDIFF, R. D., and LUND, J. K. (1977). The structure of the mouse mammary tumor virus: Isolation and characterization of the core. Virology 77, 135-148.

TRANSCRIPTASE

259

Ts’o, P. 0. P., RAPAPORT, S. A., and BOLLUM, F. J. (19661. A comparative study of polydeoxyribonucleotides and polyribonucleotides by optical rotatory dispersion. Biochemistry 54153-4170. VERMA, I. M. (19751. Studies on reverse transcriptase of RNA tumor viruses. III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNAse H. J. Viral. 15, 843-854. WEISSBACH, A. (1976). Vertebrate DNA polymerases. Cell 5, 1-6. YOUNG, L. J. T., CARDIFF, R. D., and ASHLEY, R. L. (1975). Long-term primary culture of mouse mammary tumor cells: Production of virus. J. Nat. Cancer last. 54, 12151221.