Low-Molecular-Weight Rauscher Leukemia Virus Protein with Preferential Binding for Single-Stranded RNA and DNA

Transcription

1 JOURNAL OF VIROLOGY, May 1976, p Copyright 1976 American Society for Microbiology Vol. 18, No. 2 Printed in U.S.A. Low-Molecular-Weight Rauscher Leukemia Virus Protein with Preferential Binding for Single-Stranded RNA and DNA JAMES DAVIS,' MICHAEL SCHERER, WEN PO TSAI, AND CEDRIC LONG* Flow Laboratories, Inc., Rockville, Maryland Received for publication 28 November 1975 A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNAbinding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo(dt)-cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or ph. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA. After the initial discovery of a binding protein specific for single-stranded DNA from T4 bacteriophage (1), proteins with similar properties have been isolated from a number of organisms employing DNA cellulose affinity chromatography (2, 13, 18, 24, 26). Many of these have been shown to bind cooperatively to single-stranded regions of native DNA and bring about unwinding. More recently, proteins coded for by animal tumor viruses that bind to DNA have been isolated from infected or transformed cells. Human KB and African green monkey cells infected with human adenovirus types 2 and 5 contain two new polypeptides that have a preferential affinity for single-stranded DNA cellulose (25, 31). One of the proteins has been shown to be the adenovirus tumor antigen (11). Simian virus 40 T antigen, which is believed to regulate viral DNA replication, can be recovered from the nuclei of transformed cells and has now been shown to bind preferentially to double-stranded DNA (5). Further evidence for a role of the simian virus 40 T antigen in regulating viral DNA synthesis has come from the recent finding of its binding at or near the replication point of simian virus 40 DNA (22). Among the avian and murine type C RNA tumor viruses, two proteins have been found associated with ribonucleoprotein of the viral Present address: University of Texas System, Texas Medical Center, Houston, Tex core (9). One was found to have RNA-dependent DNA polymerase (RDP) activity, and the other was a basic protein of low molecular weight. In the present report, a nitrocellulose membrane filter assay that measures retention of 125I single-stranded calf thymus DNA was employed as an assay for detection and isolation of type C viral DNA-binding proteins. By using this technique, it has been possible to purify and distinguish two single-stranded DNA-RNA binding proteins from Rauscher murine leukemia virus (R-MuLV). MATERIALS AND METHODS Virus. R-MuLV was grown in monolayer cultures of chronically infected mouse (BALB/c) bone marrow, JLS-V9 cells (33). The endogenous cat virus RD-114 was grown in human RD cells in monolayer, and the feline leukemia virus was grown in suspension in a cat lymphoblastoid cell line (27). Viruses from infected cultures were purified by sucrose density gradient centrifugation. Cell culture. All cells were grown in Eagle minimal essential medium supplemented with 10% fetal bovine serum in 32-ounce (ca liter) glass bottles at 36 C, and subculturing of cells was carried out by treatment with 0.25% trypsin in Earle balanced salt solution. Human HeLa (strain KB) cells (Flow Laboratories) were used as a source of p8 protein. These cultures were routinely checked for mycoplasma contamination and found negative by growth on agar medium (7). 709

2 710 DAVIS ET AL. Purification of p8 protein. Purified KB p8 protein was used to standardize the membrane filtration assay. The preparation of p8 protein was carried out essentially as described previously (23). Thirty bottles of exponentially growing KB cells were washed twice with phosphate-buffered saline, harvested with versene-phosphate-buffered saline, centrifuged, and resuspended in 3 ml of cold sonic treatment buffer containing 40 mm Tris-hydrochloride (ph 8.1), 10 mm MgCl2, 0.2 mm EDTA, and 1 mm mercaptoethanol. The suspension was frozen and thawed once, sonically treated for 10 s, and centrifuged at 17,000 x g for 20 min with a Beckman JA-21 rotor. The resulting supernatant was dialyzed for 48 h, with two changes, against rinse buffer containing 4 x 10 2 M NaCl, 10-3 M mercaptoethanol, 10-3 M EDTA, 2 x 10-2 M Tris-hydrochloride (ph 8.1), and 10% glycerol and centrifuged as above. The supernatant was applied directly to DNA cellulose columns, and all operations were carried out at 4 C. Singleand double-stranded DNA cellulose was prepared from calf thymus DNA by the method of Alberts and Herrick (3). The amount of DNA bound was approximately 1.5 mg/g of cellulose. Double-stranded and single-stranded DNA was taken up in rinse buffer containing 0.5 mg bovine serum albumin (BSA) per ml. Two double-stranded and one single-stranded DNA columns, each 3 by 10 cm and containing 30 mg of DNA, were prepared and maintained at 4 C. Approximately 60 mg of the above protein preparation was passed sequentially through two doublestranded DNA columns at 0.2 ml/min, and the effluent was absorbed onto a single-stranded DNA column. The column was washed with 100 ml of rinse buffer containing 0.5 mg of BSA per ml and 100 ml of rinse buffer alone. p8 protein was eluted from the column with 2.0 M NaCl. Elution was monitored by optical density at 280 nm, and 2-ml fractions were collected. The p8 protein-containing fractions were pooled and dialyzed overnight against 0.1 M ammonium acetate and concentrated to approximately 200,ug/ml by coating the surface of the dialysis bag with Sephadex G100; protein was estimated by the method of Lowry et al. (16). The concentrated material was dialyzed against the rinse buffer containing M dithiothreitol (DTT) in the place of mercaptoethanol. Electrophoresis in 9% acrylamide gels containing sodium dodecyl sulfate was carried out by the method of Laemmli (14) for p8 protein and in 15% gels for the viral proteins by the method of Weber and Osborn (32). The molecular weight was estimated using,8-galactosidase, BSA, gamma globulin (Sigma Chemical Co., St. Louis, Mo.), and RNase A (Worthington Biochemicals Corp., Freehold, N.J.) as standards. Purification of viral polymerase and DNA-binding activities. Sucrose-banded, pelleted, R-MuLV, generally amounting to 20 to 25 mg of protein, was solubilized on a rotator in 2.0 ml of 0.02 M Trishydrochloride (ph 8.0) containing 2% Triton X-100, 0.2%,8-mercaptoethanol, 1 M urea, 10% glycerol, 0.3 M KCl, and 1 mm DTT for 15 h. All the purification steps were carried out at 4 C. Solubilized virus was clarified by centrifugation at 20,000 rpm for 60 min in a Beckman 40 rotor, and the supernatant was diluted 10-fold by the addition of buffer A consisting J. VIROL. of 0.01 M Tris-hydrochloride (ph 8.0 at 4 C), 1 mm DTT, 0.1% Triton X-100, and 20% glycerol. Virus protein was loaded onto a column (2 by 15 cm) of oligo(dt)-cellulose (Collaborative Research Inc.) equilibrated with buffer A containing 0.03 M KCl at a flow rate of 15 to 20 ml/h and washed with 30 ml of the same buffer. RDP and single-stranded calf thymus DNA-binding activities were eluted from the column with buffer A containing 0.6 M KCl, collecting 1.2-ml fractions at the same flow rate. The application, wash, and high-salt eluant fractions were assayed for RDP and DNA-binding activities, and the peak active fractions were pooled. Generally, greater than 90% of the input activities bound to the column. The pooled material was dialyzed against a total of 8 liters of buffer A with four 2-liter changes, using Spectrapor-3 dialysis tubing with a 3,500-molecular-weight exclusion limit for 16 h. The dialyzed material was then applied to (100 to 200 mesh) column (1.5 x 6 cm) equilibrated with buffer A containing 0.03 M KCl at a flow rate of 20 ml/h, collecting 1.2-ml fractions. The column was washed with 20 ml of buffer A containing 0.03 M KCl and eluted with a 60-ml gradient of KCl (0.03 to 0.5 M) in the same buffer. The wash and high-salt fractions were assayed for RDP and binding activities, and the peak fractions were pooled. The binding activity that was present in the wash was dialyzed against three 1-liter volumes of buffer A containing 0.3 M KCl and 5% glycerol, with a change every 45 min. The dialyzed material was layered onto 10 to 30% glycerol gradients and centrifuged at 41,000 rpm in an SW41 Beckman rotor for 40 h. Fractions of 0.35 ml were collected from the tubes by bottom puncture and assayed for binding activity. The highsalt eluant from the DEAE-Bio-Gel column containing the RDP activity was dialyzed and fractionated on glycerol gradients in a similar fashion or further purified by chromatography on a phosphocellulose column (1.5 by 3 cm) using a 0.03 to 0.5 M gradient of KCl in the buffer A containing 1 mg of BSA per ml. Nucleic acids. For studies on the specificity of the binding proteins for nucleic acids, 1 to 2 mg/ml solutions of Escherichia coli DNA (P-L Biochemicals) and native calf thymus DNA (Miles Laboratories, Inc.) were digested with 50 jig of RNase (Worthington Biochemicals Corp.) per ml at 37 C in ammonium acetate-acetic acid buffer, ph 5.0, to remove any contaminating RNA and were further digested with 10 U of S-1 nuclease/ikg of DNA (Miles Laboratories Inc.) for 1 h at 37 C in 1.8 x 10-3 M ZnCl2-0.3 M NaCI-0.03 M sodium acetate buffer, ph 4.5. The nucleic acid preparations were then extracted twice with equal volumes of phenol and precipitated with ethanol until free of phenol at 4 C. The precipitated nucleic acids were stored at -70 C. The frozen calf thymus DNA was thawed and made up to 2 to 3 mg/ml by dissolving in 0.01 M Trishydrochloride, ph 7.4, containing 0.15 M NaCl. The single-stranded DNA was prepared from this by heating at 100 C for 20 min, dialyzed overnight against 5 volumes of sodium acetate buffer, ph 5.0, and used for iodination. The 70S RNA used for iodination was prepared from R-MuLV by the method of Tsuchida et al. (30). The 70S murine and cat viral RNAs and the QB phage RNA used for the inhibi- a DEAE-Bio-Gel A

3 VOL. 18, 1976 tion assays were kindly provided by Hiromi Okabe (Flow Laboratories). Iodination. Nucleic acids were iodinated by the method of Commerford (6) with minor modifications. A total of 500 jig of DNA was reacted at 60 C for 20 min in 500,ul of 0.1 M ammonium acetate-0.04 M acetic acid buffer (ph 5.0) containing 0.25 mm KI, 1.5 mm TlCl3 (ICN Pharmaceuticals), and 1 mci of 1251 (Amersham/Searle). The iodination was stopped by quickly cooling to 4 C and adding 25,ul of 0.1 M Na2SO3. The ph was raised to about ph 9.0 by the addition of 50,ul of 1 M ammonium acetate-0.5 M NH4OH, and the mixture was heated again at 60 C for 20 min to dissociate noncovalently bound iodine. Free and bound counts were separated on a Sephadex G25 column (1.5 by 30 cm) equilibrated with 0.1 M ammonium acetate-0.04 M acetic acid buffer, ph 5.0. Specific activities ranged from 4.5 x 104 to 5.0 x 104 counts/min per gg of nucleic acid (0.5 mci/mg). 70S viral RNA was iodinated with 1 mci of 125I as described above, except the initial reaction volume was reduced to 100,ul. All iodinated nucleic acids were dialyzed into 0.01 M Tris-hydrochloride buffer, ph 8.1, containing 0.05 M NaCl and stored at 4 C. The nucleic acids were subsequently diluted to contain 10,000 to 15,000 counts/min per 25,ul in the assay buffer used for the membrane filtration assay. Membrane filtration assay. The membrane filtration assay of Tsai and Green (29) was used with slight modifications. Membrane filters (Millipore Corp.) (catalog no. HAW P02500) were boiled in water for 20 min and soaked at room temperature in the assay buffer consisting of 0.01 M Tris-hydrochloride (ph 8.1), 0.05 M NaCl, 2 mm /8-mercaptoethanol, 1 mm EDTA, 5% glycerol, and 1% dimethyl sulfoxide until used. Both the percentage of input free nucleic acid retained on the filters and the maximum percentage of the protein-nucleic acid complexes found after washing varied with the lot of filters. Therefore, only filters with a background of less than 5% input nucleic acid and those that bound greater than 80% of the total counts in protein excess were used in the present studies. The same lot of filters was always maintained throughout each experiment. Volumes of 10 to 50,ul of protein solution were added to 0.50 ml of assay buffer, followed by 25 /.d of iodinated nucleic acid diluted to contain 10 to 15,000 counts/min. Tubes were incubated at 30 C for 5 min and then filtered under vacuum pressure. The filters were washed twice with 5 ml of assay buffer, and then the total radioactivity bound was conted in a gamma counter (Nuclear-Chicago Corp.). In some experiments an indirect binding assay was performed to measure inhibition of binding in which 20-,ul volumes of unlabeled nucleic acid were added immediately before purified binding protein and iodinated nucleic acid. Incubation and processing were the same as in the direct binding assay. RESULTS Membrane filtration assay. A sensitive nitrocellulose membrane filtration assay was developed employing iodinated DNA and a single- R-MuLV DNA PROTEIN 711 stranded DNA binding protein, p8, isolated from human KB cells. p8 protein was purified by taking advantage of its selective binding to single- and not double-stranded DNA (29). A 60-mg amount of KB cell extract was passed consecutively through two double-stranded DNA cellulose columns, and the effluent was applied to and eluted from a single-stranded DNA column with 2 M NaCl. As shown in the gel pattern of Fig. 1, the eluant from the singlestranded DNA column was found to contain mostly p8 protein. Tsai and Green (29) described a membrane filtration assay for p8 protein using 14C-labeled DNA. In this sytem approximately 3.0,ug of p8 was required for 50% maximum binding with single-stranded mouse DNA. From 15 to 20% of single-stranded DNA was retained when passed alone through the filters, whereas almost 60% of labeled DNA was retained in combination with an equivalent weight of p8. Figure 1 shows the binding profile of 25I1-labeled single-stranded DNA and p8 protein. Incubation of single-stranded DNA in the presence of increasing amounts of p8 resulted in the retention of increasing amounts of single-stranded DNA. A parallel study using increasing amounts of BSA gave only a background level of retention. Fifty percent of maximum binding was obtained with only 0.2,ug of p8 protein, and a maximum of 85% retention of input 125I single-stranded DNA was obtained with 1,g or more of p8. Retention of labeled DNA alone did not exceed 5% of the input radioactivity, and this level of binding was not increased by the addition of up to 25,ug of BSA. Purification of binding proteins from MuLV. The sensitivity of the binding assay for p8 protein indicated that this might be a unique way to examine type C viruses for nucleic acidbinding proteins. Initially, banded and disrupted Rauscher leukemia virus was found to bind a percentage of 125I single-stranded DNA comparable to purified p8 protein, with 50% of maximum binding occurring with only 2,ug of solubilized viral protein. The purification and separation of two viral proteins that bind to 125I single-stranded DNA are shown in Fig. 2. Greater than 90% of the binding and RDP activities from 23 mg of solubilized viral protein were retained during a single passage through an oligo(dt)-cellulose column. Both activities were recovered by a batchwise elution with 0.6 M KCl (Fig. 2A). After extensive dialysis against a low-salt buffer, the proteins eluted from the oligo(dt)-cellulose column were applied to a DEAE-Bio-Gel A column and washed extensively. A linear salt gradient (0.03 to 0.5 M KCl) was applied, and the wash and gradient

4 712 DAVIS ET AL a: J. VIROL. f-.w 60-i / 40W 1 II if1 A )(7- I Protein(jig 4.-A _....O FIG. 1. Sensitivity of membrane filtration assay with purified p8 protein. p8 protein was purified as described in the text. Electrophoresis in 9% acrylamide gels containing sodium dodecyl sulfate was by the method of Laemmli (14). Gels were incubated overnight in 50% trichloroacetic acid, stained with 0.1% Coomassie blue in 50% trichloroacetic acid for2 h at37 C, and destained by washing with 7% acetic acid. The membrane filtration assay was performed as in the text. Twenty-microliter volumes ofprotein solution were added to 0.50 ml of assay buffer, followed by 25 pi of iodinated nucleic acid diluted so as to contain 10 to 15,000 counts/min. Tubes were incubated at 30 C for 5 min and filtered under vacuum pressure. Filters were washed twice with 5 ml of assay buffer, and radioactivity was determined in a gamma counter. Binding is expressed as percentage of input 125I single-stranded DNA. The purity of the p8 preparation used in the binding studies is shown in the inset to the figure. Standards were run as described in the text and indicated a molecular weight of about 25,000 for the major band. L I15 fractions were assayed for binding and polymerase activities (Fig. 2B). Two peaks of binding activity were found; one appeared in the wash fractions and the other appeared in the gradient eluant and was coincident with polymerase activity. The binding activity that was not retained on the DEAE column was pooled, dialyzed, and further purified by centrifugation on 10 to 30% glycerol gradients. A single peak of single-stranded DNA-binding activity was found in the low-molecular-weight region of the gradients (Fig. 2C). Gradients run in parallel containing myoglobin (molecular weight, 17,000) as a marker showed that the binding activity was smaller than myoglobin. To determine whether the polymerase and binding activities that coeluted from the DEAE-Bio-Gel column could be further separated, peak tubes were pooled and chromatographed on a phosphocellulose column (Fig. 2D), using a 0.03 to 0.5 M KCl gradient elution. The polymerase and single-stranded DNA-binding activities coeluted midway through the gradient, further indicating a DNA-binding activity associated with the polymerase protein. Purity and size of binding protein. A peak tube of binding activity from the glycerol gradients was precipitated with 12% cold trichloroacetic acid, washed twice with 6% trichloroacetic acid, and analyzed in 15% polyacrylamide gels. One major band was found with only a faint trailing band (Fig. 3). A comparison of the

5 VOL. 18, 1976 R-MuLV DNA PROTEIN 713 _ 120- I.' X 80- i r-l a u csj x m In Cl) -2 U. A I Fraction Number Fraction Number Cl Cl. 100 (T.- t0100' x 8.280rl E I C U a 60- v*p 50- a X 40. i ] I m7 Fraction Number FIG. 2. Purification ofrdp and single-stranded-dna-binding activities. (A) Step 1. All purification steps were carried out as described in the text. Fractionation ofpolymerase and binding activities from solubilized MuLV by oligo(dt)-cellulose chromatography. (B) Step 2. Pooled polymerase and binding activities from the oligo(dt)-cellulose fractionation were chromatographed on a DEAE-Bio-Gel column, resulting in the separation of a binding activity distinct from polymerase activity. (C) Step 3. The binding activity separable from polymerase was further purified on 10 to 30% glycerol gradients. (D) Step 4. Polymerase and binding activities that coeluted during the salt gradient were further chromatographed on phosphocellulose, resulting in the further co-purification of polymerase and binding activities. Polymerase assays were carried out as essentially described previously (15), and reaction mixtures contained in a final volume of 100 pi: 10.0 MM unlabeled TTP, 0.3 pm3 HTTP (50 Cilmmol), 0.1 M Mn2+, 0.1 M glycine buffer (ph 8.3), and 25 pg of oligo(dt)-poly(a). Incubations were at 30 C for 15 min, and reactions were terminated with 10% trichloroacetic acid. Precipitates were collected on membrane filters (Millipore), and radioactivity was determined in a Beckman scintillation counter. Symbols: *, binding to 125I single-stranded calf thymus DNA; 0, RDP activity. relative migration of the binding protein with that of various standards run simultaneously in parallel gels produced a molecular weight of 9,800, suggesting a relationship to the virion structural protein p1o. Since the molecular weight of this virion-binding protein is similar to that of plo, it will be tentatively referred to as plo in this report. The band in gels showed strong red fluorescence when examined under intensive light and was also in a position identical to the fastest migrating band of whole solubilized virus Fraction Number & W c I--.-Ol s R -20 U =6-60g -0-4 w M V X I.- 30 I.- M 20 M 10 R u Influence of salt, temperature, and ph on binding. The peak tubes from the glycerol gradients containing binding protein were pooled and dialzyed into 0.02 M Tris-hydrochloride (ph 8.1), 1 mm DTT, 0.1% Triton X-100, 20% glycerol, and 0.05 M NaCl. As can be seen (Fig. 4A), the binding activity showed a strict dependence on salt concentration. Binding was reduced to less than 50% of the maximum at 0.1 M NaCl and fell to background levels at concentrations greater than 0.15 M. The optimum temperature range for binding was rather

6 714 DAVIS ET AL. J.- VIROL. T.k., I0h <..0 -e (...le> '1() 0~~~~~~~~~~~~~~~~~~~~~~ CAI'I CA~~~~~~~~~~~~~~~~~~..4I (LrS Relative NMohiIIt\ FIG. 3. Molecular weight and purity of MuLV-binding protein. A peak binding fraction from the glycerol gradient-purified binding protein was trichloroacetic acid-precipitated and electrophoresed on 15% polyacrylamide gels by the method of Weber and Osborn (32). Standards were run in parallel gels at 10 pg and included ovalbumin (OA; molecular weight, 44,000), carbonic anhydrase (CA; molecular weight, 29,000), cytochrome C (Cyt C; molecular weight, 11,700) and glucagon (Glu; molecular weight, 3,500). Gels were stained with Coomassie blue by the method of Fairbanks et al. (8). Molecular weights are plotted on a semilogarithmic scale. The arrow indicates the position of band in inset gel. The India ink line at right ofgel indicates dye front. broad, occurring between 15 and 40 C, and fell off rapidly above 50 C (Fig. 4B). The amount of binding as a function of assay ph did not vary greatly over the range of 5 to 10. ph values less than 7 gave a stimulation compared to the standard assay carried out at ph 8.0 (Fig. 40). Binding specificity. A survey was made of the specificity of purified p8 cell protein, p1o viral protein, and RDP for binding to iodinated single-stranded E. coli, single-stranded calf thymus DNA, and iodinated 70S MuLV viral RNA. When each protein was tested at a dilution that gave 40 to 70% retention of 125I singlestranded calf thymus DNA, it also produced from 35 to 70% retention with either 1251 singlestranded E. coli DNA or 125I 70S RNA (Table 1). The binding to viral, bacterial, or mammalian single-stranded nucleic acids, therefore, does not appear to be appreciably different. The binding of p1o protein was examined in more detail by measuring the competition of p1o binding to 125I single-stranded calf thymus DNA by double- and single-stranded nucleic acids. The results of the indirect-competition assay (Table 1) show a clear preference of plo binding for denatured DNA. In the case of both E. coli and calf thymus DNA, 15 to 30 times more native than denatured DNA was required to produce a 50% inhibition of binding. To determine whether p1o protein could discriminate between single-stranded RNAs, a binding inhibition assay was carried out using RNA from Rauscher leukemia virus, feline leukemia virus, and unrelated QB bacteriophage. The results in Fig. 5 show that the RNAs from all three sources compete about equally for p1o binding to 125I single-stranded calf thymus DNA, giving values close to that of unlabeled

7 VOL. 18, 1976 single-stranded calf thymus DNA required to produce 50% inhibition of binding. It therefore appears that p10 protein can recognize and bind more efficiently to single-stranded RNA and DNA than native DNA. Furthermore, using the present assay system, it does not appear that p1o can discriminate between homologous and heterologous viral RNA. Stability of binding activity. The stability of the binding activity associated with the glycerol gradient-purified MuLV p10 protein was examined under various conditions. The protein was kept (in the standard buffer) in 0.02 M Tris-hydrochloride, ph 8.0, containing 0.05 M NaCl, 2 mm DTT, 0.1% Triton X-100, and 20% glycerol. When stored in standard buffer at 4 C for 7 days, 43.0% of the initial activity remained, whereas, when stored for the same time with added 0.3 M KCl, only 3% binding activity remained. Storage under standard conditions at - 70 C for 7 days resulted in retention of 41% of the activity, and incubation for 15 min at 37 C destroyed all activity. Greater than 75% of the activity was retained over the ph range from 4 to 12 during a 30-min incubation at 4 C, and all activity was lost upon incubation at ph 2. Binding activity of purified plo protein is stable for 20 h in 6 M guanidine hydrochloride at 4 C. Therefore, an alternate means of purification could utilize a size separation, using agarose under denaturing conditions. Binding proteins from other type C viruses. To extend the presence of a small-molecularweight binding protein to other type C viruses, B t 100 so R-MuLV DNA PROTEIN 715 we partially purified single-stranded DNAbinding activities from two other mammalian viruses, the endogenous cat virus RD-114 (8) and the feline leukemia virus from FL74 cells (27). In each case approximately 10 mg of solubilized virus was chromatographed on a DEAE-Bio-Gel column, and binding activity in the wash fractions was pooled, dialyzed, and further fractionated as described previously by centrifugation on 10 to 30% glycerol gradients. In both cases a peak of binding activity was recovered with a molecular weight less than that of myoglobin, suggesting that other type C viruses possess binding proteins similar to the one described here for R-MuLV. DISCUSSION The present study has shown that Rauscher leukemia virions contain a low-molecularweight protein that binds well to 70S viral RNA and denatured DNA but has a low affinity for native DNA. This protein is distinct from the virion RDP that also binds to DNA. The bind- alo M NMCI Tenerture MC) ph FIG. 4. Influence ofsalt, temperature, and ph on binding. Reaction conditions were as described in the text except for the parameter being test. Aliquots (20 pi) ofpurified binding protein were added to 0.5 ml ofcontrol assay buffer (0.02 M Tris-hydrochloride [ph 8.1], 1 mm DTT, 0.1% Triton X-100, 20% glycerol, and 0.05 M NaCl) or test reaction mixtures at concentrations giving 60 to 80% of maximum binding. Duplicate samples were incubated for each point for 5 min and filtered through membrane filters (Millipore). Controls were done for each point with buffer incubated in place of binding protein and processed identically to the test sample. Counts retained on the membrane filters in control samples were subtracted from counts bound in test samples. An increase in background binding was evident at 5 C and also at ionic strengths greater than 0.1 M. The buffer ph was adjusted to each temperature being tested. ing activity has been followed through three purification steps by a modified nitrocellulose filter assay and gave a homogeneous final preparation by gel electrophoresis. The membrane filtration assay seems uniquely suited for the detection of virion DNA-binding proteins in combining speed and sensitivity in measuring a biological function. As shown for the KB cell p8 protein, the assay employing 125I singlestranded calf thymus DNA was about 10-fold more sensitive in detecting binding than a simi C

8 716 DAVIS ET AL. TABLE 1. Specificity for nucleic acid-binding and inhibition" Ag for Pro- Binding Nucleic acidb Percent 50% intein assay binding hibition p8 Direct I'25ss E. coli 68 I'25ss Ca Thy 71 I12570S RNA 60 RDP Direct I125sE. coli 38 I'25ss Ca Thy 44 I"2570S RNA 36 plo Direct I125sE. coli 62 I'25ss Ca Thy 63 I'25ss 70S RNA 47 p10 Indirect ss E. coli 0.04 ds E. coli 0.70 ss Ca Thy 0.05 ds Ca Thy 1.35 a The binding assay was carried out as described in the text. Twenty-five microliters of '25I-labeled nucleic acid (10 to 15,000 counts/min) was added to 500 p1 of assay buffer, followed by the addition of 20 ul of purified protein solution. In the case of the indirect assay to measure inhibition, 20 "I of cold nucleic acid was added before adding protein. Several dilutions of cold nucleic acid were tested to generate an inhibition curve and find the concentration giving 50% inhibition in the indirect assay. All cold nucleic acids were RNase and S-1 nuclease treated. b All nucleic acids were DNA, except where noted. ss, single stranded; ds, double stranded; Ca Thy, calf thymus. lar assay using '4C-labeled mouse DNA (23) and gave a higher level of maximum binding. The low-molecular-weight binding protein isolated in the present study has many features in common with previously purified plo structural proteins of MuLV (4, 9, 19). It migrates in polyacrylamide gels in a position corresponding to the fastest migrating virus protein, behaves on DEAE-Bio-Gel chromatography as a protein with a high isoelectric point, shows red fluorescence ir. intense light, and has a calculated molecular weight based on polyacrylamide gel electrophoresis of about 10,000 when compared with standard proteins. A number of proteins have now been isolated that are capable of binding to DNA. Examples of proteins that bind at specific sites and thus control gene expression are the X phage repressor (34) and the lac repressor (4). Other proteins that display preferential site binding and possess enzymatic activity are bacterial RNA polymerase, DNA methylase, and restriction enzymes (34). A second group of binding proteins does not show site specificity but can show a preferential binding to native or denatured DNA. The best-studied example in this group is the gene 32 protein of T4 bacteriophage, which binds preferentially to single-stranded DNA. It is thought to function by lowering the melting temperature of native DNA and promote unwinding by binding cooperatively to singlestranded regions (1). Proteins of this type with a selective affinity for single-stranded DNA have been isolated from a number of organisms (2, 13, 18, 24, 26). Unlike those proteins showing site specificity that are present in very small amounts per cell, the latter proteins are present in great abundance, suggesting a structural rather than an enzymatic role in DNA replication. The virion p1o DNA-RNA-binding protein in the present study has properties most like the binding proteins of the second group above. It binds equally well to single-stranded DNA from E. coli or calf thymus or to type C viral RNA. The competition experiments show that p1o protein binds about equally well to homologous Rauscher 70S viral RNA as it does to the heterologous feline leukemia virus RNA and unrelated QB phage RNA. The specificity resides in a preference for single- rather than double-stranded nucleic acids. The plo proteins of MuLV account for about 5% of total virion protein, which would suggest a structural rather than a catalytic function. The binding of p1o to single-stranded calf thymus DNA was relatively insensitive to variation in ph over the range of 5 to 10, suggesting that neither the binding to nitrocellulose filters nor the stability of the plo DNA complex is appreciably affected. Since the isoelectric point of MuLV p1o protein m z a U' U' C LO VN be 100T: has been found to be 9.5 (S. Oroszlan, personal communication), the fact that binding can occur at ph 10, when p1o should have a net negative charge, would suggest that the protein is not merely interacting with the DNA phos *o \\ 20± * ' a _---_-_ 0 J. VIROL Q25 pg Competing RNA FIG. 5. Competition-inhibition ofrauscher leukemia virus plo binding to 1251 single-stranded calf thymus DNA by single-stranded viral RNA. The binding assay was carried out as described in the text, and the competition assay was carried out as described in the footnote to Table 1. The amount of RNA was estimated by absorbance at 260 nm. Symbols: *, V9 (MuLV) RNA; 0, QB RNA; 0, feline leukemia virus.

9 VOL. 18, 1976 phate backbone. The striking dependence of binding on NaCl concentration would appear not to be a general ionic effect since KCl used during the purification did not drastically reduce binding. Routinely, the peak activity tubes from the glycerol gradients contained about 5 to 10 gg of protein, as determined from a comparison of their staining with Coomassie blue relative to known concentrations of RNase on acrylamide gels. Volumes of 10 to 20 /.l of this material were sufficient to give 50% of maximum binding. A calculation based on these figures indicates from 0.15 to 0.60,ug of purified p1o binding protein is sufficient to give 50% maximum binding to 0.3,ug of singlestranded calf thymus DNA. The biological role of plo binding protein in the replication and regulation of type C virus is at present uncertain. It may function in a manner similar to the gene 5 protein of M13 and fd bacteriophages (24). This protein binds selectively to viral single-stranded DNA and is thought to block complementary DNA synthesis by reducing the ability of the singlestranded DNA to serve as a template. By preventing the formation of double-stranded replicative form DNA, gene 5 protein would increase the pool of single-stranded DNA required for virus maturation. The biological function of plo protein in type C virus development may be similar to the repressor model presented for gene 5 protein. By binding to 70S viral RNA, p1o protein could similarly prevent complementary strand formation by reverse transcriptase. This would increase the pool of single-stranded viral RNA and favor virus maturation. Alternatively, p10 protein could function in the assembly of viral particles. Recently, it has been found that a viral protein precursor for Rauscher leukemia virus is capable of binding to single-stranded DNA cellulose (Oroszlan, Long, and Gilden, submitted for publication). By analogy with the present work, plo as a part of a large precursor molecule could provide for a protein-rna complex in the process of virion maturation. In either of the two alternatives given, inhibitors of p10 binding might prove useful as a means of blocking infectious virus formation. The low-molecular-weight proteins of mammalian type C viruses have proven valuable for the subtyping of isolates from within a species. For example, where assays for viral p30 protein do not allow a ready discrimination of viruses from the woolly monkey and gibbon ape, they can be distinguished in an assay using viral p12 protein. Among the murine viruses, Parks et al. (20) have shown that p12 and plo proteins exhibit intrastrain cross-reactivity and a high R-MuLV DNA PROTEIN 717 degree of interstrain type specificity in immunological tests. Thus, the Rauscher and Moloney strains could be readily distinguished. The ability to obtain at least one of these proteins under mild conditions that permit retention of biological activity should prove valuable for a variety of comparative functional, structural, and immunological studies. The assay method reported here may reveal the presence of nucleic acid-binding proteins in other classes of mammalian viruses. ACKNOWLEDGMENTS We thank Raymond V. Gilden for helpful discussions during this study. This study was conducted under Public Health Service contract NO1-CP within the Virus Cancer Program of the National Cancer Institute. LITERATURE CITED 1. Alberts, B. M., and L. Frey T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature (London) 227: Alberts, B. M., L. Frey, and H. Delius Isolation and characterization of gene 5 protein of filamentous bacterial viruses. J. Mol. Biol. 68: Alberts, B. M., and G. Herrick DNA-cellulose chromatography, p In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 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