Vol. 18, No. 2 Printed in USA.

JOURNAL OF ViROLoGy, May 1976, p. 418 425 Copyright © 1976 American Society for Microbiology

Further Characterization of the Friend Murine Leukemia Virus Reverse Transcriptase-RNase H Complex KARIN MOELLING' Institut fur Virologie, Bereich Humanmedizin, Justus Liebig-Universitdt 6300, Giessen, Germany

Received for publication 25 November 1975

The purified reverse transcriptase-RNase H complex from Friend murine leukemia virus consists of a single polypeptide of 84,000 molecular weight, which after mild protease treatment in vitro or after intentional degradation during the purification procedure allows the generation of several additional polypeptides. Degradation destroys the RNA-dependent DNA polymerase activity with native RNA templates and reduces RNase H but does not affect response to synthetic template primers such as poly(rA)oligo(dT). The properties of the intact murine enzyme consisting of a single polypeptide of 84,000 molecular weight are compared to those of the avian a subunit and the avian a/3 enzyme complex. The intact murine enzyme resembles the avian /3-containing enzyme complex and is different from a in the following respects: (i) it binds to native RNA templates; (ii) it transcribes native RNA templates into DNA, a reaction which can be inhibited by actinomycin D; (iii) RNase H activity behaves like a processive exonuclease; and (iv) analysis of the RNase H digestion products reveals oligonucleotides approximately four bases in length.

The RNA-dependent DNA polymerase activ- an enzyme preparation was extracted from Frity from mammalian viruses, particularly the MuLV under nonoptimal conditions for consermurine ones, have been characterized by sev- vation of the enzyme activity. Both experieral laboratories with conflicting results. As far ments (trypsin treatment and intentional degas the structure of the enzyme is concerned, the radation) generated several polypeptides. number of subunits identified on sodium dode- Structural changes were correlated with enzycyl sulfate-polyacrylamide gels after extensive matic alterations that reproduce and explain purification ranges from one to three. The en- some of the conflicting data published on the zymes from Friend murine leukemia virus (Fr- structure and enzymatic properties of mamMuLV) and from Moloney murine leukemia malian viral reverse transcriptases. virus (Mo-MuLV) have been described as a sinThe size of the largest murine viral enzyme gle polypeptide of 80,000 to 84,000 molecular polypeptide ranges between the size of the two weight (8, 12). In other reports the Fr-MuLV avian enzyme subunits, leaving open the quesenzyme has been characterized as a two-poly- tion of whether it corresponds to a or /3. There peptide complex (15), and the Mo-MuLV en- is evidence in favor of either: the ability to zyme has been shown to consist of three sub- transcribe native RNA observed in some cases units (2). The two polypeptides observed for Fr- (2, 8, 12) resembles the properties of /, whereas MuLV (15) resemble those of the hamster viral absence of RNA-dependent DNA synthesis (1, enzyme (13). 14) is similar to a. The RNase H activity has As the avian viral reverse transcriptase- been characterized as a random exonuclease (3) RNase H complex consists of two polypeptides, identical to that exhibited by the avian a fraga and /3, one of which can be shown to arise by ments (3), and the RNase H digestion products proteolytic cleavage from the other (Moell- (12) have been described as being larger than ing, Virology, in press), the question arose as to expected by analogy to a /3-containing enzyme whether some of the murine viral enzyme sub- preparation. Furthermore, it has been observed units represent degradation products as well. that protease treatment of the avian a,8 enThe answer of this question was approached in zyme complex in vitro can completely remove two ways: (i) the purified Fr-MuLV enzyme, ,/, whereas a is much more resistant to this consisting of a single polypeptide, was sub- treatment. Only more stringent conditions aljected to mild protease treatment in vitro; (ii) low digestion of a as well (Moelling, Virology, in press). The possibility exists, therefore, Present address: Max-Planck-Institut fur Molekulare that the largest murine viral protein described Genetik, D-1 Berlin-33 (West), Germany. 418

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so far is a degradation product of an even larger one. To find out whether or not the 84,000-molecular-weight polypeptide exhibits the properties of a real viral reverse transcriptase-RNase H complex, several of its enzymatic properties were compared to those of the avian a subunit, as representative of a degraded enzyme, and to the a,/ avian enzyme complex. As all previous attempts to isolate /3 alone have failed (Moelling, in press), the avian a,3 enzyme complex was chosen for comparison, since it is the best representative of a functioning reverse transcriptase-RNase H complex presently known. MATERIALS AND METHODS Virus. Avian myeloblastosis virus was isolated in part in this laboratory and was in part a generous gift of J. Beard. Fr-MuLV was grown in this laboratory. The virus was originally produced in STUmouse cells (10) that have since been established as as permanent line (Eveline cells). The cells that were grown in suspension culture were adapted to substrate-dependent growth by G. Pauli in this laboratory. Approximately 107 cells were seeded in roller bottles (5-cm diameter, 50-cm long) and grown at 37 C with 25 ml of Dulbecco modified Eagle growth medium in the presence of 5% heat-inactivated calf serum (56 C, 45 min) and 10% tryptose phosphate broth. Reagents. 3H-labeled poly(rA) and poly(dT) were obtained from Miles Laboratories. Oligonucleotides (A)A, (A)6A, (A)4A, and (A)2A were purchased from Boehringer (Mannheim, West Germany). Acrylamide and methylenebisacrylamide, electrophoresis grade, were bought from Bio-Rad Laboratories (Richmond, Calif.). Soluene-350 originated from Packard Instrument Co. (Zurich, Switzerland). Dulbecco modified Eagle medium was purchased from Flow Laboratories (Bonn, West Germany), and tryptose phosphate broth was from Difco Laboratories (Detroit, Mich.). Enzyme purification. The reverse transcriptaseRNase H complex from avian myeloblastosis virus and Fr-MuLV was isolated by published procedures (8, 9), and its purity was determined on SDS-polyacrylamide gels. Subunit a was recovered from a phosphocellulose column at 0.11 M KCl. For isolation of large amounts of a, the enzyme attached to the DEAE column was left at 4 C for 1 to 2 days before elution. The same treatment was applied to the Fr-MuLV enzyme to deliver a degraded enzyme. Enzyme activity assay. DNA polymerase and RNase H were determined as previously described (8, 9). The avian enzyme assays were performed in the presence of 8 mM MgCl2, and the Fr-MuLV assays were performed in the presence of 0.4 mM MnCl2. The specific radioactivity of one labeled deoxyribonucleotide was 5,000 counts/min per pmol. The assay for RNase H contained 10,000 counts/min of phage fd DNA-RNA hybrid, with specific activity of 3,500 counts/min per pmol of [3H]UMP. Unit of activity. A unit of enzyme activity is

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defined as the amount of enzyme required to incorporate 30 pmol of TMP at 39 C in 30 min with the synthetic template poly(rA)-oligo(dT) in a standard polymerase assay mixture, as this reaction is not affected by any degree of enzyme degradation. Comparative studies of enzyme activities were always based on identical units input. Binding of enzyme to RNA templates. In a total volume of 100 ,l, 20 tkl of the murine or avian /3containing enzymes were mixed with an excess of RNA (5 jig of 70S AMV RNA) in the presence of ions as used in enzyme assays. For binding analysis of a, 100 ,ul of enzyme was mixed with 5 ,ug of 70S AMV RNA under assay conditions in 200 ,l (final volume). Without further incubation the material was layered on top of preformed glycerol density gradients (10 to 30% glycerol in TNE [0.01 M Trishydrochloride-0.1 M NaCl-1 mM EDTA], pH 7.4-5 mM dithiothreitol-0.2% Nonidet P-40). After centrifugation for 90 min at 50,000 rpm and 4 C in a SW50. 1 Beckman rotor, the gradients were fractionated. Position of the enzyme was determined by testing an aliquot (20 ,u) of each fraction for enzyme activity by the addition of four deoxyribonucleoside triphosphates and ions to establish regular enzyme assay conditions. Alternatively, poly(rA) oligo(dT) and L3H]TTP can be added to each fraction for determination of response to synthetic template-primers (in the case of a this test has to be applied, since a does not transcribe native RNA). Chromatography of 3H-labeled poly(rA) digestion products. 3H-labeled poly(rA) oligo(dT) (5,000 counts/min per pmol of AMP) was hydrolyzed by viral RNase H activities in standard assay mixtures. After the reaction the mixture was spotted on strips (50-cm long) of Whatman no. 1 paper and chromatographed in n-propanol-ammonia-water (55:10:35) for 48 h to separate various oligomers of AMP (5). Portions (50 to 100 ,ug) of (A),A, (A)6A, (A)4A, and (A)2A were added as standards to the reaction mixtures before spotting. The paper was air dried after chromatography, the position of the standards was located by UV light, and 1-cm strips were cut. Radioactivity was determined in toluenebased scintillation fluid. The standards applied here do not have terminal phosphates and therefore do not migrate exactly like phosphorylated products of RNase H digestion. Therefore no precise assignments for the length of the oligomers were possible. Polyacrylamide gel electrophoresis. Enzyme polypeptides were analyzed by sodium dodecyl sul-

fate-polyacrylamide gel electrophoresis according to

a modified method of Shapiro et al. (11) as described by Kacian et al. (4). Before applying proteins to the gel, concentration was achieved by precipitation with 50% ethanol (for solubilization of the detergent Nonidet P-40) and 10% trichloroacetic acid (-20 C, 10 h). Protein was pelleted in a Sorvall HB4 rotor at 10,000 rpm for 30 min, dried, and suspended in sample buffer for electrophoresis. [3H]glucosamine-labeled virus (Prague A [PR-A] strain of Rous sarcoma virus) was used as internal standard (1,000 counts/min per gel). The so-called gp85 and gp37 glycoproteins migrate more slowly in the buffer system used (13) than their names indicate. The

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gels were stained with Coomassie blue, and the positions of the polypeptides were determined by a Gilford scanning apparatus at 600 nm wavelength. Afterwards the gels were frozen, sliced (1 mm), and incubated in 200 A.l of soluene for 2 h at 60 C. After the addition of 2 ml of scintillation fluid, radioactivity was determined in a liquid scintillation counter.

RESULTS Structural properties of the Fr-MuLV reverse transcriptase-RNase H complex. The reverse transcriptase-RNase H complex was isolated from purified Fr-MuLV by DEAE-cellulose and phosphocellulose column chromatography according to published procedures (8). The purified enzyme consists of a single polypeptide with an estimated molecular weight of 84,000, based on the size of the two avian enzyme subunits 13 and a being 110,000 and 70,000 (4) (Fig. 1A and E). Mild protease treatment under conditions that allow increase of a at the expense of (8 in the avian system (7) generated, from the

84,000-molecular weight polypeptide, new polypeptides of 78,000, 69,000, and 60,000 molecular weights, and a large amount of low-molecularweight material. Increasing amounts of trypsin (5 to 30 /ig/ml) predominantly resulted in an increase in the proportion of the 78,000-molecular-weight polypeptide (data not shown). Furthermore, the enzyme was extracted from the purified virus under conditions that allowed transfer of the avian enzyme to a preparation consisting predominantly of pure a. This can be achieved by binding the enzyme to a DEAE column for 1 to 2 days at 4 C before elution with a continuous salt gradient (Moelling, Virology, in press). Such a treatment caused degradation of the murine enzyme as well and gives rise to a preparation of 84,000-, 78,000-, 69,000-, and 60,000-molecular-weight polypeptides in addition to smaller material (Fig. 1C). This polypeptide pattern was obtained twice. In a third case the 78,000 polypeptide was missing (data not shown). Storage at -20 C increased the 69,000 polypeptide preferentially. Elution of the enzyme from phosphocellulose by means of a continuous salt gradient in a regular purification procedure consistently revealed a small peak of activity at a low salt concentration (0.11 M KCl) in addition to the main peak at 0.22 M KCl (not shown). The first peak corresponds to the region from which the subunit a in the avian system can be recovered (3). Its polypeptide composition (Fig. iD) consisted of 78,000and 69,000-molecular-weight bands in addition to the 84,000 and smaller ones. Purified avian af-containing enzyme is shown for control. Figure iF shows the isolated avian a subunit

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migration FIG. 1. Scanning profiles of murine and avian viral enzyme polypeptides after gel electrophoresis. (A) Purified Fr-MuLV reverse transcriptase-RNase H; (B) purified Fr-MuLV enzyme after trypsin treatment (25 H.g/ml, 15 min, 35 C); (C) purified FrMuLV reverse transcriptase after long-lasting column chromatography (attachment to DEAE-cellulose for 1 or 2 days at 4 C before elation); (D) marine material elated at 0.11 MKC1 from the phosphocellulose column (corresponding to the elation point of the avian a subunit); (E) purified avian af3 enzyme preparation; (F) avian subunit a as elated from the phosphocellulose column and also obtained after in vitro protease treatment of the purified af3 complex (40 pg of trypsin, 35 C, 20 mm). The enzyme polypeptides were subjected to electrophoresis in the presence of [3H]glucosamine-labeled virus (2,000 counts/mmn; Rous sarcoma virus, Prague A) used as internal standard in all gels except for Fr-MuLV "a." The gels were stained first and subsequently processed for determination of radioactivity as described. In (A) the dotted line indicates the radioactive marker which is only shown by arrows in (B) through (F).

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that can be recovered as such from the phosphocellulose column at low ionic strength. It can, however, also be generated from the a/3-containing enzyme complex by protease treatment, which completely removes 13 (Moelling, Virology, in press). Only more stringent digestion conditions will also digest a. This effect is shown since it demonstrates that there is a real possibility of preferentially losing one subunit and only detecting a comparatively more stable breakdown product. Binding to natural RNA and RNA transcription. The Fr-MuLV reverse transcriptaseRNase H complex was tested for its ability to bind to native RNA templates, and its binding properties were compared to those of the avian subunit a and the avian a13 complex (Fig. 2). The murine enzyme, just like the avian a,8 enzyme, bound to the high-molecular-weight 70S AMV RNA and could be sedimented through a glycerol density gradient with the viral RNA. It was then detectable by enzyme activity assay in the region of the high-molecular-weight RNA. In contrast, a could not bind to RNA and stayed on top of the gradient under these conditions. As expected from the ability of the murine enzyme to bind to native RNA, it was also able to transcribe native RNA into DNA (Fig. 3). Addition of oligo(dT) stimulated DNA synthesis severalfold. Both reactions could be inhibited by actinomycin D, with the reaction without oligo(dT) inhibited about 50%. Actinomycin D inhibition has been shown to be indicative of synthesis of double-stranded DNA (6).

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Native RNA transcription was also analyzed with the degraded murine enzyme (Fig. 1C). RNA-dependent DNA synthesis with native RNA was not seen (Table 1). Only the addition of oligo(dT) overcame this defect. Actinomycin D did not inhibit this reaction. The same phenomena have been observed for the avian a subunit (Moelling, in press) and are listed here for comparison. The properties of the undegraded murine enzyme and those of the avian a,/ complex are shown in Table 1 as well. RNase H activity of the degraded murine enzyme was reduced by about 30% (not shown) compared to the undegraded one. Mode of action of murine viral RNase H activity. The murine viral RNase H present in the 84,000-molecular-weight polypeptide has been characterized as an exonuclease (8, 12). If this RNase H behaves like the one from the avian a,3 complex it should degrade one RNA strand completely before proceeding to another one. In contrast, the RNase H of the avian a subunit attacks a new RNA strand after each bond scission (3). The RNase H activities of the Fr-MuLV enzyme consisting only of the 84,000-molecularweight polypeptide and of a degraded murine viral enzyme preparation were tested for a random or processive mode of action. Behavior of the avian a- and a/3-containing enzyme preparations are shown for comparison (Fig. 4). Radioactively labeled hybrid was hydrolyzed by RNase H from enzymes of both murine and avian viral origin. If the radioactively labeled hybrid was competed for with tenfold molar

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rVIG. 2. Binding properties of the murine enzyme and avian a and af3 to RNA. Purified enzymes were mixed with an excess of native viral RNA and sedimented without further incubation under conditions which allow the RNA to migrate two-thirds down the gradient (arrows). The gradients were fractionated, and each fraction was tested for the presence of enzyme activity by response to synthetic template primers (see Materials and Methods) in the case of AMV a and af3. RNA-dependent DNA polymerase activity in the case of Fr-MuLV was tested by the addition of four deoxyribonucleoside triphosphates (and no additional template).

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o

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FIG. 3. Kinetics of RNA transcription by purified AMV a,-containing enzyme, pure a, and purified FrMuLV reverse transcriptase-RNase H. The three enzyme preparations were incubated with 2 Mg of 70S AMV RNA in the absence and presence of oligo(dT) primers under standard assay conditions (). Furthermore, DNA synthesis of these reactions was investigated in the absence (-) and presence (+) of actinomycin D (100 pg/mi). Aliquots were withdrawn from the reaction mixtures at the times indicated and processed for determination of acid-insoluble radioactive material.

TABLE 1. RNA-dependentDNA-synthesis ofdegraded and undegradedFr-MuLV reverse transcriptase (RT) compared with that of the avian a and af3 enzymesa Template

Degraded Fr-MuLV RT

Undegraded Fr-MuLV RT

Avian a

Avian a/3

RNA 0.4 6 0.05 5.5 RNA + oligo(dT) 18 25 5.5 11 RNA + ActD 0.5 2.5 0.05 2.4 RNA + oligo(dT) + ActD 19 17 5.4 6 a Fr-MuLV reverse transcriptase was purified under nonoptimal conditions (long-lasting column chromatography), and its ability to transcribe native RNA was compared with that of undegraded Fr-MuLV reverse transcriptase (see Fig. 1) and to that of avian a- and to avian a,8-containing enzyme. Identical enzyme units, defined in Materials and Methods, were applied. A 1-Mug sample of 70S avian myeloblastosis virus RNA was used as template in each reaction, and 1 Mug of oligo(dT) and 100 Mug of actinomycin D (ActD) per ml were added to the reactions as indicated. The amount of acid-insoluble material was determined and expressed as picomoles of [3H]dGMP incorporated.

excess of unlabeled hybrid 2 min after the start of the reaction, the rate of RNA degradation by the RNase H from the intact Fr-MuLV enzyme and the avian a,8 enzyme was not affected, whereas the reactions catalyzed by the degraded murine enzyme or by the avian a were severely reduced. Addition of excess unlabeled hybrid to the reaction mixtures prior to the enzymes did not reveal any RNase H activities. The intact murine enzyme, once bound to its substrate, proceeded to hydrolyze its RNA strand, just like the avian a,3 complex, whereas a degraded murine enzyme behaved like the avian a subunit. This reaction was carried out with a synthetic hybrid, [3H~poly(rA)-poly(dT),

as well as with a phage fd 3H-labeled RNADNA hybrid (not shown), with identical results. Murine viral RNase H digestion products. L3H]poly(rA)-poly(dT) was incubated with identical units of Fr-MuLV enzyme, avian af, and a under conditions that convert 70% of the [3H]poly(rA) into acid-soluble material. The size of the digested products was analyzed by paper chromatography, which allows the separation of mononucleotides and oligonucleotides up to eight bases in length, the position of which can be estimated by the use of fluorescing standards. Fr-MuLV and the avian af3 RNase H activities gave rise to similar

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amounts of small-sized oligomers (possibly tet- radation inside the virion due to freezing and ramers; see above), whereas no monomers thawing or during purification by long-lasting could be detected (Fig. 5). In contrast, a did not column chromatography, giving rise to addigive rise to small oligonucleotides but all of the tional polypeptides of 78,000, 69,000 and 60,000 material remained at the origin, indicating a molecular weight (Fig. 1). Degradation of the size larger than eight bases. The untreated con- enzyme can also be achieved artificially by in trol also did not migrate. Solubilization of 20 or vitro treatment with trypsin, which results in a 90% of the hybrid or use of the phage fd DNA- similar polypeptide pattern. RNA hybrid gave the same results (not shown). Enzyme preparations consisting of two or three polypeptides have been described by others (2, 13, 15), with size determinations similar DISCUSSION to the ones observed here. With such degraded The Fr-MuLV reverse transcriptase-RNase murine viral enzyme preparations it was possiH complex has been previously described as a ble to reproduce some of the deficiencies desingle polypeptide of 84,000 molecular weight scribed for mammalian viral reverse transcrip(8). The present study shows that this polypep- tases; degradation of an enzyme preparation tide reveals all the properties known for the causes loss of the ability to transcribe native reverse transcriptase-RNase H complex of the RNA templates (Table 1). It can, however, be avian viruses. These properties are as follows: drastically stimulated to synthesize DNA if (i) the ability to bind to natural RNA and to oligo(dT) primers are added to the natural RNA transcribe it without the addition of synthetic template. These results obtained with a deprimers; (ii) inhibition of DNA synthesis by graded enzyme preparation explain previously actinomycin D, indicating double-stranded published results on mammalian reverse tranDNA synthesis; (iii) co-purification of an RNase scriptases (1, 13, 14). Response to synthetic H; (iv) exonucleolytic mode of action of the template primers is unaffected by any degree of RNase H activity; and (v) oligomers, approxi- enzyme degradation. Reduction of RNase H acmately tetranucleotides, as the main RNase H tivity during degradation has been observed digestion products and the absence of mononu- consistently in these studies; however, complete loss of RNase H could never be achieved cleotides. The Fr-MuLV reverse transcriptase-RNase with the RNase H assay applied here. H enzyme complex undergoes spontaneous degOnly two studies have been published show-

minutes

FIG. 4. Competition of hydrolysis of radioactively labeled hybrid by the addition of excess unlabeled hybrid. Purified reverse transcriptase-RNase H activities were incubated with [3H]poly(rA) -poly(dT) under standard assay conditions. At the times indicated portions were withdrawn from the reaction mixtures and processed for determination of acid-insoluble material (C). In a parallel experiment a 1 0-fold molar excess of unlabeled poly(rA) -poly(dT) was added 2 min after the start of the reaction (-). In a control reaction a 10-fold molar excess of unlabeled hybrid was already added to the reaction mixture prior to the enzyme (A).

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x 20

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FIG. 5. Chromatography of digestion products. Fr-MuLV RNase H digestion products obtained from a reaction that transferred 70% of the [3H]poly(rA)poly(dT) hybrid into acid-soluble material were chromatographed to separate oligomers ofAMP. The same experiment was performed with identical units of avian and af3 enzyme inputs. Untreated hybrid is shown as control. Included standards were located by UV light. a

ing a murine enzyme preparation consisting of a single polypeptide around 80,000 molecular weight (8, 12); in a third case this polypeptide appears among others (2). In these three studies natural RNA transcription has been observed. Therefore, the 84,000-molecular-weight polypeptide appears to be necessary for transcription of native RNA templates -it does not seem to be sufficient, however, since a degraded

enzyme may retain some of it without being able to transcribe RNA (Fig. 1C and Table 1). An analogous lack of RNA transcription was observed with the 84,000-molecular-weight enzyme from reticuloendotheliosis virus (9). The results obtained here with the undegraded and degraded murine viral enzyme preparations strongly resemble those obtained with the avian a,3 enzyme complex and isolated a, respectively. It was shown recently (Moelling, Virology, in press) that transcription of natural RNA can only occur in the presence of ,8. Subunit a was characterized as a degradation product of /3 that does not bind to natural RNA and is unable to transcribe it. This defect can be overcome by addition of excess oligo(dT) primers. DNA synthesis, then, appears to be predominantly single stranded as judged from insensitivity of the reaction to actinomycin D. It has been shown previously that the RNase H of the a subunit behaves like a random exonuclease (3) that is in agreement with its binding deficiency. The random mode of action of the Moloney murine viral RNase H activity and its large digestion products described by Verma (12) may then be a result of enzyme degradation as well. The 84,000-molecularweight polypeptide does not reveal these properties (Fig. 4 and 5) but behaves as one would expect, in analogy to the 18-containing avian enzyme. No longer does the murine viral reverse transcriptase-RNase H complex appear to be exceptional in its enzymatic and structural properties; instead, it behaves identically to what is considered to be characteristic of a real viral reverse transcriptase-RNase H complex, from experience with the avian enzyme as it transcribes natural RNA in the absence of synthetic primers and the RNase behaves like a processive exonuclease. It rather looks as if the avian enzyme is exceptional in respect to its structure, since it normally can only be isolated as a complex of the actual reverse transcriptase molecule, /3, accompanied by usually equal molar amounts of its degradation product, a. The reason for this effect is not understood, but it may reflect a tendency of the enzyme to form dimers or oligomers. Appearance of equal molar amounts of a prevents 8 from decaying further. Care should be taken in further enzyme characterizations not to analyze and describe the properties of a degraded enzyme preparation. Just one freezing and thawing of the virus can be sufficient to destroy the ability of the enzyme to transcribe RNA and can prevent a successful simultaneous direction assay, as was the case with reticuloendotheliosis virus (9).

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It seems very likely that the undegraded reverse transcriptase-RNase H enzyme complex exists in all RNA tumor viruses with the above mentioned enzymatic properties, possibly consisting of a single polypeptide. It will be of interest to elucidate its mode of action. ACKNOWLEDGMENT I thank H. Bauer for his support and interest in these studies. These studies were supported by the Deutsche Forschungsgemeinschaft (SFB 47). s

1.

2.

3.

4.

5.

6.

LITERATURE CITED Abrell, J. W., and R. C. Gallo. 1973. Purification, characterization and comparison of the DNA polymerase from two primate RNA tumor viruses. J. Virol. 12:431-439. Gerard, G. F., and D. P. Grangenett. 1975. Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus. J. Virol. 15:785-797. Grandgenett, D. P., and H. Green. 1974. Different mode of action of ribonuclease H in purified a and /3 RNAdirected polymerase from AMV. J. Biol. Chem. 249:5148-5152. Kacian, D. L., K. R. Watson, A. Burney, and S. Spiegelmann. 1971. Purification of the DNA polymerase of avian myeloblastosis virus. Biochim. Biophys. Acta 246:365-383. Lapidot, Y., and H. G. Khorana. 1963. Studies on polynucleotides. VVIX. The specific synthesis of C'3-C3'C,'-linked ribonucleotides. J. Am. Chem. Soc. 85:3857-3862. McDonnell, G. P., A.-C. Garapin, W. E. Levinson, N.

7.

8. 9.

10.

11.

12.

13.

14.

15.

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Quintrell, L. Fanshier, and J. M. Bishop. 1970. DNA polymerase of RSV: delineation of two reactions with actinomycin. Nature (London) 228:433-435. Moelling, K. 1974. Reverse transcriptase and RNase H: present in a murine virus and in both subunits of an avian virus. Cold Spring Harbor Symp. Quant. Biol. 39:969-973. Moelling, K. 1974. Characterization of reverse transcriptase and RNase H from Friend murine leukemia virus. Virology 62:46-59. Moelling, K., H. Gelderblom, G. Pauli, R. Friis, and H. Bauer. 1975. A comparative study on avian reticuloendotheliosis virus: relationship to murine leukemia virus and viruses of the avian sarcoma-leukosis complex. Virology 65:546-557. Schafer, W., and E. Seifert. 1968. Production of a potent complement-fixing murine leukemia virus antiserum from the rabbit and its reactions with various types of tissue culture cells. Virology 35:323-328. Shapiro, A. L., E. Vinuela, and J. V. Maizel. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815-820. Verma, J. 1975. Studies on reverse transcriptase of RNA tumor viruses. III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. J. Virol. 15:843-854. Verma, J. M., N. L. Meuth, H. Fan, and D. Baltimore. 1974. Hamster leukemia virus: lack of endogenous DNA synthesis of its DNA polymerase. J. Virol. 13: 1075-1082. Wang, L.-H., and P. H. Duesberg. 1973. DNA polymerase of murine sarcoma-leukemia virus: lack of detectable RNase H and low activity with avian viral RNA and natural DNA templates. J. Virol. 12:1512-1521. Weimann, B. J., J. Schmidt, and D. J. Wolfrun. 1974. RNA-dependent DNA polymerase and ribonuclease H from Friend virions. FEBS Lett. 43:37-44.

Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex.

Vol. 18, No. 2 Printed in USA. JOURNAL OF ViROLoGy, May 1976, p. 418 425 Copyright © 1976 American Society for Microbiology Further Characterization...
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