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Viral_RNA-dependent_DNA_polymerase - NATURE VOL 226 1209...

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Unformatted text preview: NATURE VOL. 226 JUNE 27 1970 1209 Viral RNA-dependent DNA Polymerase Two independent groups of investigators have found evidence of an enzyme in virions of RNA tumour viruses which synthesizes DNA from an RNA template. This discovery, if upheld, will have important implications not only for carcinogenesis by RNA viruses but also for the general understanding of genetic transcription: apparently the classical process of information transfer from DNA to RNA can be inverted. RNA-dependent DNA Polymerase in Virions of RNA Tumour Viruses DNA seems to have a critical role in the multiplication and transforming ability of RNA tumour virusesl. Infection and transformation by these viruses can be prevented by inhibitors of DNA synthesis added during the first 8—12 h after exposure of cells to the virus1-4. The necessary DNA synthesis seems to involve the production of DNA which is genetically specific for the infecting virus”, although hybridization studies intended to demonstrate virus< specific DNA have been inconclusivel. Also, the formation of virions by the RNA tumour viruses is sensitive to actinomycin D and therefore seems to involve DNA- dependcnt RN A synth,csis1-‘v7. One model which explains these data postulates the transfer of the information of the infecting RNA to a DNA copy which then serves as template for the synthesis of viral RNAWJ. This model requires a unique enzyme. an RNA-dependent DNA polymerase. No enzyme which synthesizes DNA from an RNA template has been found in any type of cell. Unless such an enzyme exists in uninfected cells, the RNA tumour viruses must either induce its synthesis soon after infection or carry the enzyme into the cell as part of the virion. Precedents exist for the occurrence of nucleotide pol yi'uerases in the virions of animal viruses. Vacciniawv— a DNA virus, Room-u—a double-stranded RNA virus, and vesicular stomatitis virus (VSVWV ea single-stranded RNA virus, have all been shown to contain RNA poly— merases. This study demonstrates that an RNA-depen- dent DNA polymcrasc is present in the virions of two RNA tumour viruses: Rauscher mouse leukaemia virus (R-MLV) and Rous sarcoma virus. Temin13 has also identified this activity in Rous sarcoma virus. Incorporation R-MLV A preparation of purified R-MLV was incubated in conditions of DNA polymerase assay. The preparation incorporated radioactivity from 3H-TT]? into an acid- insolublo product (Table 1). The reaction required Mg“, although Mn2+ could partially substitute and each of the four (leoxyribonucleoside triphosphates was neces- sary for activity. The reaction was stimulated strongly by dithiothreitol and weakly by NaCl (Table 1). The kinetics of incorporation of radioactivity from 3I-I-TTP by R-MLV are shown in Fig. 1, curve 1. The reaction rate accelerates for about 1 h and then declines. This time- of Radioactivity from 3H-TTP by course may indicate the occurrence of a slow activation of the polymerase in the reaction mixture. The activity is approximately proportional to the amount of added Virus. For other viruses which have nucleotide polymerases in their virions, there is little or no activity demonstrable unless the Virions are activated by heat, proteolytic enzymes or detergents“? None of these treatments increased the activity of the R-MLV DNA polymerase. In fact, incubation at 50° C for 10 min totally inactivated the R-MLV enzyme as did inclusion of trypsin (50 (Lg/ml.) in the reaction mixture. Addition of as little as 001 per cent ‘Triton N-lOl’ (a non-ionic detergent) also markedly depressed activity. Table 1. PROPERTIES OF THE RAUSCHER MOUSE LEUKAEMIA VIRUS DNA POLYMERASE pmolcs 3H-TMP Reaction system incorporated in 45 min Complete 3-31 Without magnesium acetate 0-04 Without magnesium acetate + 6 mM MnCl2 1-59 Without dithiothrcitol 0-38 \Vlthoull NaCl 2'18 Without dATP < 0-10 Without dCTP 01? Without dGTP < 0-10 A preparation of RJILV was provided by the Viral Resources Program of the National Cancer Institute. The virus had been purified from the plasma of infected Swiss mice by dill'erential centrifugation. The preparation had a titre of 104'“ spleen enlarging doses (50 per cent end point) per ml. Before use the preparation was centrifuged at 105,000g for 30 min and the pellet was suspended in 0137 M NaCleO'OOE M KCl—O-Ol M phosphate biifiCI‘ (pH 7-4)—0-6 mM ED’l‘A (PBS—EDTA) at 1/20 of the initial volume. The concentrated virus suspension contained 3-1 mg/ml. of protein. The assay mixture contained, in 0-1 1111., 5 [11110165 Tris-HCMpH 8-3) at 37° C, 0-6 ,umole magnesium acetate, 6 ,umoles NaCl, 2 umoles dithiothreitol, 0-08 ,umole each of dATP, dCTt’ and dGTP, 0-001 ymole [3H-methleeTTP (708 c.p.m. per pmole) (New England Nuclear) and 15 [lg viral protein. The reaction mixture was incubated for 45 min at 37° C. The acid—imolublc radioactivity in the sample was then determined by addition of sodium pyrophosphate, carrier yeast RNA and trichlcroacctic acid followed by filtration through a membrane filter and counting in a scintillation spectrometer, all as previously described”. The radioactivity of an unincubatcd sample was subtracted from each value (less than 7 per cent of the incorporation in the complete reaction mixture). Characterization of the Product The nature of the reaction product was investigated by determining its sensitivity to various treatments. The product could be rendered acid-soluble by either pancreatic deoxyribonuelease or micrococcal nuclease but was unaffected by pancreatic ribonuclease or by alkaline hydrolysis (Table 2). The product therefore has the pro- perties of DNA. If 50 ug/ml. of deoxyribonuclease was 1210 added to a reaction mixture there was no loss of acid-insoluble product. The product is therefore protected from the enzyme, probably by the envelope of the virion, although merely diluting the reaction mixture into 10 mM MgCl2 enables the product to be digested by deoxyribo- nuclease (Table 2). Table 2. CHARACTERIZATION OF THE POLYMERASE PRODUCT Percentage Expt. Treatment Acid-insoluble undigested radioactivity product 1 Untreated 1,425 (100) 20 Mg deoxyribonuclease 125 9 20 [lg micrococcal nuclease 69 5 20 ,ug ribonucleasc 1,361 96 2 Untreated 1,644 (100) NaOH hydrolysed 1,684 100 For experiment 1, 93 ,ug cf viral protein was incubated for 2 h in a reaction mixture twice the size of that described in Table 1, with aH-TTP having a specific activity of 1,133 c.p.m. per pmole. A 50 n1, portion of the reaction mixture was diluted to 5 ml. with 10 mM MgCl2 and 05 ml. aliquots were incubated for 15 h at 37° C with the indicated enzymes. (The sample with micrococcal nuclease also contained 5 mM CaClg.) The samples were then chilled, precipitated with trichloroacetic acid and radioactivity was counted. For experiment 2, two standard reaction mixtures were incubated for 45 min at 37° C, then to one sample was added 0-1 ml. of 1 M NaOH and it was boiled for 5 min. It was then chilled and both samples were precipitated with trichloroacetic acid and counted. In a separate experiment (unpublished) it was shown that the alkaline hydrolysis conditions would completely degrade the RNA product of the VSV virion polymerase. Localization of the Enzyme and its Template To investigate whether the DNA polymerase and its template were associated with the virions, a R-MLV sus- pension was centrifuged to equilibrium in a l5e50 per cent sucrose gradient and fractions of the gradient were assayed for DNA polymerase activity. Most of the activity was H l") N "J! 0 Di | iIii-TM? incorporated (c.p.m. x 10-3) H O Fig. 1. Incorporation of radioactivity from aH—TTP by the R-MLV DNA polymerase in the presence and absence ofribonucleasc. A 15-fold standard reaction mixture was prepared with 30 ng of viral protein and 3H—TTP (specific activity 950 c.p.m. per pmole). At various times, 20 ,ul. aliquots were added to 0-5 ml. of non-radioactive 0-1 M sodium pyrophosphate and acid insoluble radioactivity was determined”. For the preincubatcd samples, 0-06 m1. of H20 and 001 ml. of R-MLV (30 pg of protein) were incubated with or without 10 m; of pancreatic ribonuclcase at 22" (l for 20 min, chilled and brought to 015 ml. with a concentrated mixture of the components of the assay system. Iurve Tl, no treatment; curve 2, preincubated; curve 3, 10 ,ug rii‘mnudeasc added to the reaction mixture; curve 4, preincubated with 10 ng ribonuciease. NATURE VOL. 226 JUNE 27 l97O W ”’T 7 W l p=l.l6 A .l E 15 ~ - x. S o “g 10 “ 8 E? E “e a 5 ~ - — s‘ -/ ./ \o/.\o\./ \ /o" i l l \O/.\o’. 5 10 15 Fraction N0. Fig. 2. Localization of DNA polymerase activity in R-MLV by iso- pycnic centrifugation. A preparation of R-MLV containing 150 ,ug of protein in 50 Mi. was layered over a linear 52 ml. gradient of 15-50 per cent sucrose in PBS—EDTA. After centrifugation for 2 h at 60,000 r.p.m. in the Spinco ‘SW65’ rotor, 027 ml. fractions of the gradient were collected and 0-1 ml. portions of each fraction were incubated for 60 min in a standard reaction mixture. The acid-precipitable radioactivily was then collected and counted. The density of each fraction was determined from its refractive index. The arrow indicates the posit-ion of a sharp, visible band of light-scattering material which occurred at a density of 1-16. found at the position of the visible band of virions (Fig. 2). The density at this band was 1'16 g/cma, in agreement with the known density of the virions”. The polymerase and its template therefore seem to be constituents of the virion. The Template is RNA Virions of the RNA tumour viruses contain RNA but no DNA‘W". The template for the virion DNA polymerase is therefore probably the viral RNA. To substantiate further that RNA is the template, the effect of ribo- nuclease on the reaction was investigated. When 50 pg/ml. of pancreatic ribonuclcase was included in the reaction mixture, there was a 50 per cent inhibition of activity during the first hour and more than 80 per cent inhibition during the second hour of incubation (Fig. 1, curve 3). If the vii-ions were preincubated with the enzyme in water at 22° C and the components of the reaction mix- ture were then added, an earlier and more extensive inhibition was evident (Fig. 1, curve 4). Preincubation in water without ribonuclease caused only a slight in- activation of the virion polymerase activity (Fig. 1, curve 2). Increasing the concentration of ribonuclease during preincubation could inhibit more than 95 per cent of the DNA polymerase activity (Table 3). To ensure that the inhibition by ribonuclease was attributable to the enzymic activity of the added protein, two other basic proteins were preincubated with the virions. Only ribo- nuclease was able to inhibit the reaction (Table 3). These experiments substantiate the idea that RNA is the tern- plate for the reaction. Hybridization experiments are in progress to determine if the DNA is complementary in base sequence to the viral RNA. Ability of the Enzyme to Incorporate Ribonucleotides The deoxyribonucleotide incorporation measured in these experiments could be the result of an RNA poly- merase activity in the virion which can polyi‘nerize deoxyribonueleotides when they are provided in the reaction mixture. The VSV RNA polymerase and the R-MLV DNA polymerase were therefore compared. The VSV RNA polymerase incorporated only ribonucleotides. At its pH optimum of 7-3 (my unpublished observation), NATURE VOL. 226 JUNE 27 1970 in the presence of the four common ribonucleoside triphosphates, the enzyme incorporated 3H-GMP exten- sively”. At this pH, however, in the presence of the four deoxyribonucleoside triphosphates, no 3H-TMP incorpora- tion was demonstrable (Table 4). Furthermore, replace- ment of even a single ribonucleotide by its homologous deoxyribonucleotide led to no detectable synthesis (my unpublished observation). At pH 83, the optimum for the R—MLV DNA polymerase, the VSV polymerase cata- lysed much less ribonucleotide incorporation and no significant deoxyribonucleotidc incorporation could be detected. Table 3. EFFECT or RIBONUCLEASE ON THE DNA reLYMERAss ACTIVITY or RAUSCHER )Iocsn LEUKAEMIA VIRUS pmoles aH-TMP Conditions incorporation No preincubation 2-50 Preincnbated with no addition 2-20 Preincubated with 20 ug/ml. ribonuclcasc 0'69 Preincuhated with 50 yg/ml. ribonuclease 0~31 Preincubated with 200 ug/ml. ribonuclease 0‘08 Preincubatcd with no addition 369 Preincubatcd with 50 ,ug/ml. ribonuclease 0'52 Preincubatcd with 50 [lg/1111. lysozyme 3'67 Prcincubated with 50 pig/nil. cytochrome 0 3-97 In experiment 1, for the preincubation, 15 pg of viral protein in 5 ul. of solution was added to 45 ,ul. of water at 4° 0 containing the indicated amounts of enzyme. After incubation for 30 min at 22° C, the samples were chilled and 50 ul. of a 2-fold concentrated standard reaction mixture was added. The samples were then incubated at 37° C for 45 min and acidinsoluble radio- activity was measured. In experiment 2, the same procedure was followed, except that the preincubation was for 20 min at 22° C and the 37° C incuba‘ tion was for 60 min. Table 4. COMPARISON or NUCLEOTIDE INCORPORATION BY VESICULAR sronArIrrs VIRUS AND RAL‘SUHER MOUSE LEUKAEMIA VIRUS Incorporation in 45 min (pmolcs) Precursor 19H Vesicular Mouse stomatitis virus leukaemia virus SH-TTP 83 < 0-01 23 “H-TTP (omit dATI’) 8-3 ND. 006 3HTTP (omit dATP; plus ATP) 83 N.D. 0-08 “H-GTP 8-3 043 < 003 3H-GTI’ 7'3 37 < 003 When aH-TTP was the precursor, standard reaction conditions were used (see Table 1). When 3H-GTP was the precursor, the reaction mixture contained, in 01 ml., 5 ymoles Tris-HCl (p11 as indicated), 06 umoles mag- nesium acetate, 03 111110105 mercaptoethanol, 9 ”moles NaCl, 008 ymole each of ATP, GTP, UTP; and 0001 yniole 3H-GTP (1,040 c.p.m. per pmole). All VSV assays included 0-1 per cent ‘Triton N—101’ (ref. 12) and 2~5 Mg of viral protein. The R-MLV assays contained 15 ,ug of viral protein. The R-MLV polymerase incorporated only deoxyribo- nucleotides. At pH 8-3, 3I‘LTMP incorporation was readily demonstrable but replacement of dATP by ATP com- pletely prevented synthesis (Table 4). Furthermore, no significant incorporation of 3H-GMP could be found in the presence of the four ribonucleotides. At pH 7-3, the R-MLV polymerase was also inactive with ribonucleotides. The polymerase in the R-MLV Virions is therefore highly specific for dcoxyribonuclootides. DNA Polymerase in Rous Sarcoma Virus A preparation of the Prague strain of Rous sarcoma virus was assayed for DNA polymerase activity (Table 5). Incorporation of radioactivity from 31-1-TTP was demon- strable and the activity was severely reduced by omission of either Mg2+ or dATP from the reaction mixture. RNA— dependent DNA polymerase is therefore probably a con- stituent of all RNA tumour Viruses. These experiments indicate that the Virions of Rauscher mouse leukaemia virus and Rous sarcoma virus contain a DNA polymerase. The inhibition of its activity by ribo- nuclease suggests that the enzyme is an RNA-dependent DNA polymerase. It seems probable that all RNA tumour viruses have such an activity. The existence of this enzyme strongly supports the earlier suggestions“7 that 1211 genetically specific DN A synthesis is an early event in the replication cycle of the RNA tumour viruses and that DNA is the template for viral RNA synthesis. Whether the viral DNA (“provirus”)2 is integrated into the host genome or remains as a free template for RNA synthesis will require further study. It will also be necessary to determine whether the host DNA—dependent RNA poly- merase or a virus-specific enzyme catalyses the synthesis of viral RNA from the DNA. PROPERTIES OF THE Rous SARCOMA VIRUS DNA POLYMERASE pmoles aH<TMP incorporated in 120 min Table 5. Reaction system Complete 2-06 Without magnesium acetate 01 2 Without dATP 0-19 A preparation of the Prague strain (sub-group C) of Rous sarcoma virus“ having a titre 01‘5 x 10’ focus forming units per ml. was provided by Dr Peter Vogt. The virus was purified from tissue culture fluid by differential centre filgzition. Before use the preparation was centrifuged and the pellet dissolved in 1/10 of the initial volume as described for the It-MLV preparation. For each assay 15 yl. of the concentrated Rous sarcoma. virus preparation was assayed in a standard reaction mixture by incubation for 2 h. An unincu- bated control sample had radioactivity corresponding to 014 pmole which was subtracted from the experimental values. I thank Drs G. Todaro, F. Rauscher and R. Holdenreid for their assistance in providing the mouse leukaemia virus. This work was supported by grants from the US Public Health Service and the American Cancer Society and was carried out during the tenure of an American Society Faculty Research Award. DAVID BALTIMORE Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. Received June 2, 1970. 1 Green, M., Ann. Rev. Biochem, 39 (1970, in the press). 2 Temin, H. M., Virology, 23, 486 (1964). 3 Bader, J. P., Virology, 22, 462 (1964). ‘ Vigier, P., and Golde, A., Virology, 23, 511 (1964). 5 Ducsbcrg, P. H., and Vogt, P. K., Prac. US Nat. Acad. $075., 64, 939 (1969). 6 Temin. H. M., in Biology of Large RNA Viruses (edit, by Barry, 11,, and Many. B.) (Academic Press, London, 1970). 7 Tcmiu, H. M., Virology, 20, 577 (1963). 3 Katee, J. R., and McAuslan, B. 3., Proc. US Nat. Acad. Sui, 58, 13411967). 9 Munyon, W., Paoletti, E“, and Grace, J. T. J., Proc. US Nat. Acad. Sci., 58, 2280 (1967). 1“ Shatkin, A. J., and Sipe, J. D., Proc. US Nat. Acad. Sal, 61, 1462 (1968). ’3 B03368), and Graham, A. F., Blocizem. Biophys. Res. Oommun., 33, 895 12 Baltimore, 1)., Huang, A. S., and Stampfcr, M., Proc. US Nat. Acad. Sci. 66(1970, 1n the press). ‘3 Tcmin, H. 111., and Mizutani, S., Natm'e,226, 1211 (1970) (following article). 1‘ 0’?p51&e)r, T. 13., Rauscher, F. J., and Zeigel, 1%. F., Science, 144, 1141 15 Crawford, L. V., and Crawford, E. M., Virology, 13, 227 (1961). 1“ Blast-336g?) P., and Robinson, W. 8., Proc. US Nat. Acad. 305., 55, 219 ‘7 Duff, R. G., and Vogt, P. K., Virology, 39, 18 (1969). RNA-dependent DNA Polymerase in Virions of Rous Sarcoma Virus INFECTION of sensitive cells by RNA sarcoma viruses requires the synthesis of new DNA different from that synthesized in the S-phase of the cell cycle (refs. 1, 2 and unpublished results of D. Boettiger and H. M. T.); production of RNA tumour viruses is sensitive to actinc- mycin D“; and cells transformed by RNA tumour viruses have new DNA which hybridizes with viral RNA”. These are the basic observations essential to the DNA provirus hypothesisireplication of RNA tumour viruses takes place through a DNA intermediate, not ...
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