INFECriON AND IMMUNrrY, Apr. 1975, p. 815-822 Copyright i 1975 American Society for Microbiology

Vol. 11, No. 4 Printed in U.S.A.

Partial Purification and Characterization of RTG-2 Fish Cell Interferon JOHN DE SENAl* AND GUIDO J. RIO Department of Biology, St. John's University, Jamaica, New York 11432 Received for publication 9 October 1974

Interferon produced by rainbow trout gonadal cells (RTG-2) was partially purified. The physical, chemical, and biological properties of this in vitro produced fish cell interferon were studied. Purification was achieved by ultracentrifugation, molecular sieve gel chromatography, ion exchange chromatography, and polyacrylamide gel electrophoresis. The isoelectric point of RTG-2 interferon, as determined by CM-Sephadex (C-0O) chromatography, was 7.1. Filtration through Sephadex G-150 showed that RTG-2 interferon had a molecular weight of 94,000. The partially purified material was not sedimented at 105,000 x g for 2 h at 4 C. The fish cell interferon was non-dialyzable and exhibited heat and pH stability. The partially purified material was inactivated by treatment with trypsin or 2-mercaptoethanol, but was resistant to treatment with deoxyribonuclease or ribonuclease. RTG-2 interferon which was induced by infectious pancreatic necrosis virus exhibited antiviral activity against challenge with infectious hematopoietic necrosis virus or infectious pancreatic necrosis virus. Partially purified RTG-2 interferon exhibited greater species specificity than the crude material. The ability of fish cells to produce interferon or interferon-like substances has been noted in several studies. Fathead minnow cells originating from tissue of the northern fathead minnow (Pimephalus promelas) exposed to infectious pancreatic necrosis (IPN) virus (11) or reovirus type 2 (22) have been reported to elaborate an antiviral substance in vitro which possessed many of the properties of mammalian and avian interferons. Reovirus type 2 was also found to induce low levels of a viral inhibitor in swordtail cells derived from the embryonic tissues of the red swordtail (Xiphophorus helleri) (13). In an in vivo study, inoculation of the grunt fin (Haemulon sciurus) with endotoxin from Escherichia coli has been shown to result in the production of an antiviral substance of the interferon type (1). In vitro production of interferon by grunt fin cells has also been reported

(2).

Previous studies have indicated that some

biological and physicochemical properties of crude fish interferon preparations are similar to those of mammalian and avian interferons. This report presents evidence that partially purified rainbow trout gonadal (RTG-2) interferon also possesses many of the properties of interferons 'Present address: Department of Virology, The Public Health Research Institute of the City of New York, Inc., New York, N.Y. 10016.

8X15

produced by higher animals. In addition, this report offers evidence of certain biochemical and physicochemical characteristics of RTG-2

interferon. MATERIALS AND METHODS Medium. All cell cultures were grown in a modified Eagle minimum essential medium (24). Maintenance medium was the same as the growth medium, except that the fetal calf serum was reduced to a 4.4% level. Cell cultures. A stock culture of RTG-2 cells was obtained from J. Cecil (Osborn Laboratory of Marine Sciences, New York Aquarium). The RTG-2 cell line was established from normal gonads of fingerling rainbow trout (Salmo gairdneri) (26). The goldfish cell line (SJU-1) was originally established in this laboratory from tissue of the goldfish (Carassius auratus) (24). Viruses. IPN (ATCC VR299) virus used for the production of interferon was obtained from the laboratory of J. W. Rachlin (Lehman College, Bronx, N.Y.). The virus was propagated in RTG-2 cells. Infectious hematopoietic necrosis (IHN) virus used for the interferon assay was obtained from F. Amend (Western Fish Disease Laboratory, Seattle, Wash.) and was also propagated in RTG-2 cells. Interferon assay. Interferon was assayed using the neutral red dye uptake method of Finter (7). This colorimetric assay utilizes neutral red, a supravital stain, to quantitate the fraction of cells surviving after virus infection. The dye solution contained 0.0015% neutral red (Fisher Scientific Co.) and 0.85% NaCl in

816

DE SENA AND RIO

distilled water. Assays were performed using RTG-2 monolayers grown in Moderne oval prescription bottles (3 by 6 cm; Foster Forbes Glass Co., Marion, Ind.). Standard RTG-2 interferon preparations were serially diluted (0.2 log/dilution) in maintenance medium. Four assay bottles were used at each dilution, and each bottle received 2.0 ml of interferon. To obtain maximal interferon activity, it was necessary to incubate cells with interferon at 20 C for 24 h before the addition of challenge virus (IHN virus). At the end of this period, the interferon was decanted and each replicate culture received 102 83 mean tissue culture infective doses/0.1 ml of IHN virus plus 0.1 ml of lx Earle balanced salt solution (BSS). The assay bottles were then placed at 20 C for 1 h. After a 1-h adsorption period, the virus was decanted and 2 ml of maintenance medium was added to each assay bottle. After 72 h at 20 C, the medium was discarded and the monolayers were washed twice with 2.0 ml of physiological saline. Two milliliters of dye solution was then added to each assay bottle. The bottles were placed in the dark at 20 C for 1 h. At the end of this time, residual neutral red was removed by washing the cultures three times with 2.0 ml of physiological saline. The dye was extracted from the cells in each bottle with 2.0 ml of a mixture containing equal parts of absolute ethanol and 0.1 M NaH2PO4 (pH 4.5). After a 5-min extraction period, all of the neutral red taken up by the cells was released into the overlaying solution. The absorbance of the extracted dye was determined at a wavelength of 540 nm employing an Hitachi Perkin-Elmer spectrophotometer. To estimate the interferon titer graphically, the mean absorbancy readings at 540 nm obtained for the resulting dye solutions were plotted against the corresponding log dilutions of interferon. This plot resulted in a sigmoid curve, with an intermediate linear portion which ranged from 25 to 705% dye uptake. From the linear portion of the curve, the interferon titer was calculated in 50% dye uptake (DU) units per milliliter by locating the interferon dilution which gave dye uptake, as measured by absorbance midway between the mean absorbance of virus-infected cultures which were not protected by interferon and the mean absorbance of the control cultures which were not treated with interferon or infected with virus. The reciprocal of the interferon dilution which gave 50% of the dye absorbancy of control cells was defined as the titer. The base-line was the dye absorbancy of virusinfected cells. The virus-infected cultures were treated basically the same as the interferon-protected cells. The only modification was the substitution of maintenance medium (interferon diluent) for interferon. The control cells also received maintenance medium instead of interferon. At the time of challenge, the control cells received 0.2 ml of 1 x BSS (virus diluent) instead of the virus. With the exception of these two modifications, the control cells were treated the same as the interferon-protected cells. Interferon production. Five-day-old monolayers of RTG-2 cells grown in 12.6- by 4.0-cm stock bottles were used to produce crude interferon. In a typical harvest of RTG-2 crude interferon, the growth media

INFECT. IMMUN.

from 20 bottles of cells were decanted. After being washed three times with 2.0 ml of lx BSS, each bottle received 1.0 ml of lx BSS containing 105 78 mean tissue culture infective doses of IPN virus. The bottles were placed at 20 C for 1 h. After this period of adsorption, the cells were gently rinsed four times with 2.0 ml of lx BSS (pH 5.5). One milliliter of a sodium bicarbonate-buffered lx BSS (pH 7.2) was added to all the bottles. This 1.0-ml volume of solution was sufficient to completely immerse an entire monolayer. The bottles were placed at 20 C for 24 h. The overlay fluids were then pooled and placed into 25-ml vials. The pooled crude interferon preparation was brought to pH 2 by the addition of concentrated HCl and placed at 4 C for 48 h. The pH of the crude interferon was brought back to pH 7.2 by the dropwise addition of saturated NaOH. The interferon preparation was clarified by centrifugation at 70,000 x g for 2 h at 4 C in a Beckman preparative ultracentrifuge (model L2-65B) using the type 65 fixed-angle rotor. The upper two-thirds of the supernatant from each centrifuge tube was used as the starting material for gel chromatography. The Sephadex G-1l0-purified material was used as the starting material for polyacrylamide gel electrophoresis and carboxymethyl (CM)-Sephadex (C-S0) ion exchange chromatography. Gel chromatography. The chromatography techniques used were direct applications or modifications of techniques described by Fischer (8). Sephadex G-1l0 (Pharmacia Fine Chemicals, Inc., Uppsala, Sweden) columns (30 by 1.5 cm) were equilibrated with chromatographic buffer [0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, containing 0.05 M NaCl] at 5 C. Clarified interferon samples (1.0 ml) were eluted at a hydrostatic pressure of 4.0 cm. In the molecular weight determinations, 0.a-ml samples of clarified interferon were used as the starting material. The void volume (VO/) was determined with Blue Dextran 2000 (Pharmacia Fine Chemicals, Inc.). The columns were calibrated with known-molecular-weight markers (Pharmacia Fine Chemicals, Inc.). Four 0.4% marker protein solutions of aldolase (molecular weight, 158,000), ovalbumin (molecular weight, 45,000), chymotrypsinogen A (molecular weight, 25,000), and ribonuclease A (molecular weight, 13,700) were separately prepared. From the marker solutions, two samples were prepared. Sample 1 consisted of a mixture of 0.25 ml of aldolase and 0.25 ml of chymotrypsinogen A. Sample 2 consisted of a mixture of 0.25 ml of ovalbumin and 0.25 ml of ribonuclease A. Each of the two samples was separately passed through the column. To construct a standard curve (Fig. 2), the log of the molecular weights of the protein standards was plotted against their partition coefficients (K8.) which are defined by the following formula: - K., = (V./Vo)/ (Vt - VO), where V. is the elution volume of the protein. VO is the void volume, and Vt is the bed volume. The molecular weight of RTG-2 interferon was estimated by locating its K,, value on the standard curve and finding the corresponding molecular weight. CM-Sephadex (C-50) chromatography. The

VOL. 11, 197'a

RTG-2 FISH CELL INTERFERON

method of Lampson and Tytell (15) was used for the isoelectric point determination. Gel chromatographically purified interferon samples were pooled and dialyzed for 48 h against 0.1 M sodium phosphate buffer (pH 6.0). After dialysis, 15.0 ml of interferon was applied to a CM-Sephadex (C-50) column (1.5 by 10 cm) equilibrated with the same buffer. Interferon was eluted by successive addition of 5.0-ml amounts of 0.1 M sodium phosphate buffer with increments of 0.2 pH units. The absorbance at 280 nm, the pH, and activity of each fraction were measured. The deviations of elution pH of known proteins from their reported isoelectric points were averaged, and the average deviation was added to the elution pH of RTG-2 interferon to approximate its isoelectric point. The known proteins used in this study were hemoglobin (2x crystallized, Nutritional Biochemicals Co.), globin (purified, Nutritional Biochemicals Co.), ribonuclease (crystallized and chromatographically pure, Schwarz/Mann), delta-chymotrypsin (3x crystallized, Nutritional Biochemicals Co.), and lysozyme (3x crystallized, Sigma Chemical Co.) Polyacrylamide gel electrophoresis. The alternative Davis (3) procedure for polyacrylamide gel electrophoresis was used. The formation of a sample gel was bypassed, and the sample was layered directly on top of the spacer gel before electrophoresis. Electrophoresis was performed on the Buchler polyacrylamide vertical electrophoresis apparatus (Buchler Instruments, Fort Lee, N.J.). In a typical electrophoretic run, a constant current of 4 mA/gel was used. In each run, of the 10 gels (0.5 by 7.0 cm) that were processed, two were stained with amido black (Eastman) and eight remained unstained. Unstained gels (7 cm long) were cut into 2-mm-long sections. Each section was homogenized by means of a hypodermic syringe. After homogenization, the homogenate from each section was mixed with 4.0 ml of maintenance medium and incubated at 4 C for 24 h. The fluid was removed from the mixture, and its total volume was adjusted to 5.0 ml by the addition of maintenance medium. Each 5.0-ml sample was then assayed for activity by the dye uptake method. Other procedures. Protein content was assayed by the method of Lowry et al. (18). The additional biological, biochemical, and biophysical procedures

employed the text.

are

817

described in the appropriate section of

RESULTS Partial purification of RTG-2 interferon. The purification procedures are summarized in Table 1. Gel chromatography. Gel chromatography of crude RTG-2 interferon on Sephadex G-150 resulted in the elution of interferon (activity and protein) in one peak (Fig. 1). The interferon in this peak corresponded to a molecular weight of 94,000 (Fig. 2). 0.50

t 0.40

10

C,,

8 0x

-

4~~~~~~~~~~ E

°0.30

6

cv

c

0

0.10

-2c ~~~~~~z

c..0-

12 Vo

..J

20

16

24

28

FRACTION NUMBER

FIG. 1. Gel chromatography of RTG-2 interferon. A one-ml amount of crude RTG-2 interferon (9,441 interferon DU units and 0.85 mg of protein) was chromatographed on Sephadex G-150 at 5 C. A solution of 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4) containing 0.05 M NaCI was used as the eluent, with a flow rate of 1.0 ml/h. Fractions containing 1.0 ml were collected and assayed for interferon activity in RTG-2 cells using the dye uptake method. Protein concentrations in effluent fractions were determined by absorbancy in a Beckman DU spectrophotometer operated at a wavelength of 280 nm.

TABLE 1. Partial purification of RTG-2 interferon Total protein Purification step

mg

Units

Sp act (units/mg of protein)

Vity

Vol (ml)

Purification factor ( ePer CumulCu la Pe tive step

Tissue culture supernatant

22.5

38.25

212,423

(5,554)

pH 2 x 48 h, 70,000 x g for 2 h

15.0

12.75

141,615

11,107

2.0

2.0

66.7

Gel chromatography of multiple 1-ml samples (total, 15 ml) on Sephadex G-150

15.0

3.30

133,080

40,322

3.63

7.26

62.7

Pooled peak fractions (15) placed on CMSephadex (C-50) column

5.0

0.16

13,260

85,023

2.19

15.87

6.2

a Interferon

activity is defined in DU units.

100

DE SENA AND RIO

818 20

pH stability. Crude and Sephadex G-150purified RTG-2 interferon preparations were fairly stable at pH values of 1, 2, 7, and 10 (Table 3). These interferon preparations lost amounts of at pH 11. The significant gel chromatographically activity purified interferon exhibited a greater degree of sensitivity to pH treatment than the crude interferon preparation. Species specificity. Using the assay system described above, RTG-2 interferon was assayed

ALDOLASE

_RTG-2 \

---RTG-2 1010 IN TERFERON

c OVALBUMIN

OVALBUMIN

x

\4

in RTG-2 cells and goldfish (SJU-1) cells. Crude

and gel chromatographically purified RTG-2 interferon preparations exhibited a significant

CHYMO TRYPSINOGEN A

degree of species specificity (Table 4). These interferon preparations were more active in RTG-2 cells than they were in SJU-1 cells. Crude interferon more active in heterologous cells than gel was chromatographically purified

RIBONUCLEASE AX 0

0.2

0.4

INFECT. IMMUN.

0.6

0.8

1.0

Kav IG. 2. Molecular weight determination of RTG-2 inte?rferon by gel filtration through Sephadex G-150. F

The? logs of the molecular weights of known proteins plotted against their respective Ka, values. The molfecular weight of RTG-2 interferon was determined fronn this standard curve. weroe

/M-Sephadex (C-50) chromatography. Chiromatography of Sephadex G-1150-purified RT 'G-2 interferon on CM-Sephadex (C-50) resultted in the spreading of RTG-2 interferon (ac tivity and protein) over several fractions (Taable 2). The most active RTG-2 interferon fra(ction contained 31.5 ,ug of protein and 2,652 int4 erferon DU units/ml (Table 2). The specific actiivity of 85,023 represented a 2.2-fold purificat ion of Sephadex G-150-purified interferon (Tatble 1). Adding the average isoelectric point devTiation of +0.4, which was calculated from the elution pH of known proteins passed thr ough CM-Sephadex (C-50) columns, to the pH of this peak activity fraction (pH 6.7) gave an estimated isoelectric point of pH 7.1 for RT 'G-2 interferon. 'olyacrylamide gel electrophoresis. Activity coincided with two of the four bands given by Sephadex G-150-purified RTG-2 interferon (Fi1 g. 3 and 4). Efeat stability. Crude and Sephadex G-150pur *ified RTG-2 interferon preparations were fair.*ly stable at 40 and 56 C (Table 3). These intE arferon preparations lost significant amounts of Eactivity at 65 and 75 C. The gel chromatogra Lphically purified interferon exhibited a grekater degree of sensitivity to heat treatment at 65a C than the crude interferon preparation.

interferon. Trypsin, ribonuclease, and deoxyribonuclease sensitivity of RTG-2 interferon. Exposure of gel chromatographically purified RTG-2 interferon to 0.2 mg of trypsin per ml at 37 C for 2 h destroyed 88% of its biological activity (Table 5). Exposure of this material to 0.1 mg of ribonuclease or deoxyribonuclease per ml for 2 h at 37 C did not have any inhibitory effect on interferon activity. Additional characteristics of Sephadex G-150-purified RTG-2 interferon. Fortyeight hours of dialysis at pH 7.2 did not result in a significant loss of interferon activity. The material was not sedimented by centrifugation at 105,000 x g for 2 h at 4 C. Analysis of the upper two-thirds of the supernatant indicated that ultracentrifugation did not significantly result in the loss of interferon activity. RTG-2 interferon was inactivated by exposure to 0.1 M 2-mercaptoethanol. Direct incubation of RTG-2 interferon with 102 83 mean tissue culture infective doses of IHN virus per ml at 4 C for 48 h did not result in the loss of virus activity. RTG-2 interferon afforded RTG-2 cells protection against infectivity with IHN or IPN virus. The interferon activity exhibited against IPN virus was observed to be greater than the activity exhibited against IHN virus. DISCUSSION

The studies presented here demonstrate that RTG-2 cells infected by IPN virus produce an antiviral substance (interferon) which exerts its activity intracellularly. This non-dialyzable, non-sedimentable (105,000 x g) substance was observed to be both heat and pH stable. The destruction of RTG-2 interferon activity by trypsin but not by ribonuclease or

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RTG-2 FISH CELL INTERFERON

VOL. 11, 197'a

TABLE 2. Elution from a CM-Sephadex (C-50) column of Sephadex G-150-purified RTG-2 interferon by means of a rising pH gradient in 0.1 M phosphate buffer pH

Interferon activity (DU units/ml)

15

6.0

8,840

50 40 20

6.0 6.0 60

Partial purification and characterization of RTG-2 fish cell interferon.

INFECriON AND IMMUNrrY, Apr. 1975, p. 815-822 Copyright i 1975 American Society for Microbiology Vol. 11, No. 4 Printed in U.S.A. Partial Purificati...
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