Isolation and Properties of Poliovirus Minus

JOURNAL OF VIROLOGY, Nov. 197, p. 64-69 Copyright ( 197 American Society for Microbiology Vol. 6, No. 5 Prinzted in U.S.A. Isolation and Properties of Poliovirus Minus Strand Ribonucleic Acid POLLY ROY AND D. H. L. BISHOP Inistitutte of Canlcer Research, Columbia Unziversity, New York, New York 132 Received for publication 23 June 197 Poliovirus minus strands were purified from double-stranded polio ribonucleic acid. The minus strands have a base ratio complementary to that of the viral ribonucleic acid and are not infectious. The complementary (minus) strand to the Q3 bacteriophage genome (18) is not only an excellent template for the Qf replicase, but also is an intermediate in the in vitro replication of Q3 ribonucleic acid (RNA; 11, 21, 22). Without host factors (12, 21), Q3 replicase cannot be templateduplicated by the viral RNA, although it will use minus strands very efficiently (1). This suggests that depletion of host factors in an infected cell could be a regulatory mechanism allowing only production of plus strands late in infection. Free minus strands are produced in vitro before free plus strands, although later predominantly plus strands are synthesized (21, 22). It is not known whether free minus strands can template-duplicate the enzymes responsible for replicating the genomes of RNA viruses of higher organisms. With this in mind, we describe here the isolation and characterization of poliovirus minus strands. MATERIALS AND METHODS The methods employed to obtain poliovirus minus strands were similar to those described by Pollet et al. (18) for the isolation of bacteriophage Qfl minus strand RNA. Modifications of their procedure were developed to accommodate problems specific to handling poliovirus RNA. The basic procedure involved melting double-stranded poliovirus RNA and annealing the separated strands to an excess of poliovirus RNA fragments. The hybrid so formed was isolated by agarose chromatography and the denaturation-annealing process was repeated. After a final denaturation, full-length minus strands were separated from plus strand fragments by centrifugation. The minus strands so obtained were contaminated, as judged by annealing studies, with less than 5%', plus strand RNA, and, from infectivity assays, with less than.1% full-length plus strands. Preparation of poliovirus and poliovirus RNA fragments. Poliovirus was obtained from infected HeLa S3 cells by employing the observations of Ehrenfeld et al. (1) that detergent lysis of HeLa cells solubilizes the cellular membranes, liberating the intracellular 64 virus but leaving the nucleic intact. Log-phase cells were infected with wild-type (ts+) Sabin poliovirus at a multiplicity of infection of 1 to 3 plaque-forming units (PFU) per cell. The cells were grown under standard conditions (5, 14) and were harvested after 8 hr. Infected cells, 18 cells per ml of TSM buffer [.1 M tris(hydroxymethyl)aminomethane (Tris), ph 7,.5 M NaCl,.5 M MgCI2], were lysed by freeze-thawing thrice in detergents (1; 1% NP-4,.5sc deoxycholate), and nuclei were removed by centrifugation at 8 X g for 5 min. The lysate was adjusted to contain.5%c, sodium dodecyl sulfate (SDS; 15) and was centrifuged at 4, rev/min for 2.5 hr at 1 C in a Spinco SW41 rotor over a bottom 1-ml pad of CsCl (p 1.4) to collect the virus and a 1.5-ml pad of 47C%/G sucrose to retain membrane residues. The virus band was removed, the CsCl concentration was adjusted to give a density of 1.34 g/ml, and the preparation was centrifuged for 36 hr at 4, rev/min and 5 C in a Spinco SW41 rotor. The virus band from this equilibrium gradient centrifugation was collected and usually yielded virus having a 2:1 particle to PFU ratio. RNA was extracted from virus by procedures described previously (6) and was dissolved in.5 M ethylenediaminetetraacetate (EDTA), ph 7, at a concentration of 1 mg/ml. Viral RNA fragments were obtained by adding 2pliters of 1 M NaOH per ml ofrna, incubating at room temperature for 5 min, and neutralizing with I M HCI. The fragments so obtained were centrifuged in a glycerol gradient (1 to 3% glycerol,.1 M LiCl,.1 M Tris buffer,.5 M EDTA, ph 7) for 6 hr at 4, rev/min and 5 C, by use of a Spinco SW41 rotor (see Fig. 1). The low molecular weight viral RNA fragments (average, 12S) were collected by alcohol precipitation and dissolved in.5 M EDTA, ph 7, to give a concentration of 1 mg/ml. Preparation of labeled double-stranded (DS) and multistranded (MS) poliovirus RNA. Poliovirus-infected HeLa cells were labeled from 5 min postinfection with 3H-uridine (1 to 5,uCi/ml) or 32P-orthophosphate (5 uci,ml) and were grown for 6 hr; the cells were then lysed by detergents as described above. After removal of the nuclei, the total RNA was extracted, and the bulk of the single-stranded RNA was precipitated by I NI NaCl (3, 4, 8). Both the DS- and

VOL. 6, 197 POLIO MINUS STRAND RNA 65 I5. 3 OD,2 5 VOID 5 1 A 2-2 (L 3I 5 1 15 2 25 FRACTION FIG. 2. Separation of RNA from infected cells on 2% agarose. A column of 2% Agarose (approximately 2 liters in volume, 14 cm in height) was prepared as described in Materials and Methods. Infected-cell RNA (1 mg) in 4 ml of column buffer was loaded and 7.5-ml fractions were collected. 1 2 3 F RACTION FIG. 1. Poliovirus fragments. Fragments of poliovirus RNA were prepared as described in Materials and Methods. The fragmenits were centrifuged for 6 hr on a 1 to 3% glycerol gradienit with 3H-labeled 28S HeLa ribosomal RNA. The indicated fractions were pooled and used for annealing studies. z A 4-' a~, I - B MS-RNA as well as transfer RNA (trna) were alcohol-precipitated from the supernatant and loaded on a 2% agarose column (3, 17). Agarose columns were run in.1 M NaCl,.1 M Tris buffer,.5 M EDTA, and.1% SDS, ph 7; several days prior to use, they were washed in the same buffer saturated with diethylpyrocarbonate (DEPC) to remove all traces of nuclease (2). The DEPC was washed out with fresh buffer in view of the observation by Oberg (16) that DEPC destroys the infectivity of poliovirus RNA. Agarose chromatography of the total infected-cell RNA is shown in Fig. 2. The predominantly ribonuclease-resistant DS-RNA and MS-RNA were recovered in the void volume (Fig. 2) and were subsequently further purified by glycerol gradient centrifugation (see Fig. 4). No deoxyribonucleic acid was detectable in the postglycerol RNA preparations. Denaturation of DS and MS poliovirus RNA. No attempt was made to separate the DS- from the MS- RNA in the 18S postglycerol fractions. The ribonuclease resistance of these RNA preparations was usually between 8 and 9%. Several conditions for denaturation of the RNA were examined. Incomplete melting was the greatest difficulty encountered. If labeled RNA was melted at 1 or 1 ug per ml of.1 M EDTA, ph 7, by heating at 1 C for 9 sec, then, no matter what concentration it was diluted to, it reannealed to recover 7 to 8% of the label in a ribonuclease-resistant form. This concentration-independent reannealing indicates that the strands during this (melting) procedure were not completely separated and consequently reassociated with each other when incubated at 7 C in.4 M NaCl. This occurred even with a 1-fold excess of unlabeled fragments in the annealing cocktail (Fig. 3, line A). Heating at 1 C for longer periods gave similar results. Heating at 1 5 1 5 IC 5 1 RNA /ig / ml FIG. 3. Anntealing poliovirus double-strantded RNA after melting at different concentrations. Poliovirus double-stranded RNA was heated in.1 Mi EDTA at 1 C for 9 sec at 1,ug/ml (curve A), diluted to the anniealing concentration indicated, and incubated at 7 C in.4 M NaCl for 15 min prior to determination of the ribonuclease resistance. Alterniatively (curve B), thle RNA was heated at 1 C at the annzealinzg concentration, cooled, brought to 7 C and.4 m NaCl, and annealed for 15 min before determintationt of the ribonuclease resistance. these concentrations under pressure at above 1 C gave good melting, as judged by low ribonuclease resistance, but resulted in a recovery of only 1% of the RNA as full-length single strands (compare Fig. 4B). However, melting at RNA concentrations below 1,ug/ ml resulted in strand separation, as judged by the observation that reannealing was concentration-dependent (Fig. 3, line B). This concentration-dependent reannealing indicates that the strands during the melting procedure were completely separated. The reason that melting is better at 1,ug/ml than 1,g/ml could be that dilution of some contaminating divalent cation occurs. Alternatively, it could be an expression of some primary sequences reannealing at a fasterthan-normal rate, even at 1 C. As annealing is a concentration-, temperature-, and time-dependent process, dilution of the RNA for melting therefore favors strand separation. The procedure adopted was, therefore, as follows. RNA was added to.1 M EDTA, ph 7, previously equilibrated at 1 C in a boiling-water bath, to give a

66 ROY AND BISHOP J. VIROL. concentration of 1,ug/ml or less. After 9 sec, the solution was rapidly cooled in iced water. The ribonuclease resistance was then determined by incubating a sample for 2 min at 37 C in.4 M NaCl with (per ml) 1,ug each of ribonuclease A and T1. The resistance of melted RNA was 5%. Single-stranded poliovirus RNA has a ribonuclease-resistant core of 4% under these conditions. Glycerol gradient separation of melted and unmelted native DS-RNA is shown in Fig. 4. Melting RNA in dimethyl sulfoxide (13) gave essentially the same results and a similar recovery of full-length RNA from the DS- and MS-RNA preparations. Annealing with poliovirus RNA fragments. To melted DS-RNA (at 1. to.1 pg/ml), poliovirus 15 A RNA fragments were added to give a concentration of 5,ug/ml; the mixture was adjusted to 7 C and was made.4 M NaCl. After 15 min, the solution was cooled and the RNA was collected by alcohol precipitation. The time course of annealing with or without fragments is shown in Fig. 5. Usually 4 to 5% of the label became ribonuclease resistant as compared to an increased resistance of 4% without fragments. Isolation of the annealed RNA. The RNA was chromatographed through 4% agarose, and the annealed RNA was recovered from the void volume. Under thse conditions, single-stranded poliovirus RNA and poliovirus fragments are recovered in the included volume of the gel (see Fig. 6). The annealed RNA so obtained was 96% ribonuclease-resistant. Subsequent minus strand purification. The hybrid of 1 5 1 2 FRACTION 3J N Ox a. : LY z cr 5 / WITH FRAGMENTS WITHOUT FRAGMENTS --- ----- * -3-- 2 _ 1 2 3 T M E (MIN) FIG. 5. Annealinig melted poliovirus double-stranded RNA to poliovirus fragments. Poliovirus 3H-labeled double-stranded RNA was melted at a concentration of.1 ug per ml and annealed with or without 5 ug of viral 3 fragments per ml for the times indicated at 7 C and.4.a NaCI as described in Materials and Methods. The ribonuclease resistance was determined as described in Materials and Methods. 1* 2 POOL I in 5 1 2 3 FRACT ION FIG. 4. Gradient centrifuigation of native (A) and melted (B) poliovirus double-stranded RNA. (A) 32p_ labeled poliovirus double-stranded RNA and 3H-labeled poliovirus RNA were prepared and centrifuged on a glycerol gradient, as described in Materials and Methods. (B) Double-stranded 32P-labeled poliovirus RNA was heated together with 3H-labeled poliovirus RNA in.1 M EDTA at I Mg per ml and 1 C for 9 sec and was similarly centrifuiged. Ceentrifugation throughl 1 to 3% glycerol in.1 M NaCl,.1 m Tris,.5 vr EDTA was for 6 hr at 5 C in a Spinco SW41 rotor. a. Io, 6 a.3 N: 1 5L : 5 3 6 FRAC T I Or FIG. 6. Agarose (4%1) chromatography of poliohybrid RNA. The synthetic hybrid of 32P-labeled minus strands and unlabeled polio fragments (see Materials and Methods) was chromatographed with 3H-labeled viral RNA on a 4% agarose column (9 by.9 cm). The indicated fractions were pooled for subsequent minuls strand purification. *e

VOL. 6, 197 POLIO MINUS STRAND RNA 67 labeled minus strands and unlabeled viral fragments was melted and reannealed again to a 2-fold excess of viral fragments as described above. This second hybrid, after agarose chromatography, was denatured and centrifuged on a glycerol gradient (Fig. 7); the indicated fractions were pooled and used to determine the properties of poliovirus minus strands. Base-ratio determination, infectivity assays. The base ratios of 32P-labeled RNA were determined as described previously (7). Infectivity of single-stranded RNA and DS- and MS-RNA, with the use of DEAEdextran, was determined by the agar suspension technique described by Bishop and Koch (8). Stocks and materials. HeLa S3 and the Sabin poliovirus strains were kindly given to us by D. Summers and E. Ehrenfeld. Agarose was obtained from Bio- Rad Laboratories, Richmond, Calif., and DEPC, from Naftone Inc., New York, N.Y. RESULTS Purity of the poliovirus minus strands. Four methods were used to determine the purity of poliovirus minus strands prepared as detailed in Materials and Methods. (i) Contamination of minus strands by original plus strand RNA. It has been shown in Materials and Methods (Fig. 5) that added plus strand fragments anneal to minus strands in the annealing cocktail. To demonstrate the loss of original plus strand RNA from the DS-RNA preparation, 32P-labeled viral plus strands were added to 3Hlabeled DS-RNA, and minus strands were pre- 4 3 I,~ I2 In 1 FRACTION 2 Io x 4 E FIG. 7. Glycerol gradient centrifugation of melted 32P-labeled poliovirus hybrid RNA. Poliovirus hybrid RNA (containing 32P-labeled polio minus st?anzds and unlabeled poliovirus fragments) was melted and centrifuged with 3H-labeled 28S HeLa ribosomal RNA on a 1 to 3%X glycerol gradient for 6 hr. The indicated 35S fractions were pooled for further analysis. CY) pared as described in Materials and Methods. The purified minus strands contained less than.8% of the original 32P-labeled viral plus strands-equivalent to 1% plus strand RNA in the final minus strand preparation (Table 1). (ii) Contamination of minus strands with plus strand RNA. The extent of fragmented and complete plus strand contamination of the minus strand preparation was determined by selfannealing at a concentration of 5,ug of RNA per ml (see Materials and Methods). The ribonuclease resistance before and after annealing was 4 and 5%, respectively. Furthermore, annealing with added, unlabeled, plus strand fragments gave 95% resistance of the label. Consequently, there was less than 5% plus strand contamination of the minus strand preparation. (iii) Infectivity of the minus strand preparation. The infectivity of purified viral plus strand RNA, the initial DS-RNA (containing DS- and MS- RNA), and the minus strand RNA preparation was determined by use of DEAE-dextran as described by Bishop and Koch (8). The results, recorded in Table 2, indicated that minus strands are not infectious and that there was less than.1% contamination of the preparation with infectious plus strands. The infectivity of the DS-RNA, which is slightly greater than would be expected from its plus strand content, probably reflects the better survival of DS-RNA during the plating procedure (8). (iv) Base ratios of purified plus and minus strands as well as DS-RNA. The base ratios of 32p_ labeled plus and minus strand RNA, as well as that of the DS 18S RNA, are given in Table 3. The minus strands were essentially complementary in base composition to the plus strand preparation, and the base ratio of the DS-RNA is as expected (2, 8, 9, 19, 23). DISCUSSION Two forms of experiment are possible with purified minus strands. Although minus strands are not infectious, it is possible, if the minus strand genome was replicated by the replicase coded for in the plus strand genome, that in the presence of plus strands the minus strand genome could be expressed. DS poliovirus RNA is infectious (8); consequently, it should be possible to hybridize plus and minus strands of different genotypes, purify the DS-RNA, and test its infectivity with regard to the expression of the two genotypes. Advantage can also be taken of the observation that DEPC destroys the infectivity of single-stranded RNA but not DS-RNA (16). The second form of experiment in which minus

68 ROY AND BISHOP J. VIROL. TABLE 1. Loss of original plus stranids from original double-stranded poliovirus RNAa Double-stranded Single-stranded 3H-RNA 32P-RNA Calculated Calculated labeled Prepn minus strand (9H and 32p) content plus strand content Counts/min Amt Counts/min Amt Ag lg pg pg Original 2 X 15 1 6 X 14 1 45 65 First Agarose 4 X 14 2 5 X 13.8 15 5 Second Agarose... 2 X 14 1 6.1 1.7 Postglycerol gradient. 1. 2 X 14 6 <4 <.8 6 <.4 a 3H-labeled double-stranded RNA was mixed with 32P-labeled single-stranded 35S poliovirus RNA and minus strands were isolated as described in Materials and Methods. For the first and second annealing, 5 and 4,Ag of viral fragments were used, respectively. The minus strand content of doublestranded RNA (column 5) was calculated from the label rendered ribonuclease-resistant after annealing a small portion of melted double-stranded RNA to a 1-fold excess of unlabeled fragments. This figure is only a rough estimate. It was assumed that the unannealable portion of the label was poliovirus plus strand RNA (column 6). TABLE 2. Intfectivity ofdouble-strantded and purified poliovirus single-stranded minus and viral (plus) RNAa Prepn PFU per pg of RNA Native double-stranded RNA 18, Poliovirus minus strand RNA... Poliovirus RNA... 24, a RNA samples (1.,.1, and.1,ug) in DEAEdextran were incubated with HeLa cells according to the procedure described by Bishop and Koch (8). RNA samples were prepared as described in Materials and Methods. TABLE 3. Base ratios of double-stranded (DS) anzd purified poliovirus single-stranided minius anid viral (plus) RNAa Component Component Poliovirus Poliovirus Poliovirus DS RNA minus RNA RNA Cytosine... 23.2 23.3 24.1 Adenine... 26.8 23.2 3.3 Guanine... 23.1 23.3 22.4 Uridine... 26.9 3. 23.3 a 32P-labeled RNA samples were prepared and purified as described in Materials and Methods. Base ratios (mole %7) after alkali hydrolysis were obtained as described by Bishop, Mills, and Spiegelman (7). strands can be used involves the purification of polio replicase. A polio replicase preparation which, without template addition, synthesizes single-stranded 35S and DS 18S poliovirus RNA has been recently isolated from infected HeLa cells by Ehrenfeld et al. (1). This enzyme, in our hands, apparently synthesizes only plus strand RNA, even in the 18S DS product. This suggests, therefore, that the enzyme has bound to it minus strand templates. If so, then it is logical to look for dissociated enzyme with minus strands as templates, especially in view of the observation that Q3 replicase purified from host factors can be template-duplicated by minus, but not by plus, strands (1). ACKNOWLEDGMENTS We appreciate the advice and encouragement of Sol Spiegelman and the expert technical assistance of Ulrike Stadler. This investigation was supported by Public Health Service research grant CA-2332 from the National Cancer Institute. LITERATURE CITED 1. August, J. T., A. K. Banerjee, L. Eoyang, M. T. Franze de Fernandez, K. Hori, C. H. Kuo, M. Rensing, and L. Shapiro. 1968. Synthesis of bacteriophage Q6 RNA. Cold Spring Harbor Symp. Quant. Biol. 33:73-81. 2. Baltimore, D. 1966. Purification and properties of poliovirus double-stranded RNA. J. Mol. Biol. 18:421-428. 3. Baltimore, D. 1968. Structure of the poliovirus replicative intermediate RNA. J. Mol. Biol. 32:359-368. 4. Baltimore, D., and M. Girard. 1966. An intermediate in the synthesis of poliovirus RNA. Proc. Nat. Acad. Sci. U.S.A. 56:741-748. 5. Baltimore, D., M. Girard, and J. E. Darnell. 1966. Aspects of the synthesis of poliovirus RNA and the formation of virus particles. Virology 29:179-189. 6. Bishop, D. H. L., J. R. Claybrook, and S. Spiegelman. 1967. Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26:373-387. 7. Bishop, D. H. L., D. R. Mills, and S. Spiegelman. 1968. The sequence of the 5'-terminus of a self-replicating variant of Q3-RNA. Biochemistry 7:3744-3753. 8. Bishop, J. M., and G. Koch. 1967. Purification and characterization of poliovirus-induced infectious double stranded ribonucleic acid. J. Biol. Chem. 242:1736-1743. 9. Bishop, J. M., D. F. Summers, and L. Levintow. 1965. Characterization of ribonuclease-resistant RNA from poliovirus-infected HeLa cells. Proc. Nat. Acad. Sci. U.S.A. 54:1273-1281. 1. Ehrenfeld, E., J. V. Maizel, and D. F. Summers. 197. Soluble RNA polymerase complex from poliovirus-infected HeLa cells. Virology 4:84-846. II. Feix, G., R. Pollet, and C. Weiss-nann. 1968. Replication of

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