Large-scale isolation and partial purification of type C RNA viruses on hydroxyapatite

ANALYTICAL

86, 252-263 (1978)

BIOCHEMISTRY

Large-Scale

Isolation and Partial Purification RNA Viruses on Hydroxyapatite 1. Biochemical

of Type C

Characterization

RICHARD GUY SMITH*~'AND SIN A. LEE? *Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesdu. Maryland 20014, and THEM Research, Inc., 5451 Randolph Road. Rockville, Maryland 20852

Received March 1. 1977; accepted November 14, 1977 A chromatographic procedure utilizing common laboratory equipment and based on the batchwise adsorption of type C RNA virus onto hydroxyapatite for the concentration and partial purification of viruses from large volumes of tissue culture fluid has been developed. This procedure provides an alternative to the use of elaborate and expensive high-speed zonal ultracentrifuge equipment. The viruses obtained by this procedure have a buoyant density of 1.16 g/cm”, contain 70 S RNA, an RNA-directed DNA polymerase (reverse transcriptase), surface glycoproteins (GP69/71), and the internal viral specific polypeptides p10 to 15 and p27 or ~30.

Calcium phosphate in the brushite form (CaHPO,*2H,O) has been used in a number of laboratories for the small-scale purification of several viruses, including influenza (1,2), Newcastle disease (3), and vaccinia, encephalomyocarditis, Coxsackie, and poliomyelitis type III (4). Hydroxyapatite, Ca,,(PO,),(OH),, has been used extensively for the purification of proteins (5-7) and for the separation of native from denatured deoxyribonucleic acid (DNA) (8). Hydroxyapatite has also been used for the isolation of bacteriophage (9). In an effort to obtain large quantities of type C viruses of high quality for biochemical studies, we have developed a batch adsorption method for their isolation directly from tissue culture fluids. We report here the use of hydroxyapatite (HAP) for the one-step isolation and partial purification of type C RNA viruses from large volumes (10 liters or more) of virus-containing tissue culture fluids with recoveries of almost 100% in some cases. This procedure has allowed us to concentrate and partially purify virus from volumes of tissue culture media ranging from 50 ml up to 15 liters. Our experience suggests the possibility of processing volumes of 50 to 100 liters with little ’ R.G.S. was supported by a postdoctoral fellowship from the National Cancer Institute

(No. 5F22CA02128-02). 0003.2697/78/0861-0252$02.00/O Copyright All rights

Q 1978 by Academic Press, Inc. of reproduction in any form reserved.

252

VIRUS

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ON

HYDROXYAPATITE

253

difficulty. Our experience with brushite showed it to be unsuitable for the isolation of RNA viruses directly from tissue culture fluids by the batch adsorption method. The viruses isolated by this method have been shown to contain all of the known viral-related proteins (with relatively low levels of contaminating serum proteins), viral 70 S RNA, and RNA-directed DNA polymerase (reverse transcriptase). The method described here was optimized for the isolation of biochemically pure virus, and not for the preservation of biological activity. In most cases the recovery of biological activity was below that obtained by other, small-scale methods, but comparable to virus obtained by large-scale ultracentrifuge techniques (ca. l-.5%). A separate study is underway to optimize the technique for the recovery of biologically active viruses and will appear elsewhere (10). MATERIALS

AND METHODS

Preparation of hydroxyapatite. Hydroxyapatite (HAP) was prepared in two ways: (1) by the procedure of Siegelman and Firer (11) or (2) by the Tiselius method (7). Both methods involved the initial preparation of brushite, CaHPO.ZH,O, by standard procedures (7,ll). In method 1 the brushite was partially converted to the hydroxyapatite form by titration with 2.0 M KOH to a pH of 11. The mixture was allowed to stand without stirring until the pH had dropped to about 8 and was again titrated to pH 11. This procedure was repeated until the pH stabilized above 9. Fine particles were removed by differential settling in 0.2 M NaCl, 0.001 M phosphate, pH 7. To ensure rapid flow rates this material was made fresh every 4 weeks. In method 2, the brushite was converted to hydroxyapatite by Bernardi’s modification (12) of the Tiselius et al. procedure (7). Viruses. The type C viruses isolated by the HAP method are listed in Table 1. Growth of virus: Radiolabeled Virus. All viruses containing tissue culture fluids were prepared at HEM Research, Inc., Rockville, Maryland. Virus-producing cells were grown in glass roller bottles with Dulbecco’s minima1 essential medium (D-MEM), supplemented with 10% fetal calf serum (FCS) (Reheis Corp., Phoenix, Arizona) and 1% glutamine. When the cell density reached about 70 to 80% confluency, the fluids were replaced with fresh medium containing 50 &i/ml of [5-3H]uridine and cell growth continued for an additional 16 hr. In some experiments labeled medium was replaced every 2 hr and pooled. In all cases, the pooled medium was clarified by centrifugation at 5001: for 10 min prior to virus isolation. Unlabeled virus. Conditions of culture were the same as described above except that the uridine-labeling step was omitted. Hydroxyapatite chromatography of type C RNA viruses. After

254

SMITH

AND LEE

TABLE VIRUSES

PURIFIED

BY HYDROXYAPATITE

1 AND

THEIR

Virus Murine leukemia virus, MuLV-Rauscher

HOST

CELLS

Host cell JLS-VIO,

mouse embryo fibroblast

(253) Avain myeloblastosis virus, AMV (27)

Chicken, viremic plasma

Woolly monkey sarcoma virus, SiSV/SiSAV (28)

71AP1, marmoset HF, marmoset NRK, normal rat kidney K-NRK: NRK, cells transformed with murine sarcoma virus-Kirsten NC37, human lymphoblastoid

Gibbon ape lymphosarcoma virus, GALV (29)

NC37, human lymphoblastoid

Putative human leukemia virus, HL23V-1; HL23V-5”

A204, human rhabdomyosarcoma CF2Th, normal dog thymus NRK, normal rat kidney CCL88, bat lung (BL88) DBS-FRhL-1,

rhesus monkey lung (DBS)

HEL-12b

Human embryonic lung

Murine leukemia virus, MuLVAKR (30)

BALB/3T3

mouse

a C-type virus released from human myeloid leukemic cells, transmitted to the indicated secondary cells (3 1). * C-type virus isolated from human embryonic lung cells (32).

clarification by low-speed centrifugation, the virus-containing fluids were adjusted to 0.15 M potassium phosphate, pH 7.2 (KPB 7.2) by addition from a 2 M stock solution. Five milliliters of a 1: 1 (v/v) suspension of HAP in 0.15 M KPB 7.2 was added per 100 ml of fluid and the suspension was agitated at 4°C for at least 3 hr, the length of exposure depending upon the virus titer in the starting material. (The medium obtained from the SW-l(71APl) cell line contained approximately 2.5 x lo5 FFU/ml, and kinetics of attachment studies indicated a minimum exposure time of 3 hr for this titer. To ensure maximum recovery, overnight exposure was routinely employed.) All subsequent operations were performed at 4°C in a cold room. HAP was separated by sedimentation at 5OOg for 10 min and the

VIRUS

PURIFICATION

ON TABLE

2

PURIFICATION OF HL23V-5

Fraction Clarified medium HAP pellet

Volume (ml) 10,000 5.5

Reverse transcriptase activity (total cpm)’ 1.29 x 10”’ 5.27 x 10y

255

HYDROXYAPATITE

(BL88)

Total protein “50 x 10’ b6. 1

Specific activity’ 2.5 8.65

x IO4 x IO*

Purification 1 35,000

r( Fetal calf serum has 40 mg/ml of serum protein; the protein concentration in D-MEM is I mgiml. Therefore, the equivalent protein concentration in D-MEM + 10% FCS is 5 mgiml. b Protein content is estimated according to the method of Lowry et al. (33). c Total (A);(dT)s directed reverse transcriptase activity divided by total protein.

medium was discarded. The HAP slurry was then washed into a column (height to diameter ratio of 2:l) with 0.15 M KPB 7.2 and washed extensively with the same buffer. Virus was eluted from the HAP by washing with 5 to 10 column volumes of 0.5 M KPB 7.2 (sodium phosphate buffers will precipitate at this concentration and temperature). Column fractions were routinely monitored either for trichloracetic acid (TCA)precipitable radioactivity, when purifying labeled virus, or for viral reverse transcriptase activity. Active fractions were pooled and concentrated by centrifugation at 100,OOOg for 90 min. Pellets were redissolved in TNE (0.15 M Tris, pH 7.4,O. 1 M NaCl, 0.001 M EDTA). and dialyzed against the same buffer. Hereafter, virus preparations isolated and concentrated by the HAP method are designated as HAP viruses. Reverse transcriptase assay. Viral reverse transcriptase activity was assayed by measuring the incorporation of [3H]dTMP into TCAprecipitable material directed by synthetic homopolymeric RNA and DNA. A 5-~1 aliquot of the viral preparation was preincubated with 10 ~1 of solution containing 0.3% Triton X-100,0.68 M KCl, 2.5 pg of bovine serum albumin, and4pgofeither(A);(dT)rj or(dA);(dT)sfor 15 min at 4°C; the rest of the reaction components were then added in a volume of 85 ~1, bringing the final 100~~1 reaction volume to 40 mM Tris-HCl, pH 7.8, 15 mM dithiothreitol. 0.5 mM MnCl,, and 8 PM [3H]TTP (16 Ciimmol, New England Nuclear). After incubation at 37°C for 30 min the reaction was stopped by the addition of an equal volume of 20% TCA. The samples were placed on ice for 10 min and acid-precipitable products were collected on glass fiber or nitrocellulose filters. After washing with 10% TCA, the filters were dried and the radioactivity counted in a scintillation cocktail containing 6 g of PPO and 75 mg of POPOP per liter of toluene. Preparation of viral RNA. Viral 70 S RNA was prepared by the oligo(dT)-cellulose procedure of Smith et al. (13).

256

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AND

LEE

Analytical centrifugation of virus and RNA. HAP virus was centrifuged to equilibrium in a linear sucrose-density gradient (20-50%, w/w, in TNE) at 4°C and 100,OOOg for 4 hr. Viral RNA was centrifuged into a glycerol gradient (lo-30%, v/v. in 0.01 M Tris, 0.1 M LiCI, 1 mM EDTA, 0.2% sodium dodecyl sulfate (SDS) at 4°C and 200,OOOg for 4 hr. Gradient fractions were collected from the bottom of the centrifuge tube. Polyacrylamide -gel electrophoresis of viral proteins. HAP virus was solubilized in 0.1% (w/v) SDS and subjected to electrophoresis in polyacrylamide gels according to the procedure of Maize1 (14). RESULTS Virus Isolation

A typical isolation and partial purification for the putative human virus, HL23V-5, is shown in Table 2. Two fractions are obtained from the hydroxyapatite column: (1) intact virus in the 0.5 M phosphate eluate and (2) degraded virus containing 70 S RNA, reverse transcriptase, and other viral proteins from a wash of the column with nonionic detergent. This second fraction is the result of apparent irreversible binding of a population of the virus particles in the starting fluids. The relative size of this fraction appeared to depend upon the quality of the virus in the starting fluids, but we have not quantitated this effect. The 0.5 M KPB 7.2 eluates from the HAP column were assayed for exogenously templated reverse transcriptase activity as described under

FRACTIOli

FIG. I. Elution column. Growth

NUHBER

profile of [3H]uridine-labeled SSV-1 (7lAPl) virus from a hydroxyapatite and labeling of virus as described under Materials and Methods.

VIRUS

PURIFICATION

ON TABLE

COMPARISON

OF

YIELDS

OF VIRUSES

ISOLATED

3 BY HYDROXYAPATITE

Hydroxyapatite

Starting volume (liters)

Virus

Final volume (ml)

257

HYDROXYAPATITE

AND

Zonal Total polymerase activity (cpm)”

Starting volume (liters)

CENTRIFUGATION

ultracentrifuge

(ml)

HL23V-1 (BL88)

10

3.2

5.1 x 10”

24

I2

HL23V-5 (BL88)

IO

3.2

3.2 x 10”

IO

5

(1 Viral

polymerase

activity;

see Materials

Total polymerase activity (cpm)”

Final volume

4.8 x 10” (2.0 x 10” per 10 liter) 3.0 x II”

and Methods.

Materials and Methods. Fractions containing the highest activity (Fig. 1) were pooled and concentrated by centrifugation at 100,OOOg for 90 min. There have been reports from a few laboratories (15,16; S. Mayasi, personal communication) that repeated exposure of type C viruses to high concentrations of sucrose yields a virus lacking or largely deficient in the surface glycoprotein gp69/71. For this reason we have avoided the use of preparative sucrose density gradients as a means of purification and/or concentration of virus. TABLE RECOVERY

VlWS HL23V-5 lBL88l HL23V-5 (BL88)” HL23V-5(CF2Th) HL23V-5lCF2Thl HL23V-5 (DBS) HLZSV-I (BL88) HL23V.I (BL88)” SSV (HF) SSV (NC371 GaLV (NC37) MuLV,m

Starting volume (liters) 9 IO IO 12.5 4 10 10 5 8 9 4.5

OF FItA volume (ml) 2.2 3.1 2.2 2.2 2.2 3.0 3.0 2.2 2.5 3.1 2.0

VIRUS

FROM

4 TISSUE

Reverse

x x x x x x x x x x x

lo5 108 106 lo8 IO5 IO” lo5 105 I@ IP l@

FLUIDS

transcriptax? (dA);(dT)ir

(A):(dTk 4 5 2 5 7.2 8 7.6 3.4 3 4.94 6.25

CULTURE

3650 I940 3780 3410 1650 1830 I880 ml 9094 9210 1249

Rat& III.5 2577 529. I 666.7 438 437 405 I708 33 54 5cKNl

Proteinb (m&d) 0.76 1.85 1.30 2.20 0.32 1.50 0.92 1.20 1.1 2.2 ND

PelWlt?& FXOVery’ ND’ 40 90 75 40 ND 90 29.8 78 90 ND

a Reverse transcriptax activity in 5 ~1 of the final concentrate (see Methods). ’ Protein determinations performed by the method of Lowry e’f al. (33) on whole virus lysed by exposure to IOO”C for 5 min. c Thirty milliliters of starting fluids was centrifuged at loO,OOOg for 90 min; the pellet was suspended m 100 pl of TNE: and 5 @I of this solution was used lo assay for reverse transcriptase activity as described under Materials and Methods. This input value is used to compute percentage recovery. d Virus prepared from a separate lot of medium. ’ Ratio of (A);(dTk lo (dA):(dT)rjdirected polymerase activity. ‘Q.D.. not determined.

258

SMITH AND LEE

1B 16

-1.22

14

-1.20

12

-1.18

10 8 m-

6

,"

4

x

2

: " > F ;

0

0

2

4

6

8

10

12

14

16

18

20

: ‘; "

22

; : 0

18

2 CL

14

.ZO

12

.18

10

.16

8

.14

6

.12

4

.lO

16

2 0 2

4

6

8

10

12

FRACTION

14

16

18

20

22

NUMBER

FIG. 2. Equilibrium density sedimentation of [3H]uridine-labeled SSV-l(71APl). Virus was layered over a 20 to 50% (w/w) linear sucrose gradient, and centrifuged at 100,OOOgfor 4 hr at 4°C. (A) Equilibrium density sedimentation of virus pelleted from tissue culture media. Ten milliliters of cell-free medium was pelleted by centrifugation at 100,OOOgfor 1 hr at 4°C. The pellet was washed twice by resuspension in 10 ml of TNE and recentrifuging as above in order to reduce the level of included soluble [3H]uridine. The final pellet was suspended in a total volume of 200 ~1 and layered onto the density gradient. (B) Equilibrium density sedimentation of HAP virus. Five microliters of the final product from hydroxyapatite concentration; purification was diluted to 100 ~1 with TNE, and layered onto the density gradient.

In two experiments a direct comparison was made of the hydroxyapatk procedure with commercial methods employing large-volume zonal ultracentrifuges (17). Table 3 presents the results of these experiments. As shown in this table, the recovery of the viruses was approximately 2-fold [HL23V-5 (BL88)] to lo-fold [HL23V-1 (BL88)] higher when the media were processed by the hydroxyapatite procedure. Buoyant Density of the HAP Viruses Type C RNA viruses have a buoyant density of 1.16 to 1.18 g/cm” in solutions of high osmotic pressure (e.g., sucrose or potassium tartrate)

VIRUS

PURIFICATION

ON

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259

(18). Viruses isolated on hydroxyapatite, when centrifuged to equilibrium in 20 to 50% sucrose gradients, exhibit very sharp density profiles (Fig. 2B) with the activity peak at 1.16 g/cm3. When viruses were pelleted directly from tissue culture fluids the shape of the virus-containing zone was broad and complex. (Fig. 2A). The profiles shown in Fig. 2 were obtained on SSV-1 (71APl), but are typical of all viruses tested. Biochemical

Properties

of the HAP Viruses

Type C RNA viruses contain an RNA with a molecular weight of 3 to 6 x IO6 (19) and several proteins, including the reverse transcriptase (20) and structural and envelope glycoproteins (21). The reverse transcriptases of HAP viruses have been analyzed for their template-primer specificities in the presence of Mn2+ and were found to prefer the synthetic template-primer complex (A);(dT)s over (dA);(dT),-, by at least lOO:l, a characteristic of all known type C RNA viruses (20,21). Table 4 presents template specificity data and recovery of virus from several isolations from large quantities of starting fluids. Viral structural proteins were analyzed on SDS-polyacrylamide gels (Fig. 3). All viruses tested were found to contain the surface glycoprotein(s)

MIGRATION

w

FIG. 3. Electrophoresis of whole SSV-1/71APl virion proteins in polyacrylamide gels in the presence of SDS. Total viral protein (250 Fg) was analyzed on each gel. Coomassie blue staining was used for the visualization of total protein, and the Schiff’s base stain, ANS (8anilino-1-naphthalene sulfonic acid) was used for the specific visualization of glycoprotein (see Refs. 14 and 22). Marker proteins are phosphorylase u (MW 94.000), bovine serum albumin (BSA) (MW 68,000), ovalbumin (MW 43,000), and chymotrypsinogen (MW 25.000) (29). Total protein stain, - - -; glycoprotein stain, -.

260

SMITH AND LEE

28s

18s

A 2800 2400 2000

2

2 t zI

220_

z

200

4

6

8 10

12

14

16

18

20

22

24

26

TOP

4

6

8 10

12

14

16

18

20

22

24

26

TOP

8.

180 160 140 120 100 80 60 40 20 2

FRACTION

NUMBER

FIG. 4. Velocity sedimentation analysis of [3H]uridine-labeled RNA extracted from SSV-1 (71APl) virions isolated on hydroxyapatite. Isolation of RNA followed the procedure of Ref. 13. (A) Viral RNA cenrrifuged into a linear 10 to 30% glycerol velocity gradient. (B) RNA isolated from the 70 S region of gradient A was heated at 80°C for 5 min in 60% formamide. The RNA was precipitated from ethanol and centrifuged into a 10 to 30% glycerol gradient as above.

gp69/71 (detected by specific staining with periodic acid-Schiff reagent) (22) as well as the structural proteins p27 or p30 and the plO- 15 complex. Purified viral RNA (13) was found to sediment as a 60 to 70 S species in SDS-glycerol velocity gradients (Fig. 4A), and when prepared from 2-hr harvest virus was dissociated into a species of 30 to 40 S following incubation in 60% formamide at 80°C (Fig. 4B). DISCUSSION

The large-scale isolation of type C RNA viruses has been performed almost exclusively with the aid of continuous-flow zonal ultracentrifuges

VIRUS

PURIFICATION

ON

HYDROXYAPATITE

261

(17). The product obtained by this method often requires extensive additional purification because of contamination by high levels of serum protein, broken cell debris, and inactive viral structures. These preparations may also contain considerable ribonuclease and protease activity, resulting in poor yields during the purification of subviral components. The isolation and partial purification method described here was developed in response to a need for large quantities of biochemically pure type C viruses. In general, as shown in Table 4, this one-step process satisfied this requirement. The procedure is fast (20 to 50 liters of tissue culture fluid can be processed in 24 hr) and uses biochemical reagents and equipment easily and economically available. The procedure allows complete containment during all stages of production, thus assuring sterility of the product and safety for the worker. As mentioned under Results there were two fractions routinely obtained from the hydroxyapatite column. The first fraction was eluted with 0.5 M phosphate and contained virus particles of homogenous buoyant density (see Fig. 2B). (A comparison of Figs. 2A and 2B shows the relative heterogeneity of particles sedimented directly from virus-containing fluids.) The second fraction was released only with detergent and contained 70 S RNA as well as other viral components. It is our feeling that isolation and chromatography on hydroxyapatite separates intact virus from damaged particles and other ribonucleoprotein complexes and that these latter components are those which are tightly bound to the crystal, i.e., not elutable by phosphate concentrations up to 2 M. Stewart et al. (23) have reported a similar observation in their studies using concanavalin A for the precipitation of type C viruses. In their studies they observed a selective precipitation from tissue culture fluids of infective Friend murine leukemia virus exhibiting a buoyant density of 1.16 g/cm3, whereas the untreated media contained virus-like particles of more heterogenous density. The quality of viruses concentrated from tissue culture fluids is largely a function of (1) the amount of cellular contaminant in the fluids; (2) the method used to obtain virus containing fluids; and (3) the handling of the fluids prior to isolation of the virus (18). To minimize contamination by cellular components, one can accumulate virus containing fluids at frequent intervals during cell growth, for example every 2 to 4 hr. The virus in this early harvest medium can be concentrated by precipitation with (NH&SO, or polyethyleneglycol and further purified in the ultracentrifuge. This procedure is suitable for obtaining relatively small quantities of virus, but becomes quite cumbersome with volumes of fluids in excess of a few liters. The use of hydroxyapatite therefore provides an acceptable alternative for the isolation of good quality viruses in this case and should be especially useful to those laboratories having limited ultracentrifuge capabilities. This process also offers the obvious advantage of concentrating and partially purifying viruses from many different tissue culture lines

262

SMITH AND LEE

simultaneously. Several 5- to 20-liter quantities of media can be processed in parallel, thus avoiding the decontamination procedure between isolations associated with the use of continuous flow ultracentrifuges. Also, this process can be adapted to the continuous flow isolation of early harvest virus by interfacing the continuous-feed roller bottle system (24) with a device designed to meter phosphate buffer into the collecting vessel containing a premeasured weight of HAP. ACKNOWLEDGMENTS We thank H. Schetters and V. Kalyanaraman for their contribution to the electrophoretic analyses described here. We also wish to thank R. C. Gallo, D. H. Gillespie, M. Robert-Guroff, M. G. Samgadharan, and N. R. Miller for helpfuldiscussions. A portion ofthis work was supported by Litton Bionetics, Inc., Bethesda, Maryland.

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24. Smith, R. E., and Quade. K. (1976) Anal. Eioch. 70, 354-358. 25. Rauscher, F. J. (1962) J. Nat. Cancer Inst. 29, 515-543. 26. Wright, B. S., O’Brien, P. A., Shibley, G. P., Mayyasi. S. A., and Lasfargues. J. C. (1967) Cancer Res. 27, 1672- 1677. 27. Langlois, A. J., Bolognesi, D. P.. Fritz, R. B., and Beard, J. W. (1969) Proc. SOL.. Exp. Biol. Med. 131, 138-143. 28. Wolfe, L. G., Deinhardt, F.. Theilen. G. H., Rabin, H., Kawakami, T.. and Bustad, L. K. (1971)J. Nat. CancerInst. 47, 1115-1120. 29. Giard, D. J.. Aaronson, S. A., Todaro. G. J., Arnstein, P., Kersey. J. H.. Dosik, J. H.. and Parks, W. P. (1973) J. Nat. Cancer Inst. 51, 1417-1423. 30. Kirsten, W. H.. and Platz, C. E. (1964) Cancer Res. 24, 1056-1062. 31. Teich. N. M., Weiss, R. A., Salahuddin. S. Z., Gallagher. R. E., Gillespie, D. H.. and Gallo, R. C. (1975) Nature 256, 551-555. 32. Panem, S.. Prochownik, E. V.. Reale, F. R., and Kirsten, W. H. (1975) Science 189, 297-298. 33. Lowry, 0. H.. Rosebrough, N. J.. Farr. A. L.. and Randall. R. (1951)J. Biol. Chem. 193, 265-276.