Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 620-624, February 1979
Biochemistry
Effect of benzo[a]pyrene-diolepoxide on infectivity and in vitro translation of phage MS2 RNA (polycyclic aromatic hydrocarbons/infectious phage RNA/RNA binding by benzo[alpyrene-diolepoxide/chemical carcinogenesis)
DAPHNA SAGHER*, RONALD G. HARVEY*, WEN-TAH HSUtt,
AND
SAMUEL B. WEISS*tt
tThe Franklin McLean Memorial Research Institute, the *Departments of Biochemistry and Microbiology, and *The Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, Illinois 60637
Communicated by Elwood V. Jensen, November 6,1978
ABSTRACT Previous studies have shown that alkylation of MS2 RNA by certain derivatives of polycyclic aromatic hydrocarbons renders it noninfectious. Since phage RNA serves as a template for translation and transcription, either of these RNA-directed processes, or both, could be responsible in vivo for the inhibition of phage replication by metabolically activated hydrocarbons. The present study correlates the degree of inhibition of MS2 RNA infectivity, at various levels of alkylation by (-I-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpyrene, with the translation efficiency in vitro of the same alkylated RNA for the synthesis of viral synthetase and of maturation and coat proteins. The results indicate that dihydroxyepoxy-tetrahydrobenzola pyrene modification of MS2 RNA impairs its template capacity for the synthesis of phagespecific proteins; this inhibition is insufficient, however, to account for the loss of RNA infectivity at lower molar ratios of alkylation. For the three viral proteins synthesized in vitro, the translation of RNA synthetase is much more sensitive to MS2 RNA modification than either coat or maturation protein synthesis. Our results also indicate that the loss of viral RNA infectivity follows a single-hit inactivation mechanism, whereas several alkylation events in the viral RNA synthetase cistron may be necessary to block translation of this gene product.
Carcinogenic polycyclic aromatic hydrocarbons (PAH) inhibit the replication of single-stranded RNA and DNA bacterial viruses (1-3). We have used this inhibitory activity to evaluate the relative potency of different PAH and their oxidized metabolites and derivatives (4). Among the compounds examined, (+)-trans - 7,8 - dihydroxy-anti -9,10-epoxy-7,8,9, 10- tetrahydrobenzo[a]pyrene (anti-BzPDE) was the most active inhibitor of phage replication (4). This compound is the principal metabolite of benzo[a ]pyrene (BzP) bound to DNA in mouse cells (5) and in bovine and human bronchial explants (6, 7). It is also mutagenic in animals (8, 9) and bacteria (10, 11) and carcinogenic in mice (12-14), and has been suggested to be the ultimate carcinogenic metabolite of BzP. Studies with R17 bacteriophage (a single-stranded RNAcontaining virus) (3) and with infectious 4X174 DNA (15) have indicated that an average of one molecule of bound hydrocarbon derivative per molecule of viral nucleic acid is sufficient to block phage replication. The work by Hsu et al. (15) also showed that conjugation of OX DNA with BzPDE impairs its function as a template for the synthesis of double-stranded DNA replicative intermediates, which is a necessary first step in the replication of OX phage. The replication of single-stranded RNA phage is different from that of single-stranded DNA phage in that the infectious RNA serves first as a template for the translation of viral proteins and then as a template for the transcription of new viral RNA molecules. The inhibition of RNA phage replication by activated PAH derivatives could occur at either of these RNA-directed synthetic processes.
Purified RNA from the related bacteriophages f2, R17, and MS2 functions as a template, in cell-free systems, for the directed synthesis of phage-specific RNA synthetase and of maturation and coat proteins (16-18). The present study shows that alkylation of MS2 RNA with anti-BzPDE inhibits its template function for the synthesis of RNA synthetase; this inhibition is significantly less pronounced, however, than the inhibition of MS2 RNA infectivity. METHODS MS2 RNA. MS2 RNA was isolated Infectious of Preparation from CsCI-purified MS2 phage by lysis in 1% sodium dodecyl sulfate (NaDodSO4) and repeated extraction with phenol and finally with phenol/chloroform 1:1 (vol/vol) containing 4% isoamyl alcohol. One-tenth volume of 20% potassium acetate (pH 5) was added, and the RNA was precipitated with ethanol. The precipitation process was repeated, and the RNA was finally dissolved in sterile water and stored in aliquots at -700C. Assay for Infectious MS2 RNA. MS2 RNA was assayed for infectivity by a described procedure (1). MS2 RNA was mixed with spheroplasts prepared from Escherichia coil K-12W1485 (male strain). After 5 min of incubation at 25°C, the infectious mixture was plated with intact E. coil K-12W1485 and incubated overnight at 37°C for plaque development. Under our assay conditions, 1 ,tg of freshly prepared MS2 RNA produced t1000 plaque-forming units. Treatment of RNA with BzP-Diolepoxide. Hydrocarbon was reacted with nucleic acid by the "pretreatment" method (4). RNA (100 ,ug/ml) and various amounts of anti-BzPDE were incubated at 25°C for 10-30 min in 50 mM Tris-HCl (pH 7.5). Stock solutions of anti-BzPDE were made in dimethylformamide; the concentration of this solvent in the reaction mixture did not exceed 6%. After incubation, the RNA was precipitated with ethanol at -20°C and successively with acetone and 95% ethanol. This washing procedure is effective in removing unreacted hydrocarbon (4). The RNA precipitate was suspended in water, and aliquots were taken for measurements of its A26o and infectivity content and for the biosynthetic reactions described in the text. anti-BzPDE, prepared as reported elsewhere (19-21), was of the highest attainable purity. When [3H]BzPDE was used in the above reaction, the extent of RNA alkylation was determined from radioactivity measurements of the dissolved precipitate, after washing. Protein Synthesis In Vitro. A 30,000 X g supernatant extract (S30) was prepared from E. coli Q13 (cells in middle logarithmic phase) by the method of Nirenberg (22), with the following minor modifications: 1 mM dithiothreitol replaced 2-mercapAbbreviations: PAH, polycyclic aromatic hydrocarbons; BzP, benzo[a pyrene; anti-BzPDE, (+)-trans-7,8-dihydroxy-anti-9,10epoxy-7,8,9,10-tetrahydro-BzP; NaDodSO4, sodium dodecyl sul-
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fate.
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Biochemistry: Sagher et al. toethanol in all buffers, and the S30 supernatant was preincubated and dialyzed before small portions were frozen at -70'C. The S30 preparation contained 97 A280 units/ml. The conditions for polypeptide synthesis in vitro were modified from those described by Capecchi (17). A 50-Al reaction mixture contained 50 mM Tris-HC1 (pH 7.8), 11.6 mM Mg(OAc)2, 40 mM NH4Cl, 7.5 mM phosphoenolpyruvate, 1 /ig of pyruvate kinase, 3 mM ATP, 2 mM GTP, 5 mM dithiothreitol, 5 ,ug of leucovorin, 40 ,uM each of 19 amino acids, 6 ,M of the missing radioactive amino acid (e.g., [3H]valine, 2 gCi), 2 ,ug of MS2 RNA, and 10 Ail of the preincubated S30 extract. Incubation was at 35°C for 30 min. Assay for Protein Synthesis. Protein synthesis was followed by the incorporation of radioactive amino acids into an acidprecipitable form, and by gel electrophoresis. Aliquots of the in vitro incubation mixtures were dried on filter paper discs, washed in cold 10% trichloroacetic acid, and heated at 90°C in 5% trichloroacetic acid. Radioactivity was measured in a Nuclear Chicago Mark III scintillation spectrometer in the presence of a Triton X-100/toluene scintillant mixture. To obtain the size pattern of the polypeptide products, we boiled aliquots of the incubation mixture for 5 min in the presence of 5% 2-mercaptoethanol/3% NaDodSO4 and subjected them to electrophoresis, together with protein markers, on polyacrylamide slab gels that contained 25% acrylamide monomer and 0.067% bisacrylamide (23). Electrophoresis was carried out at constant currents of 10 mA and 15 mA (per gel) for 1.5- and 2.0-mm-thick gels, respectively, for 18-20 hr at room temperature. After electrophoresis, the gels were fixed and stained in 50% methanol/7% acetic acid/0.25% Coomassie blue and destained in 20% methanol/7% acetic acid. For locating the radioactive polypeptides, the gels were either subjected to fluorography (24) or they were cut into 5-mm slices and dissolved in 30% H202 at 60°C and radioactivity was measured in a liquid scintillation spectrometer.
Radioisotopes. [3H]Valine (14.6 Ci/mmol) was purchased
from New England Nuclear. anti-[3H]BzPDE (specific activity, 822 cpm/ng) was synthesized under National Cancer Institute Contract CP-33387 by Midwest Research Institute (Kansas City, MO) and stored in dimethylformamide at 4°C. This material had a chemical purity of more than 90% (Shen Yang, National Cancer Institute, Bethesda, MD, personal communication). EXPERIMENTAL RESULTS Fig. 1A illustrates the binding of labeled anti-BzPDE to MS2 RNA. As the concentration of [3H]BzPDE is increased in the reaction mixture, the amount of labeled hydrocarbon bound to viral RNA increases linearly; at the same time, the infectivity of the viral nucleic acid is inactivated (see the fi curve). Under the conditions used for binding, the infectious activity of MS2 RNA is reduced by about 70% with 0.05 ,g of hydrocarbon in the reaction mixture; this corresponds to a molar alkylation ratio of 1 mole of BzPDE bound per mole of viral RNA. Fig. 1B shows the fraction of viral RNA inactivated (fi) as a function of the molar ratio of BzPDE bound to MS2 RNA, which closely follows the theoretical Poisson distribution for inactivation of nucleic acid bound by one or more moles of BzP (f,1BzP)- These results suggest, therefore, that a single molecule of bound hydrocarbon is sufficient to inactivate a molecule of MS2 RNA. In the E. coli S30 cell-free system used for polypeptide synthesis, MS2 RNA serves as an effective template for the incorporation of [3H1valine into an acid-precipitable form. Fig. 2 shows that, when BzPDE-bound MS2 RNA is used as a template in this system, its template efficiency for polypeptide synthesis is reduced significantly; the more BzPDE is bound to MS2 RNA,
Proc. Natl. Acad. Sci. USA 76 (1979)
[3H]BzPDE
in binding reaction, jig
621
Molar ratio of BzP bound to MS2 RNA
FIG. 1. Binding of BzPDE to MS2 RNA and its effect on infectivity. (A) Reaction mixture (0.1 ml) contained 10 ,g of MS2 RNA and various amounts of [3HJBzPDE. After incubation, the [3H]BzP-RNA complex was isolated and its RNA content, radioactivity, and infectivity were determined. f;, Fraction of infectious MS2 RNA that lost infectivity after reaction with the diolepoxide. (B) Fraction of MS2 RNA inactivated (ft) plotted as a function of the molar ratio (MR) of the average number of BzP molecules bound per molecule of MS2 RNA, calculated from the radioactivity and the RNA content of the isolated [3H]BzP-RNA complex. f4inzp and f>2BzP were derived from the expressions 1 - Po and 1 - Po - P1, respectively, in which Po = e-MR and Pi = MR x e-MR.
the lower the incorporation of radioactive valine. However, at a molar binding ratio of 4, which suppresses MS2 RNA infectivity by about 99% (Fig. 1B), incorporation of labeled valine is inhibited only 20%. The kinetics of [3H]valine incorporation for unmodified and modified MS2 RNA at two molar ratios of bound hydrocarbon is illustrated in Fig. 3A. Maximum incorporation occurs after 30-40 min of incubation with both types of templates; however, the amount of labeled protein formed at each time point is significantly less with modified MS2 RNA. Fig. 3B shows that, during relatively short reaction times (10 min), the amount of label incorporated is directly related to the amount of MS2 RNA present in the reaction mixture for both unmodified and modified templates. In this system, saturation with template was not achieved at the highest concentrations examined. Fig. 4 is a radioautograph of the protein products synthesized in the S30 system with [3H]valine, under the direction of untreated and modified MS2 RNA, and subjected to gel electrophoresis. Four distinct bands of varied radioactive intensity appeared, each migrating at a different rate. Band A was most intense, moved fastest, and was estimated to have a molecular size of 13,000 daltons; band B was the least intense, with an estimated size of approximately 17,000 daltons; bands C and D migrated in the gel corresponding to sizes of 39,000 and 64,000 daltons, respectively. The molecular sizes for the radioactive bands corresponding to A, C, and D agree very closely with those reported for MS2 coat, maturation, and RNA synthetase proteins (25); the nature and origin of band B radioactivity are not known. When [3H]histidine replaced radioactive valine in the S30 system, no label was found in the band A region (data not shown); this information supports the molecular size data which indicate that band A represents viral coat protein, since this protein is deficient in histidine (26). In Fig. 4, sample 1 shows the distribution of radioactive polypeptides assembled with unmodified MS2 RNA, and samples 2-5 represent the labeled protein products made under the direction of BzPDE-modified MS2 RNA templates; sample 6 is a control lacking a template. As the extent of BzPDE alkylation in MS2
Biochemistry: Sagher et al.
622
Proc. Natl. Acad. Sc. USA 76 (1979)
I
IR
BSA
41 0
4 A
3
4
5 6 D
E a,
C
0
4-6a 0
0
STI Q
B
C 0
0 C
c
Cyt ce
IL
A
0I
Molar ratio of BzP bound to MS2 RNA
FIG. 2. Effect of alkylation of MS2 RNA on its template function for protein synthesis. Untreated and BzPDE-modified MS2 RNA samples were used as templates in the S30 translational system in vitro, derived from E. coli. After 30 min of incubation, 10-,ll aliquots were taken from each reaction mixture and the amount of [3H]valine incorporated into an acid-precipitable form was determined. In the above system, t70,000 total counts were incorporated into trichloroacetic acid-precipitable material when 2 gg of unmodified MS2 RNA served as template; in the absence of added RNA, incorporation was less than 3% of the complete system, and this endogenous background activity was subtracted from all experimental results.
RNA is increased (from sample 2 to sample 5), there is a slight, but detectable, decrease in the amount of radioactivity incorporated into coat and maturation proteins (bands A and C); incorporation of isotope into band D (RNA synthetase), however, is abolished almost completely. In order to quantitate the relative amounts of the three viral Io
A
20
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20
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30
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40
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80
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.C. E
c,
M
0
40
20
30
Reaction time,
RXI0
40
min
MS2 RNA,
jig/ml
FIG. 3. Effect of BzP alkylation on the kinetics of MS2 RNAdirected protein synthesis. (A) Reaction system was as described under Methods, except that 1 jig of untreated or modified MS2 RNA (molar ratios of 4 and 10) served as template. At the time intervals
indicated, 10-jgl aliquots were removed and processed for trichloroacetic acid-precipitable radioactivity. (B) Reaction system was the same as for A, except that the amount of MS2 RNA (untreated and modified) was varied and the reaction was terminated after 10 min of incubation. MR, molar ratio.
FIG. 4. Gel electrophoresis of 3H-labeled proteins synthesized under the direction of MS2 RNA. After reaction, the incubation mixtures were heated in a boiling water bath for 5 min in the presence of 5% 2-mercaptoethanol/3% NaDodSO4 and then subjected to gel electrophoresis on 25% polyacrylamide containing 0.1% NaDodSO4. After electrophoresis, the gels were stained and dried, and radioactivity was detected. Molecular-weight markers: bovine serum albumin (BSA), Mr 68,000; soybean trypsin inhibitor (STI), Mr 23,500; and cytochrome c (Cyt c), Mr 12,400. MS2 RNAs used as templates were: 1, untreated; 2, 3, 4, and 5, treated with BzPDE containing molar ratio of BzP:MS2 RNA of 1, 2, 4, and 10, respectively; 6, no RNA control.
proteins made in vitro, we sliced the gels and determined their radioactivity. Fig. 5 shows the radioactivity profile of the labeled proteins assembled in the S30 system under the direction of unmodified and modified MS2 RNA. In Fig. 5A, the ratio of coat:synthetase:maturation protein synthesized was determined to be 1:0.27:0.08, which is comparable to the ratio of 1:0.3:0.06 for f2 phage RNA-directed protein products made in a similar in vitro system (27). With alkylated MS2 RNA as template (molar ratio of 10), RNA synthetase formation was almost completely absent (Fig. 5B), even though synthesis of maturation and coat proteins was affected only slightly. We routinely found distortions in the radioactive profile of the coat protein peak when BzPDE-MS2 RNA served as template (Fig. 5B), suggesting the formation of incomplete phage protein products. In Table 1, the infectivity of MS2 RNA is compared with its capacity in vitro to direct the synthesis of phage proteins as the amount of BzP binding to viral RNA is increased. With increasing molar ratios of bound hydrocarbon, both the infectivity of MS2 RNA and the translation of viral RNA synthetase were inhibited; however, RNA infectivity was considerably more sensitive to alkylation than is the RNA-directed synthesis of viral proteins. At a molar ratio of bound BzPDE to MS2 RNA of 1, infectivity was reduced by about 73%, whereas RNA synthetase formation was reduced by only about 30%. At a molar ratio of 4, the infectious activity of MS2 RNA was abolished almost completely; nevertheless, the synthesis of RNA synthetase under the direction of this alkylated template was still substantial (36%). Appreciable inhibition of maturation and coat protein synthesis was observed only at molar ratios greater than 4.
Biochemistry: Sagher et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
623
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0
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--
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FIG. 6. Relationship between RNA synthetase formed in vitro and the extent of BzP binding to the MS2 RNA synthetase cistron. The values for the inhibition of RNA synthetase formation (fA) were obtained from Table 1. The molar ratios of BzP bound to the synthetase cistron were estimated by multiplication of the molar ratios shown in Table 1 by the fraction of the total MS2 genome occupied by the synthetase cistron (0.46). f>lzp and f>2BzP were derived as described in the legend of Fig. 1.
X
C~~~~~~e slc (N
0
2
4
6
8 10 12 14 16 18 20 22 Gel slice
FIG. 5. Profiles of MS2 RNA-directed synthesized [3Hlproteins subjected to gel electrophoresis. Protein synthesis and gel electrophoresis of translation products in vitro were as described in the legend of Fig. 4. After electrophoresis, gels were sliced and the radioactivity was determined. (A) Unmodified MS2 RNA and (B) modified MS2 RNA (molar ratio of BzP:RNA = 10) served as templates in the protein synthesis reaction in vitro. Counts shown in A and B have been adjusted so that they are equivalent in terms of total radioactivity recovered from the gels. S, M, and C represent the peak positions for viral synthetase, maturation, and coat proteins.
Bacteriophage MS2 RNA is 3569 nucleotides long; the RNA synthetase cistron accounts for 1635 nucleotides or 46% of the total viral genome (28). If one assumes that the alkylation of MS2 RNA is random, there is a 46% chance for the binding of a single molecule of BzP to the synthetase cistron when the molar ratio of bound BzPDE to MS2 RNA is one. Fig. 6 illustrates the fraction of RNA synthetase inactivated (fi) as a Table 1. Relationship between alkylation of MS2 RNA and its effect on infectivity and phage protein synthesis % inhibition of infectivity and of phage mol BzPDE proteins synthesized RNA Maturation Coat bound/mol protein MS2 RNA Infectivity synthetase protein 0 0 0 None 0 1 2 4 10
73 86 99 100
30 40 64 92
1 0 0 21
4 2 3 Molar ratio of BzP bound to synthetase cistron
0 2 13 40
MS2 RNA was treated with BzPDE to provide various degrees of BzP alkylation. Samples of the untreated and modified viral RNAs were assayed for infectivity and for their template capacity in the in vitro translation system. Translation products were subjected to gel electrophoresis, and the distribution of radioactivity in synthetase, maturation, and coat proteins was determined as described in the legend for Fig. 5. The percent inhibition for each viral protein made with modified MS2 RNA templates was calculated from the decrease in radioactivity recovered in each protein region compared to untreated MS2 RNA translation products.
function of the molar ratio of BzPDE bound to the synthetase cistron; the values for fi and the molar ratios were taken from Table 1, except that the molar ratios were adjusted by a factor of 0.46. The experimental points for fi, at relatively low molar ratios, fall between the two theoretical Poisson curves f>1BzP and f¢2Bzp; thus, one or several alkylation events in the synthetase cistron may be required to block translation of the synthetase gene. DISCUSSION Certain PAH metabolites are inhibitory for bacteriophage replication. A single alkylation event within the genome of single-stranded phages is sufficient to suppress infectivity completely (3, 15). The experiments described here are consistent with these findings. We have attempted to define more precisely the mechanism of infectious RNA inactivation since single-stranded viral RNAs participate both in translation and transcription during virulent phage production. Grunberger and Weinstein (29) had shown that the binding of N-2-acetylaminofluorene residues to poly(U3G) blocks translation of this RNA copolymer. The present study shows, in addition, that the alkylation of MS2 RNA by anti-BzPDE inhibits its capacity to direct polypeptide synthesis; however, the inhibition of the messenger function of viral RNA is much less sensitive to PAH modification than its infectivity. At an average binding molar ratio of 1, the infectivity of the alkylated viral RNA is suppressed by 70%, whereas the synthesis of phage-specific polymerase (RNA synthetase) is reduced by only 30%. Thus, the reduced translational capacity of BzPDEmodified MS2 RNA, at relatively low molar ratios of bound hydrocarbon, is insufficient to account for the loss of infectious RNA activity, suggesting that alkylation blocks some other RNA-directed step; i.e., RNA synthesis. Leffler et al. (30) reported that the modification of calf thymus DNA with BzPDE inhibits its transcription with E. coli DNA-dependent RNA polymerase. Hsu et al. (15) showed that BzPDE groups on OX174 DNA block its conversion to a duplex replicative form by E. coli DNA polymerase. The studies by both groups indicate that nucleotide polymerization by RNA and DNA polymerases in vitro is impaired by alkylated sites on DNA templates; thus,
624
Biochemistry: Sagher et al.
chain elongation stops and incomplete complementary fragments are formed. It is not unreasonable to assume that phage-specific RNA polymerase is also blocked during transcription at BzP-conjugated sites in viral RNA and that the inability to form intact RNA-RNA replicative intermediates is directly responsible for the loss of infectivity observed. Unpublished results in our laboratory have indicated that similar alkylation of Qf3 RNA prevents the synthesis de novo of infectious Qf3 RNA in vitro by Qf3 RNA polymerase (31). The synthesis in vitro of RNA synthetase was affected much more adversely by the modification of MS2 RNA than the synthesis of either maturation or coat protein. Since the synthetase cistron has a considerably greater nucleotide length than the other two genes, one would expect a higher frequency of BzPDE binding in the synthetase region of the phage genome on the basis of random alkylation. It is not completely clear whether one or several alkylation sites within the synthetase cistron are required to block translation of this phage protein. At low molar ratios of bound hydrocarbon in the synthetase cistron, the reduction in synthetase formation follows a single-hit inactivation mechanism, but at higher molar ratios, a significant deviation from the theoretical f,1Bzp curve was observed (Fig. 6). It is possible that these deviations reflect some inaccuracy in our determination of viral synthetase formed and in the assumption that alkylation is random throughout the viral RNA chain; each of these factors would affect the estimated molar ratios of BzP bound to the synthetase cistron. It is also possible that some miscoding occurred with the BzPDE-modified viral template during translation, resulting in the formation of complete, but abnormal, polypeptides with electrophoretic properties similar to those of phage synthetase. In the translation experiments of Grunberger and Weinstein (29), however, inactivation, but not miscoding, was found with acetylaminofluorene-modified synthetic templates. The inactivation of single-stranded infectious RNA by PAH binding can be summarized as follows: At low molar ratios of PAH binding (4 or less), the translational synthesis of phage proteins decreases somewhat; this decrease is insufficient, however, to account for the loss of RNA infectivity, assuming that the polypeptides identified by our assay procedure represent functional phage proteins. Most probably, the loss of infectivity can be attributed to the inability of phage polymerase to transcribe PAH-modified viral RNA. At higher molar ratios, translation of phage polymerase is minimal so that, if the modified viral RNA could be transcribed, the amount of phage enzyme synthesized would be insufficient to catalyze RNA replication. The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the United States Department of Energy under Contract EY-76-C-02-0069. This study was partly supported by a grant from the American Cancer Society (BC-132A) and by funds from Environmental Protection Agency Subagreement 77BAN. D.S. is supported by Training Grant CA-09183 from the National Cancer Institute. 1. Hsu, W.-T., Moohr, J. W. & Weiss, S. B. (1965) Proc. Natl. Acad. Sci. USA 53,517-524.
Proc. Natl. Acad. Sci. USA 76 (1979) 2. Hsu, W.-T., Moohr, J. W., Tsai, A. Y. M. & Weiss, S. B. (1966) Proc. Natl. Acad. Sci. USA 55, 1475-1482. 3. Dipple, A. & Shooter, K. V. (1974) Biochim. Biophys. Acta 374, 392-399. 4. Hsu, W.-T., Harvey, R. G., Lin, E. J. S. & Weiss, S. B. (1977) Proc. Natl. Acad. Sci. USA 74, 1378-1382. 5. King, H. W. S., Osborne, M. R., Beland, F. A., Harvey, R. G. & Brookes, P. (1976) Proc. Natl. Acad. Sci. USA 73,2679-2681. 6. Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H. & Nakanishi, K. (1976) Science 193, 592-595. 7. Jeffrey, A. M., Weinstein, I. B., Jennette, K. W., Grzeskowiak, K., Nakanishi, K., Harvey, R. G., Autrup, H. & Harris, C. (1977) Nature (London) 269,348-350. 8. Huberman, E., Sachs, L., Yang, S. K. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73, 607-611. 9. Newbold, R. F. & Brooks, P. (1976) Nature (London) 261,52-
54. 10. Malaveille, C., Bartsch, H., Grover, P. L. & Sims, P. (1975) Biochem. Biophys. Res. Commun. 66,693-700. 11. Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., Yagi, H., Hernandez, O., Jerina, D. M. & Conney, A. H. (1976) Biochem. Biophys. Res. Commun. 68,1006-1012. 12. Levin, W., Wood, A. W., Yagi, H., Jerina, D. M. & Conney, A. H. (1976) Proc. Nati. Acad. Sci. USA 73,3867-3871. 13. Slaga, T. J., Viaje, A., Bracken, W., Berry, D. L., Fischer, S. M., Miller, D. R. & LeClerc, S. M. (1977) Cancer Lett. 3,23-30. 14. Kapitulnik, J., Levin, W., Conney, A. H., Yagi, H. & Jerina, D. M. (1977) Nature (London) 266,378-380. 15. Hsu, W.-T., Lin, E. J. S., Harvey, R. G. & Weiss, S. B. (1977) Proc. Natl. Acad. Sci. USA 74, 35-3339. 16. Nathans, D., Notani, G., Schwartz, J. & Zinder, N. (1962) Proc. Natl. Acad. Sci. USA 48,1424-1431. 17. Capecchi, M. (1966) J. Mol. Biol. 21,173-193. 18. Vinuela, E., Salas, M. & Ochoa, S. (1967) Proc. Natl. Acad. Sci. USA 57, 729-734. 19. Beland, F. A. & Harvey, R. G. (1976) J. Chem. Soc. Chem. Commun. 84-85. 20. Fu, P. P. & Harvey, R. G. (1977) Tetrahedron Lett. 415-418. 21. Harvey, R. G. & Fu, P. P. (1978) in Polycyclic Hydrocarbons and Cancer: Chemistry, Molecular Biology and Environment, eds. Gelboin, H. V. & Ts'o, P. 0. P. (Academic, New York), Vol. 1, pp.131-162. 22. Nirenberg, M. W. (1963) Methods Enzymol. 6,17-23. 23. Blatter, D. P., Garner, F., Van Slyke, K. & Bradley, A. (1972) J. Chromatogr. 645 147-155. 24. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 25. Capecchi, M. R. & Webster, R. E. (1975) in RNA Phages, ed. Zinder, N. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 279-299. 26. Lin, J.-W., Tsung, C. M. & Fraenkel-Conrat, H. (1967) J. Mol. Biol. 24, 1-14. 27. Lodish, H. F. (1968) Nature (London) 220,345-350. 28. Fiers, W., Contreras, R., Duerinck, F., Haegeman, G., Iserentant, D., Merregaert, J., Min-Jou, W., Molemans, F., Raeymaekers, A., Van den Berghe, A., Volckaert, G. & Ysebaert, M. (1976) Nature (London) 260,500-507. 29. Grunberger, D. & Weinstein, I. B. (1971) J. Biol. Chem. 246, 1123-1128. 30. Leffler, S., Pulkrabek, P., Grunberger, D. & Weinstein, I. B.
(1977) Biochemistry 16,3133-3136.
31. Spiegelman, S., Haruna, I., Holland, I. B., Beaudreau, G. & Mills, D. (1965) Proc. Natl. Acad. Sci. USA 54,919-927.
Biochemistry
Effect of benzo[a]pyrene-diolepoxide on infectivity and in vitro translation of phage MS2 RNA (polycyclic aromatic hydrocarbons/infectious phage RNA/RNA binding by benzo[alpyrene-diolepoxide/chemical carcinogenesis)
DAPHNA SAGHER*, RONALD G. HARVEY*, WEN-TAH HSUtt,
AND
SAMUEL B. WEISS*tt
tThe Franklin McLean Memorial Research Institute, the *Departments of Biochemistry and Microbiology, and *The Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, Illinois 60637
Communicated by Elwood V. Jensen, November 6,1978
ABSTRACT Previous studies have shown that alkylation of MS2 RNA by certain derivatives of polycyclic aromatic hydrocarbons renders it noninfectious. Since phage RNA serves as a template for translation and transcription, either of these RNA-directed processes, or both, could be responsible in vivo for the inhibition of phage replication by metabolically activated hydrocarbons. The present study correlates the degree of inhibition of MS2 RNA infectivity, at various levels of alkylation by (-I-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpyrene, with the translation efficiency in vitro of the same alkylated RNA for the synthesis of viral synthetase and of maturation and coat proteins. The results indicate that dihydroxyepoxy-tetrahydrobenzola pyrene modification of MS2 RNA impairs its template capacity for the synthesis of phagespecific proteins; this inhibition is insufficient, however, to account for the loss of RNA infectivity at lower molar ratios of alkylation. For the three viral proteins synthesized in vitro, the translation of RNA synthetase is much more sensitive to MS2 RNA modification than either coat or maturation protein synthesis. Our results also indicate that the loss of viral RNA infectivity follows a single-hit inactivation mechanism, whereas several alkylation events in the viral RNA synthetase cistron may be necessary to block translation of this gene product.
Carcinogenic polycyclic aromatic hydrocarbons (PAH) inhibit the replication of single-stranded RNA and DNA bacterial viruses (1-3). We have used this inhibitory activity to evaluate the relative potency of different PAH and their oxidized metabolites and derivatives (4). Among the compounds examined, (+)-trans - 7,8 - dihydroxy-anti -9,10-epoxy-7,8,9, 10- tetrahydrobenzo[a]pyrene (anti-BzPDE) was the most active inhibitor of phage replication (4). This compound is the principal metabolite of benzo[a ]pyrene (BzP) bound to DNA in mouse cells (5) and in bovine and human bronchial explants (6, 7). It is also mutagenic in animals (8, 9) and bacteria (10, 11) and carcinogenic in mice (12-14), and has been suggested to be the ultimate carcinogenic metabolite of BzP. Studies with R17 bacteriophage (a single-stranded RNAcontaining virus) (3) and with infectious 4X174 DNA (15) have indicated that an average of one molecule of bound hydrocarbon derivative per molecule of viral nucleic acid is sufficient to block phage replication. The work by Hsu et al. (15) also showed that conjugation of OX DNA with BzPDE impairs its function as a template for the synthesis of double-stranded DNA replicative intermediates, which is a necessary first step in the replication of OX phage. The replication of single-stranded RNA phage is different from that of single-stranded DNA phage in that the infectious RNA serves first as a template for the translation of viral proteins and then as a template for the transcription of new viral RNA molecules. The inhibition of RNA phage replication by activated PAH derivatives could occur at either of these RNA-directed synthetic processes.
Purified RNA from the related bacteriophages f2, R17, and MS2 functions as a template, in cell-free systems, for the directed synthesis of phage-specific RNA synthetase and of maturation and coat proteins (16-18). The present study shows that alkylation of MS2 RNA with anti-BzPDE inhibits its template function for the synthesis of RNA synthetase; this inhibition is significantly less pronounced, however, than the inhibition of MS2 RNA infectivity. METHODS MS2 RNA. MS2 RNA was isolated Infectious of Preparation from CsCI-purified MS2 phage by lysis in 1% sodium dodecyl sulfate (NaDodSO4) and repeated extraction with phenol and finally with phenol/chloroform 1:1 (vol/vol) containing 4% isoamyl alcohol. One-tenth volume of 20% potassium acetate (pH 5) was added, and the RNA was precipitated with ethanol. The precipitation process was repeated, and the RNA was finally dissolved in sterile water and stored in aliquots at -700C. Assay for Infectious MS2 RNA. MS2 RNA was assayed for infectivity by a described procedure (1). MS2 RNA was mixed with spheroplasts prepared from Escherichia coil K-12W1485 (male strain). After 5 min of incubation at 25°C, the infectious mixture was plated with intact E. coil K-12W1485 and incubated overnight at 37°C for plaque development. Under our assay conditions, 1 ,tg of freshly prepared MS2 RNA produced t1000 plaque-forming units. Treatment of RNA with BzP-Diolepoxide. Hydrocarbon was reacted with nucleic acid by the "pretreatment" method (4). RNA (100 ,ug/ml) and various amounts of anti-BzPDE were incubated at 25°C for 10-30 min in 50 mM Tris-HCl (pH 7.5). Stock solutions of anti-BzPDE were made in dimethylformamide; the concentration of this solvent in the reaction mixture did not exceed 6%. After incubation, the RNA was precipitated with ethanol at -20°C and successively with acetone and 95% ethanol. This washing procedure is effective in removing unreacted hydrocarbon (4). The RNA precipitate was suspended in water, and aliquots were taken for measurements of its A26o and infectivity content and for the biosynthetic reactions described in the text. anti-BzPDE, prepared as reported elsewhere (19-21), was of the highest attainable purity. When [3H]BzPDE was used in the above reaction, the extent of RNA alkylation was determined from radioactivity measurements of the dissolved precipitate, after washing. Protein Synthesis In Vitro. A 30,000 X g supernatant extract (S30) was prepared from E. coli Q13 (cells in middle logarithmic phase) by the method of Nirenberg (22), with the following minor modifications: 1 mM dithiothreitol replaced 2-mercapAbbreviations: PAH, polycyclic aromatic hydrocarbons; BzP, benzo[a pyrene; anti-BzPDE, (+)-trans-7,8-dihydroxy-anti-9,10epoxy-7,8,9,10-tetrahydro-BzP; NaDodSO4, sodium dodecyl sul-
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fate.
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Radioisotopes. [3H]Valine (14.6 Ci/mmol) was purchased
from New England Nuclear. anti-[3H]BzPDE (specific activity, 822 cpm/ng) was synthesized under National Cancer Institute Contract CP-33387 by Midwest Research Institute (Kansas City, MO) and stored in dimethylformamide at 4°C. This material had a chemical purity of more than 90% (Shen Yang, National Cancer Institute, Bethesda, MD, personal communication). EXPERIMENTAL RESULTS Fig. 1A illustrates the binding of labeled anti-BzPDE to MS2 RNA. As the concentration of [3H]BzPDE is increased in the reaction mixture, the amount of labeled hydrocarbon bound to viral RNA increases linearly; at the same time, the infectivity of the viral nucleic acid is inactivated (see the fi curve). Under the conditions used for binding, the infectious activity of MS2 RNA is reduced by about 70% with 0.05 ,g of hydrocarbon in the reaction mixture; this corresponds to a molar alkylation ratio of 1 mole of BzPDE bound per mole of viral RNA. Fig. 1B shows the fraction of viral RNA inactivated (fi) as a function of the molar ratio of BzPDE bound to MS2 RNA, which closely follows the theoretical Poisson distribution for inactivation of nucleic acid bound by one or more moles of BzP (f,1BzP)- These results suggest, therefore, that a single molecule of bound hydrocarbon is sufficient to inactivate a molecule of MS2 RNA. In the E. coli S30 cell-free system used for polypeptide synthesis, MS2 RNA serves as an effective template for the incorporation of [3H1valine into an acid-precipitable form. Fig. 2 shows that, when BzPDE-bound MS2 RNA is used as a template in this system, its template efficiency for polypeptide synthesis is reduced significantly; the more BzPDE is bound to MS2 RNA,
Proc. Natl. Acad. Sci. USA 76 (1979)
[3H]BzPDE
in binding reaction, jig
621
Molar ratio of BzP bound to MS2 RNA
FIG. 1. Binding of BzPDE to MS2 RNA and its effect on infectivity. (A) Reaction mixture (0.1 ml) contained 10 ,g of MS2 RNA and various amounts of [3HJBzPDE. After incubation, the [3H]BzP-RNA complex was isolated and its RNA content, radioactivity, and infectivity were determined. f;, Fraction of infectious MS2 RNA that lost infectivity after reaction with the diolepoxide. (B) Fraction of MS2 RNA inactivated (ft) plotted as a function of the molar ratio (MR) of the average number of BzP molecules bound per molecule of MS2 RNA, calculated from the radioactivity and the RNA content of the isolated [3H]BzP-RNA complex. f4inzp and f>2BzP were derived from the expressions 1 - Po and 1 - Po - P1, respectively, in which Po = e-MR and Pi = MR x e-MR.
the lower the incorporation of radioactive valine. However, at a molar binding ratio of 4, which suppresses MS2 RNA infectivity by about 99% (Fig. 1B), incorporation of labeled valine is inhibited only 20%. The kinetics of [3H]valine incorporation for unmodified and modified MS2 RNA at two molar ratios of bound hydrocarbon is illustrated in Fig. 3A. Maximum incorporation occurs after 30-40 min of incubation with both types of templates; however, the amount of labeled protein formed at each time point is significantly less with modified MS2 RNA. Fig. 3B shows that, during relatively short reaction times (10 min), the amount of label incorporated is directly related to the amount of MS2 RNA present in the reaction mixture for both unmodified and modified templates. In this system, saturation with template was not achieved at the highest concentrations examined. Fig. 4 is a radioautograph of the protein products synthesized in the S30 system with [3H]valine, under the direction of untreated and modified MS2 RNA, and subjected to gel electrophoresis. Four distinct bands of varied radioactive intensity appeared, each migrating at a different rate. Band A was most intense, moved fastest, and was estimated to have a molecular size of 13,000 daltons; band B was the least intense, with an estimated size of approximately 17,000 daltons; bands C and D migrated in the gel corresponding to sizes of 39,000 and 64,000 daltons, respectively. The molecular sizes for the radioactive bands corresponding to A, C, and D agree very closely with those reported for MS2 coat, maturation, and RNA synthetase proteins (25); the nature and origin of band B radioactivity are not known. When [3H]histidine replaced radioactive valine in the S30 system, no label was found in the band A region (data not shown); this information supports the molecular size data which indicate that band A represents viral coat protein, since this protein is deficient in histidine (26). In Fig. 4, sample 1 shows the distribution of radioactive polypeptides assembled with unmodified MS2 RNA, and samples 2-5 represent the labeled protein products made under the direction of BzPDE-modified MS2 RNA templates; sample 6 is a control lacking a template. As the extent of BzPDE alkylation in MS2
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Proc. Natl. Acad. Sc. USA 76 (1979)
I
IR
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4 A
3
4
5 6 D
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C
0
4-6a 0
0
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B
C 0
0 C
c
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IL
A
0I
Molar ratio of BzP bound to MS2 RNA
FIG. 2. Effect of alkylation of MS2 RNA on its template function for protein synthesis. Untreated and BzPDE-modified MS2 RNA samples were used as templates in the S30 translational system in vitro, derived from E. coli. After 30 min of incubation, 10-,ll aliquots were taken from each reaction mixture and the amount of [3H]valine incorporated into an acid-precipitable form was determined. In the above system, t70,000 total counts were incorporated into trichloroacetic acid-precipitable material when 2 gg of unmodified MS2 RNA served as template; in the absence of added RNA, incorporation was less than 3% of the complete system, and this endogenous background activity was subtracted from all experimental results.
RNA is increased (from sample 2 to sample 5), there is a slight, but detectable, decrease in the amount of radioactivity incorporated into coat and maturation proteins (bands A and C); incorporation of isotope into band D (RNA synthetase), however, is abolished almost completely. In order to quantitate the relative amounts of the three viral Io
A
20
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80
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M
0
40
20
30
Reaction time,
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MS2 RNA,
jig/ml
FIG. 3. Effect of BzP alkylation on the kinetics of MS2 RNAdirected protein synthesis. (A) Reaction system was as described under Methods, except that 1 jig of untreated or modified MS2 RNA (molar ratios of 4 and 10) served as template. At the time intervals
indicated, 10-jgl aliquots were removed and processed for trichloroacetic acid-precipitable radioactivity. (B) Reaction system was the same as for A, except that the amount of MS2 RNA (untreated and modified) was varied and the reaction was terminated after 10 min of incubation. MR, molar ratio.
FIG. 4. Gel electrophoresis of 3H-labeled proteins synthesized under the direction of MS2 RNA. After reaction, the incubation mixtures were heated in a boiling water bath for 5 min in the presence of 5% 2-mercaptoethanol/3% NaDodSO4 and then subjected to gel electrophoresis on 25% polyacrylamide containing 0.1% NaDodSO4. After electrophoresis, the gels were stained and dried, and radioactivity was detected. Molecular-weight markers: bovine serum albumin (BSA), Mr 68,000; soybean trypsin inhibitor (STI), Mr 23,500; and cytochrome c (Cyt c), Mr 12,400. MS2 RNAs used as templates were: 1, untreated; 2, 3, 4, and 5, treated with BzPDE containing molar ratio of BzP:MS2 RNA of 1, 2, 4, and 10, respectively; 6, no RNA control.
proteins made in vitro, we sliced the gels and determined their radioactivity. Fig. 5 shows the radioactivity profile of the labeled proteins assembled in the S30 system under the direction of unmodified and modified MS2 RNA. In Fig. 5A, the ratio of coat:synthetase:maturation protein synthesized was determined to be 1:0.27:0.08, which is comparable to the ratio of 1:0.3:0.06 for f2 phage RNA-directed protein products made in a similar in vitro system (27). With alkylated MS2 RNA as template (molar ratio of 10), RNA synthetase formation was almost completely absent (Fig. 5B), even though synthesis of maturation and coat proteins was affected only slightly. We routinely found distortions in the radioactive profile of the coat protein peak when BzPDE-MS2 RNA served as template (Fig. 5B), suggesting the formation of incomplete phage protein products. In Table 1, the infectivity of MS2 RNA is compared with its capacity in vitro to direct the synthesis of phage proteins as the amount of BzP binding to viral RNA is increased. With increasing molar ratios of bound hydrocarbon, both the infectivity of MS2 RNA and the translation of viral RNA synthetase were inhibited; however, RNA infectivity was considerably more sensitive to alkylation than is the RNA-directed synthesis of viral proteins. At a molar ratio of bound BzPDE to MS2 RNA of 1, infectivity was reduced by about 73%, whereas RNA synthetase formation was reduced by only about 30%. At a molar ratio of 4, the infectious activity of MS2 RNA was abolished almost completely; nevertheless, the synthesis of RNA synthetase under the direction of this alkylated template was still substantial (36%). Appreciable inhibition of maturation and coat protein synthesis was observed only at molar ratios greater than 4.
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Proc. Natl. Acad. Sci. USA 76 (1979)
623
c
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--
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,
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FIG. 6. Relationship between RNA synthetase formed in vitro and the extent of BzP binding to the MS2 RNA synthetase cistron. The values for the inhibition of RNA synthetase formation (fA) were obtained from Table 1. The molar ratios of BzP bound to the synthetase cistron were estimated by multiplication of the molar ratios shown in Table 1 by the fraction of the total MS2 genome occupied by the synthetase cistron (0.46). f>lzp and f>2BzP were derived as described in the legend of Fig. 1.
X
C~~~~~~e slc (N
0
2
4
6
8 10 12 14 16 18 20 22 Gel slice
FIG. 5. Profiles of MS2 RNA-directed synthesized [3Hlproteins subjected to gel electrophoresis. Protein synthesis and gel electrophoresis of translation products in vitro were as described in the legend of Fig. 4. After electrophoresis, gels were sliced and the radioactivity was determined. (A) Unmodified MS2 RNA and (B) modified MS2 RNA (molar ratio of BzP:RNA = 10) served as templates in the protein synthesis reaction in vitro. Counts shown in A and B have been adjusted so that they are equivalent in terms of total radioactivity recovered from the gels. S, M, and C represent the peak positions for viral synthetase, maturation, and coat proteins.
Bacteriophage MS2 RNA is 3569 nucleotides long; the RNA synthetase cistron accounts for 1635 nucleotides or 46% of the total viral genome (28). If one assumes that the alkylation of MS2 RNA is random, there is a 46% chance for the binding of a single molecule of BzP to the synthetase cistron when the molar ratio of bound BzPDE to MS2 RNA is one. Fig. 6 illustrates the fraction of RNA synthetase inactivated (fi) as a Table 1. Relationship between alkylation of MS2 RNA and its effect on infectivity and phage protein synthesis % inhibition of infectivity and of phage mol BzPDE proteins synthesized RNA Maturation Coat bound/mol protein MS2 RNA Infectivity synthetase protein 0 0 0 None 0 1 2 4 10
73 86 99 100
30 40 64 92
1 0 0 21
4 2 3 Molar ratio of BzP bound to synthetase cistron
0 2 13 40
MS2 RNA was treated with BzPDE to provide various degrees of BzP alkylation. Samples of the untreated and modified viral RNAs were assayed for infectivity and for their template capacity in the in vitro translation system. Translation products were subjected to gel electrophoresis, and the distribution of radioactivity in synthetase, maturation, and coat proteins was determined as described in the legend for Fig. 5. The percent inhibition for each viral protein made with modified MS2 RNA templates was calculated from the decrease in radioactivity recovered in each protein region compared to untreated MS2 RNA translation products.
function of the molar ratio of BzPDE bound to the synthetase cistron; the values for fi and the molar ratios were taken from Table 1, except that the molar ratios were adjusted by a factor of 0.46. The experimental points for fi, at relatively low molar ratios, fall between the two theoretical Poisson curves f>1BzP and f¢2Bzp; thus, one or several alkylation events in the synthetase cistron may be required to block translation of the synthetase gene. DISCUSSION Certain PAH metabolites are inhibitory for bacteriophage replication. A single alkylation event within the genome of single-stranded phages is sufficient to suppress infectivity completely (3, 15). The experiments described here are consistent with these findings. We have attempted to define more precisely the mechanism of infectious RNA inactivation since single-stranded viral RNAs participate both in translation and transcription during virulent phage production. Grunberger and Weinstein (29) had shown that the binding of N-2-acetylaminofluorene residues to poly(U3G) blocks translation of this RNA copolymer. The present study shows, in addition, that the alkylation of MS2 RNA by anti-BzPDE inhibits its capacity to direct polypeptide synthesis; however, the inhibition of the messenger function of viral RNA is much less sensitive to PAH modification than its infectivity. At an average binding molar ratio of 1, the infectivity of the alkylated viral RNA is suppressed by 70%, whereas the synthesis of phage-specific polymerase (RNA synthetase) is reduced by only 30%. Thus, the reduced translational capacity of BzPDEmodified MS2 RNA, at relatively low molar ratios of bound hydrocarbon, is insufficient to account for the loss of infectious RNA activity, suggesting that alkylation blocks some other RNA-directed step; i.e., RNA synthesis. Leffler et al. (30) reported that the modification of calf thymus DNA with BzPDE inhibits its transcription with E. coli DNA-dependent RNA polymerase. Hsu et al. (15) showed that BzPDE groups on OX174 DNA block its conversion to a duplex replicative form by E. coli DNA polymerase. The studies by both groups indicate that nucleotide polymerization by RNA and DNA polymerases in vitro is impaired by alkylated sites on DNA templates; thus,
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chain elongation stops and incomplete complementary fragments are formed. It is not unreasonable to assume that phage-specific RNA polymerase is also blocked during transcription at BzP-conjugated sites in viral RNA and that the inability to form intact RNA-RNA replicative intermediates is directly responsible for the loss of infectivity observed. Unpublished results in our laboratory have indicated that similar alkylation of Qf3 RNA prevents the synthesis de novo of infectious Qf3 RNA in vitro by Qf3 RNA polymerase (31). The synthesis in vitro of RNA synthetase was affected much more adversely by the modification of MS2 RNA than the synthesis of either maturation or coat protein. Since the synthetase cistron has a considerably greater nucleotide length than the other two genes, one would expect a higher frequency of BzPDE binding in the synthetase region of the phage genome on the basis of random alkylation. It is not completely clear whether one or several alkylation sites within the synthetase cistron are required to block translation of this phage protein. At low molar ratios of bound hydrocarbon in the synthetase cistron, the reduction in synthetase formation follows a single-hit inactivation mechanism, but at higher molar ratios, a significant deviation from the theoretical f,1Bzp curve was observed (Fig. 6). It is possible that these deviations reflect some inaccuracy in our determination of viral synthetase formed and in the assumption that alkylation is random throughout the viral RNA chain; each of these factors would affect the estimated molar ratios of BzP bound to the synthetase cistron. It is also possible that some miscoding occurred with the BzPDE-modified viral template during translation, resulting in the formation of complete, but abnormal, polypeptides with electrophoretic properties similar to those of phage synthetase. In the translation experiments of Grunberger and Weinstein (29), however, inactivation, but not miscoding, was found with acetylaminofluorene-modified synthetic templates. The inactivation of single-stranded infectious RNA by PAH binding can be summarized as follows: At low molar ratios of PAH binding (4 or less), the translational synthesis of phage proteins decreases somewhat; this decrease is insufficient, however, to account for the loss of RNA infectivity, assuming that the polypeptides identified by our assay procedure represent functional phage proteins. Most probably, the loss of infectivity can be attributed to the inability of phage polymerase to transcribe PAH-modified viral RNA. At higher molar ratios, translation of phage polymerase is minimal so that, if the modified viral RNA could be transcribed, the amount of phage enzyme synthesized would be insufficient to catalyze RNA replication. The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the United States Department of Energy under Contract EY-76-C-02-0069. This study was partly supported by a grant from the American Cancer Society (BC-132A) and by funds from Environmental Protection Agency Subagreement 77BAN. D.S. is supported by Training Grant CA-09183 from the National Cancer Institute. 1. Hsu, W.-T., Moohr, J. W. & Weiss, S. B. (1965) Proc. Natl. Acad. Sci. USA 53,517-524.
Proc. Natl. Acad. Sci. USA 76 (1979) 2. Hsu, W.-T., Moohr, J. W., Tsai, A. Y. M. & Weiss, S. B. (1966) Proc. Natl. Acad. Sci. USA 55, 1475-1482. 3. Dipple, A. & Shooter, K. V. (1974) Biochim. Biophys. Acta 374, 392-399. 4. Hsu, W.-T., Harvey, R. G., Lin, E. J. S. & Weiss, S. B. (1977) Proc. Natl. Acad. Sci. USA 74, 1378-1382. 5. King, H. W. S., Osborne, M. R., Beland, F. A., Harvey, R. G. & Brookes, P. (1976) Proc. Natl. Acad. Sci. USA 73,2679-2681. 6. Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H. & Nakanishi, K. (1976) Science 193, 592-595. 7. Jeffrey, A. M., Weinstein, I. B., Jennette, K. W., Grzeskowiak, K., Nakanishi, K., Harvey, R. G., Autrup, H. & Harris, C. (1977) Nature (London) 269,348-350. 8. Huberman, E., Sachs, L., Yang, S. K. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73, 607-611. 9. Newbold, R. F. & Brooks, P. (1976) Nature (London) 261,52-
54. 10. Malaveille, C., Bartsch, H., Grover, P. L. & Sims, P. (1975) Biochem. Biophys. Res. Commun. 66,693-700. 11. Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., Yagi, H., Hernandez, O., Jerina, D. M. & Conney, A. H. (1976) Biochem. Biophys. Res. Commun. 68,1006-1012. 12. Levin, W., Wood, A. W., Yagi, H., Jerina, D. M. & Conney, A. H. (1976) Proc. Nati. Acad. Sci. USA 73,3867-3871. 13. Slaga, T. J., Viaje, A., Bracken, W., Berry, D. L., Fischer, S. M., Miller, D. R. & LeClerc, S. M. (1977) Cancer Lett. 3,23-30. 14. Kapitulnik, J., Levin, W., Conney, A. H., Yagi, H. & Jerina, D. M. (1977) Nature (London) 266,378-380. 15. Hsu, W.-T., Lin, E. J. S., Harvey, R. G. & Weiss, S. B. (1977) Proc. Natl. Acad. Sci. USA 74, 35-3339. 16. Nathans, D., Notani, G., Schwartz, J. & Zinder, N. (1962) Proc. Natl. Acad. Sci. USA 48,1424-1431. 17. Capecchi, M. (1966) J. Mol. Biol. 21,173-193. 18. Vinuela, E., Salas, M. & Ochoa, S. (1967) Proc. Natl. Acad. Sci. USA 57, 729-734. 19. Beland, F. A. & Harvey, R. G. (1976) J. Chem. Soc. Chem. Commun. 84-85. 20. Fu, P. P. & Harvey, R. G. (1977) Tetrahedron Lett. 415-418. 21. Harvey, R. G. & Fu, P. P. (1978) in Polycyclic Hydrocarbons and Cancer: Chemistry, Molecular Biology and Environment, eds. Gelboin, H. V. & Ts'o, P. 0. P. (Academic, New York), Vol. 1, pp.131-162. 22. Nirenberg, M. W. (1963) Methods Enzymol. 6,17-23. 23. Blatter, D. P., Garner, F., Van Slyke, K. & Bradley, A. (1972) J. Chromatogr. 645 147-155. 24. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 25. Capecchi, M. R. & Webster, R. E. (1975) in RNA Phages, ed. Zinder, N. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 279-299. 26. Lin, J.-W., Tsung, C. M. & Fraenkel-Conrat, H. (1967) J. Mol. Biol. 24, 1-14. 27. Lodish, H. F. (1968) Nature (London) 220,345-350. 28. Fiers, W., Contreras, R., Duerinck, F., Haegeman, G., Iserentant, D., Merregaert, J., Min-Jou, W., Molemans, F., Raeymaekers, A., Van den Berghe, A., Volckaert, G. & Ysebaert, M. (1976) Nature (London) 260,500-507. 29. Grunberger, D. & Weinstein, I. B. (1971) J. Biol. Chem. 246, 1123-1128. 30. Leffler, S., Pulkrabek, P., Grunberger, D. & Weinstein, I. B.
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31. Spiegelman, S., Haruna, I., Holland, I. B., Beaudreau, G. & Mills, D. (1965) Proc. Natl. Acad. Sci. USA 54,919-927.
