JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1987, p. 1735-17400095-1137/87/091735-06$02.00/0Copyright © 1987, American Society for Microbiology

Induction and Measurement of 2',5'-Oligoadenylate Synthetase inMadin-Darby Bovine Kidney Cells and in Cattlet

EVERETT C. SHORT, JR.,1* AND ROBERT W. FULTON2

Departments ofPhysiological Sciences' and Veterinary Parasitology, Microbiology and Public Health,2 College ofVeterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078

Received 27 January 1987/Accepted 8 June 1987

2',5'-Oligoadenylate [2',5'-oligo(A)J was separated from 14C-labeled nucleosides produced in the 2',5'-oligo(A) synthetase assay by using 100-,ul columns of Dowex 1. No detectable nucleoside remained on thecolumn after elution with 20 column volumes of water, whereas less than 1% of oligonucleotides were elutedfrom the column. At least 99% of oligonucleotides were eluted from the column with 1 M NaCl, pH 2. Themajor product had properties consistent with 2',5'-oligo(A). Exposure to alpha-1 bovine interferon (IFN)caused an increase in cellular 2',5'-oligo(A) synthetase activity which was proportional to the concentration ofIFN in the medium up to 104 U of IFN per mi and then leveled off at about 15 x control activity. Under the assayconditions used, 2',5'-oligo(A) synthetase activity was directly proportional to the amount of cell extract overa 10-fold range. Cattle inoculated with IBR/BVD/PI-3 modified live virus vaccine showed an increase in2',5'-oligo(A) synthetase activity in peripheral blood mononuclear leukocytes which persisted for at least 3 dayspostvaccination. Intramuscular injection of cattle with IFN caused a similar increase in 2',5'-oligo(A)synthetase activity. Changes in 2',5'-oligo(A) synthetase activity should be of value in (i) assessing the responseof cattle to experimental viral infections or inoculations with viral vaccines or IFN or (ii) indicating a possibleviral etiology in disease.

Interferons (IFNs), which are proteins produced andsecreted by virus-infected cells, can cause the establishmentof an antiviral state in uninfected cells (1). Because IFN isformed primarily in response to viral infection, its detectionin the body can be a useful diagnostic aid in viral diseases.However, the formation of IFN in cells is a short-termprocess (43) and its clearance from the blood is rapid (9),making direct measurement of IFN in serum difficult. Con-sequently, the sera of a large proportion of humans ordomestic animals with viral infections may not containdetectable IFN. All three types of IFN (cx, P, and -y) have thecapacity to induce the enzyme 2',5'-oligoadenylate [2',5'-oligo(A)] synthetase, which was discovered by Kerr et al.(19) and which is important in the development of anantiviral state in cells (33). The enzyme 2',5'-oligo(A) syn-thetase is considered a reliable marker of IFN action in vitro(8, 10, 13, 14, 18, 19, 32, 35, 45) and in vivo (4, 17, 21, 30, 31,34, 38, 39). Schattner et al. (38) have shown that elevation ofthe enzyme level in the peripheral blood mononuclear leu-kocytes (PBML) increases early in viral infection and re-mains elevated for a prolonged time. It has been shown that2',5'-oligo(A) synthetase activity is increased in PBMLduring virus infection (2, 4, 7, 20, 22, 37, 38) or treatmentwith IFN (20, 23, 26) and that the increased activity persistsfor at least 24 h after administration of IFN (26). Activity of2',5'-oligo(A) synthetase has therefore been measured todetermine whether humans and other animals have beeninfected with virus or are responding to IFN (4, 7, 12, 23, 25,26, 37, 40, 44). Viral diseases in cattle cause major economiclosses throughout the world. Present diagnostic tests fordetecting viral infections in cattle are time-consuming andcostly. A method of early and rapid detection of suchdiseases would be of considerable value in veterinary med-

* Corresponding author.t Journal article 5146 from the Oklahoma Agricultural Experi-

ment Station.

icine. In addition, IFN is being tested as a prophylactic agentfor bovine respiratory diseases. An assay of the biochemicalresponse of animals given IFN would be a useful adjunct totheir clinical responses to administration of IFN. Assays for2',5'-oligo(A) synthetase in PBML have been shown to bemore rapid, sensitive, and reliable than measurements ofIFN in the sera of humans. We have, therefore, examined2',5'-oligo(A) synthetase activity in Madin-Darby bovinekidney cells and in PBML of cattle to test its potential valueas an indicator of viral infection or response to IFN in cattle.

MATERIALS AND METHODS

Abbreviations used in this work are IFN, interferon;PBML, peripheral blood mononuclear leukocytes; SVPD,snake venom phosphodiesterase; AP, alkaline phosphatase;MDBK, Madin-Darby bovine kidney; PBS, phosphate-buffered saline; 2',5'-oligo(A), 2',5'-oligoadenylate; CE, cellextract; and ApA, adenylyl(3'--5')adenosine.

Materials. Agarose-poly(I)- poly(C) and poly(I) poly(C)were purchased from Pharmacia P-L Biochemicals, Pis-cataway, N.J. [8-'4CJATP was purchased from ICN Phar-maceuticals Inc., Irvine, Calif. 3',5'[8-3H]poly(A) was pur-chased from Amersham Corp., Arlington Heights, Ill.Bovine IFN-a,1, synthesized in Escherichia coli, was ob-tained from Genentech, Inc., South San Francisco, Calif.Bacterial AP, Pl nuclease, and SVPD (Crotalus atrox) wereobtained from Sigma Chemical Co., St. Louis, Mo. Dowex1x2, 100 to 200 mesh, was washed alternately with 20 bedvolumes of 1 N HCl-water, and 1 N NaOH-water, and thewash cycle was repeated five times (29). A final wash with 1N HCI followed by distilled water converted the resin intoCl- form.

Animals. Calves of mixed breeding, weighing 100 to 300 kgeach, were obtained from commercial sources. They weremaintained on free-choice hay and grain supplement andwere clinically normal throughout each experiment.

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TABLE 1. Susceptibility of assay product to enzymatic digestion

'7 of assay proditct eluted from I)owex 1with CE and client:

Enzyme utsedin digestion MDBK ceils wAithout IFN MIDBK ceils wvith IFN

HO HCI-NaCI H`O HCI-NaCI

AP 98.1 1.9 53.8 46.2SVPI) 98.8 1.2 98.7 1.2Pi nuclease 98.( 2.0 54.2 45.8

Cell extracts. Confluent monolayer cultures of MDBKcells were grown in Eagle minimum essential medium withEarle salt solution, 2 mM glutamine, 10% fetal bovine serum,100 IU of penicillin per ml, and 100 .'g of streptomycin perml. Confluent monolayers were exposed to IFN for 24 h.IFN was added in the same medium as above, except thatfetal bovine serum was reduced to 5%c. Before harvestingwas done, monolayers were washed twice with 100 p.I ofPBS per cm . Monolayer cells were removed by adding 50 x1d

of PBS per cm2 of monolayer and scraping. The culturevessel was rinsed once with the same volume of PBS, andthe washings were added to the scraped-cell suspension.Cells were sedimented at 300 x g for 3 min, suspended in an

Eppendorf microtest tube in 10 to 100 of PBS per cm2 ofmonolayer, and resedimented at 300 x g for 3 min. Cellswere lysed by suspension in buffer containing the detergentNonidet P-40, as described by Schattner et al. (38), andstanding at 4°C for 30 min. After centrifugation at 12,000 X

g for 5 min, the CE was removed and used immediately forthe enzyme assay or kept frozen at -10°C.Bovine PBML were collected from 2 ml of blood as

described by Schattner et al. (38), except that centrifugationon the Ficoll-Hypaque cushion was at 800 x g for 30 min.CE was prepared as described above at a concentration of107 cells per ml.

Activity of 2',5'-oligo(A) synthetase. Assay conditions were

as described by Schattner et al. (38) except that [8-14C]ATPinstead of [K 3r2P]ATP was used as the substrate. Afterincubation for about 20 h, digestion with AP, and dilutionwith 25 p.l of water, assay vessel contents were applied to a

column of Dowex 1 (CI-) 3 mm in diameter and 13 mm inheight. The column was eluded with 2 ml of water, and theeluent was collected and counted as a single fraction in 15 mlof Scintiverse (Fisher Scientific Co., Pittsburgh, Pa.). Thecolumn was then eluded with 2 ml of 0.01 M HCI-i M NaCI,which was collected and counted as described above.

Oligonucleotide digestion. Enzyme digestions were doneunder the conditions described by Brownlee (3) for SVPDand by Rose (36) for Pi nuclease.

Analytical procedures. Cells were counted by using a

hemacytometer. Dinucleoside monophosphates and nucleo-sides were measured by A,57 at pH 2.

RESULTS

Chromatography on Dowex 1. ApA and adenosine weredissolved separately in medium used for assay of 2',5'-oligo(A) synthetase (38) to give concentrations of 10 and 2.5mM, respectively. ApA (10 pi) and adenosine (10 pl) were

applied separately to 100-pLi columns of Dowex 1. Eachcolumn was eluded with 2 ml of water followed by 2 ml of0.01 M HCI-i M NaCi. The amount of dinucleoside mono-phosphate or nucleoside in each fraction was measured byabsorbance at pH 2 (the millimolar extinction coefficient at

257 nmn was taken as 14.6 for adenosine and 30.2 for ApA).Less than 1% of ApA (A'57, 0.003) was eluted with water,and 99%c was eluted with HCI-NaCl. Adenosine was 99.9%eluded with water, and no detectable concentration (AI57-O.001) was eluded with HCl-NaCI.Susceptibility of 2',5'-oligo(A) assay product to enzymatic

digestion. Assays were done in duplicate as described inMaterials and Methods, using in each vessel CE from 105MDBK cells not exposed to IFN or MDBK cells exposed toIFN for 24 h. After incubation for 20 h, individual assayvessels were subjected to one of the following enzymatictreatments. (i) AP was added, and incubation was continuedfor 1 h according to standard assay procedure. (ii) SVPD(0.02 U) was added along with 5 p.mol of Tris hydrochloride,pH 7.5, and 2.5 ,umol of MgClI; incubation was continued for3 h, AP was then added, and incubation was continued for 1h. (iii) Pi nuclease (1 p.g) was added along with 0.7 itmol ofBis-Tris hydrochloride, pH 6.0; incubation was continuedfor 3 h, AP was then added, and incubation was continuedfor 1 h. Ail vessels were adjusted to a final volume of 80 iiland kept on ice until the contents were analyzed by chro-matography on Dowex 1 as described under activity of2',5'-oligo(A) synthetase in Materials and Methods. Theresults are shown in Table 1.

In a parallel experiment, 6.13-fig (4.5-nCi) portions of3',5'-poly[8-3H]adenylate (average degree of polymeriza-tion, 80) were subjected to digestion by AP alone and APwith SVPD or Pi nuclease. The digests were subsequentlychromatographed on Dowex 1 under the conditions de-scribed above. In this experiment, AP converted no measur-able amount of the 3',5'-poly(A) to nucleosides, whereas APwith SVPD converted 87%s to nucleosides and AP with Pinuclease converted 87%7! to nucleosides.

Effect of IFN on 2',5'-oligo(A) synthetase activity in MDBKcells. Assay vessels were prepared by using CE frommonolayers of MDBK cells not exposed to IFN and MDBKcells exposed to 1(5 U of IFN per ml of medium. Radioac-tivity of contents of assay vessels was measured directly orthe contents were subjected to chromatography on Dowex 1,and the radioactivity of water- and HCI-NaCl-eluted frac-tions was measured. Detectable radioactivity retained on thecolumns was less than 0.1% of that applied (15 pCi). CE from105 MDBK cells catalyzed the synthesis of 20 ± 8 pmol of2',5'-oligo(A) per h (n = 4). CE from MDBK cells exposed toIFN for 24 h catalyzed the synthesis of 373 + 26 pmol of2', 5'-oligo(A) per h (n = 4).

Effect of concentration of IFN on induction of 2',5'-oligo(A)synthetase. MDBK cells were exposed to IFN at concentra-tions of 2 x 10"' to 2 x I07 U/ml of culture medium. IFNcaused 2',5'-oligo(A) synthetase activity of cells to increaseas a function of IFN concentration (Fig. 1). Each data pointrepresents the average of duplicate experiments except thepoint for unexposed cells, which represents four replicates.The relative increase in 2',5'-oligo(A) synthetase of IFN-treated cells compared with untreated cells ranged from3-fold at 2 U of IFN per ml to 15-fold at 2 x 104 U of IFN perml.

Relationship between 2',5'-oligo(A) synthesis and theamount of CE. MDBK cells were exposed to IFN at aconcentration of 2 x 105 U/ml of culture medium. Theamount of 2',5'-oligo(A) produced under the conditions ofthe assay was proportional to the amount of CE added, up toan incorporation rate of 500 pmol/h (Fig. 2), which corre-sponds to incorporation of about 35% of the ATP substratein the assay vessel during an 18-h incubation. The rate ofincorporation of 2',5'-oligo(A) synthesized per microliter of

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500LE 450

400o 350 fE

9 300250 f

o 200

-- 150o

I 100Ln

50

-°o 1.3 3.3 5.3 7.3IFN (log U/ml)

FIG. 1. Effect of concentration of IFN on 2',5'-oligo(A) synthe-tase activity of MDBK cell extracts. Cells grown on 24-well plates(201 mm2 per well) were treated with the indicated amount of IFNfor 20 h. The contents of two wells were combined, and cells werewashed twice with 200-pi portions of PBS and lysed with 25 pli oflysis buffer. Assays were done by using 20 p1 of CE in each vessel.Incubation was for 17 h. Products were separated and measured asdescribed in Materials and Methods.

CE was reduced at higher rates of incorporation. In fourdifferent experiments, the slope of the line relating theamount of CE and synthesis of 2',5'-oligo(A) was linearbelow levels of incorporation of 30 to 35%. Correlationcoefficients were 0.985 to 0.999. The slope of the lineobtained by using CE prepared from MDBK cells notexposed to IFN was about 10% of that obtained by using CEfrom cells exposed to IFN (Fig. 2).

2',5'-Oligo(A) synthetase levels in PBML of cattle inocu-lated with modified live virus vaccine or IFN. Two animalswere injected intravenously with a commercial IBR/BVD/PI-3 modified live virus vaccine; two animals served as con-trols. Samples of blood were withdrawn into heparinizedtubes from both animals 24 h before the injection of vaccine,at the time of injection, and at 24-h intervals after injectionfor measurement of 2',5'-oligo(A) synthetase. Blood (2 ml)was mixed with 2 ml of PBS, layered on a 3-ml cushion ofFicoll-Hypaque, and centrifuged at 800 x g for 30 min atroom temperature. Cells at the interface were collected,washed twice with 5-ml portions of PBS, and sedimentedeach time by centrifugation at 500 x g for 5 min. The cellpellet was suspended in 100 pi of PBS. A 10-pul portion of thesuspension was used for counting cells. The remaining cellswere pelleted as above and Iysed in 200 pul oflysis buffer (38).Assays for 2',5'-oligo(A) synthetase were done as describedabove. Results are shown in Fig. 3. Standard deviations forpaired samples are shown for all samples except for the 24-hvaccinated group; the second sample for that group was lostduring laboratory preparation of CE.Two animals were injected intramuscularly with 106 U of

IFN per kg of body weight. One animal served as a control.CE was prepared from leukocytes, as described above, fromblood which was withdrawn at 0, 24, 48, and 96 h afterinjection. Results are shown in Fig. 4.

DISCUSSION

Dowex 1 ion-exchange resin was selected for separation of2',5'-oligo(A) from nucleosides in this work because it

900

800

- 700* I/O 600rE

',-e 500

O 400

o 300

Ln 200

100

0 5 10 15 20 25 30 35 40Cel1 Extract (pl)

FIG. 2. Relationship between synthesis of 2',5'-oligo(A) andamount of CE. CE was prepared from MDBK cells not exposed toIFN (M) and MDBK cells exposed to IFN (El). Assay vesselvolumes were adjusted with medium to compensate for variations inthe volumes of CE.

separates oligonucleotides from nucleosides produced by thephosphatase treatment of ATP and hence permits the use of'4C-labeled nucleotide precursors of oligo(A). Chromatogra-phy on alumina (24), which is used extensively in the2',5'-oligo(A) synthetase assay, separates 32P released by thephosphatase treatment from the phosphatase-resistant coresof oligo(A)s. Dowex 1 retained no detectable nucleosidesand passed less than 1% of dinucleotides with water as theeluent. Less than 1% of radioactivity applied to columns

50

° 45rl_.o 40's..(n 35

30o: 25

20

3-' 15oID 10-J0 5

u.)o`

-1 0 1 2TIME (Days)

3

FIG. 3. Effect of vaccination of cattle with IBR/BVD/PI-3 mod-ified live virus vaccine on 2',5'-oligo(A) synthetase activity ofPBML. CE was prepared from PBML of unvaccinated controlanimals (A) and vaccinated animals (El).

Injection

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1738 SHORT AND FULTON

--

o

t1)

ovc-'

on

Ec:

Lfl(N

60555045403530252015105O0

O 1 2TIME (days)

3

FIG. 4. Effect of intramuscular injection of lFN into cattle on

2',5'-oligo(A) synthetase activity of PBML. CE was prepared fromPBML of uninjected control animals (A) and animals injected with1FN (O).

during regular assay was retained after elution with waterand HCl-NaCl, pH 2. Under the conditions used in theseexperiments, Dowex 1 effectively separated nucleosidesfrom oligonucleotides. Chromatographic properties of largeroligonucleotides were not examined because they bind more

strongly than ApA to Dowex 1 and are eluted by 0.01 MHCl-1 mM NaCl (29).Exposure of MDBK cells in tissue culture to 105 U of IFN

per ml of medium increased the level of 2',5'-oligo(A)synthetase about 20-fold over the level in unexposed cells.This increase is comparable to that described for HeLa cells(6, 15) and for Georgia bovine kidney cells (28).The retention of the AP-resistant material from the assay

on Dowex 1 columns combined with its susceptibility todegradation by SVPD and its resistance to degradation by Pinuclease are consistent with a reaction product of 2',5'-oligo(A) (16, 19). When similarly tested on 3',5'-poly(A), APalone converted no measurable amount to nucleosides,whereas AP combined with either SVPD or Pi nucleaseconverted most of it to nucleosides, thus demonstrating thatthe enzyme preparations were active on 3',5'-linked poly-mers.

Cell extracts prepared from bovine kidney cells exposedto different concentrations of IFN (2 to 2 x 107 U/ml)contained different amounts of 2',5'-oligo(A) synthetaseactivity, exhibiting a rough proportionality between concen-

tration of IFN and amount of 2',5'-oligo(A) synthetaseactivity up to about 2 x 104 U of IFN per ml of medium.These results are consistent with those of Minks et al. (27)and Vanden Broecke (41), except that the proportionalitybetween concentration of IFN and amount of 2',5'-oligo(A)synthetase appeared to continue to a higher concentration ofIFN (2 x 104 U/ml in this work, compared with 1 x 102 to 1x 103 in the cited works, respectively). No attempt has been

made to determine the cause for the differences; they could,however, be caused by the different cell lines that were used.The proportionality between the amount of cell extract usedin assays and the amount of 2',5-oligo(A) synthesized dem-onstrates that enzyme activity can be comparably measuredover a severalfold range of enzyme concentrations.

The level of 2',5'-oligo(A) synthetase has been shown torise significantly in PBML and granulocytes of animalsinfected with viruses (38) or injected with IFN (42) and inpeople suffering from viral, bacterial, or autoimmune dis-eases (4, 7, 22, 25, 44) or receiving IFN therapy (22, 23, 25,26, 39). An assay of 2',5'-oligo(A) synthetase has beensuggested for use in assessing the state of disease andrecovery of patients suffering from viral or bacterial infec-tions (4, 7, 44); for confirming or excluding a possible viraletiology in acute diseases for which alternative nonviral ornoninfectious diseases could be considered (7, 37, 38, 44);for indicating a possible viral or autoimmune etiology indiseases of obscure pathogenesis (37); for monitoring re-sponse to IFN therapy (22, 25, 26, 37); and as an aid indetermining which patients are likely to respond to IFNtherapy (22). In addition, 2',5'-oligo(A) synthetase assaysshould be of value in assessing prophylactic use of IFN. Weare especially interested in the 2',5'-oligo(A) synthetasesystem for experimental evaluation of IFN for prophylaxisagainst bovine respiratory diseases and for early and rapiddetection of possible viral infections of cattle. Although the2',5'-oligo(A) synthetase system is not specific for virusdiseases, the relative magnitude of the response to virusinfection (7, 11, 37) and the rapid determination of results(less than 24 h), combined with history and clinical signs,should make this a useful diagnostic aid (4, 7, 37, 38).PBML of calves inoculated with a modified live virus

vaccine showed an increase in 2',5'-oligo(A) synthetasewhich persisted for at least 3 to 4 days. These results areconsistent with those reported by others (37, 38, 42). Anal-ysis of variance for combined data for postinoculation days1, 2, and 3 showed that the difference between control andinoculated animals was highly significant (P < 0.01). Theaverage increase in 2',5'-oligo(A) synthetase activity inPBML of calves inoculated with modified live virus vaccinewas threefold.

Calves injected with IFN showed an average 1.6-foldincrease in PBML 2',5'-oligo(A) synthetase activity over thecontrol calf. Although the difference between the control andexperimental calves on a given day may not appear to besignificant, analysis of variance for combined data forpostinoculation days 1, 2, and 3 showed a significant differ-ence (P < 0.05). Injection with larger amounts of IFN mayhave increased the degree of response; the amount chosen,however, was selected because it corresponds to the dosagecurrently suggested for clinical applications in cattle. Fur-thermore, this dose rate is comparable to dose rates thathave been shown by Merritt et al. (25) to induce 2.3- to 5.7-fold increases in 2',5'-oligo(A) synthetase activity in PBMLof human subjects. Observations similar to those reportedherein have been reported for virus-infected mice (38, 39),and human patients with virus infections (4, 7, 22, 37) orsubjected to treatment with IFN (23, 26). All animals in theIFN experiment evidenced somewhat higher 2',5'-oligo(A)synthetase levels than did those in the vaccine experiment.On the basis of our experience, we believe that two factorsmay have contributed to the difference. (i) The IFN experi-ment was done during a period of very hot weather, withdaily temperatures above 39°C, which caused stress to theanimals. (ii) Animals in the IFN experiment may have beenmore distressed by handling. Glucocorticoids, which areproduced during stress, have been shown to affect IFN-induced enzymes (5).

This study suggests that the 2',5'-oligo(A) synthetaseassay may be useful in confirming or excluding a possibleviral etiology in acute diseases and in evaluating response of

IfnJected

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BOVINE 2',5'-OLIGO(A) SYNTHETASE 1739

cattle to prophylactic use of interferon or vaccines as well asto viral challenge.

ACKNOWLEDGMENTS

We thank Lurinda J. Burge, Frederick Frye, Jr., and Louis J.Perino for their excellent technical assistance.

This work was supported by the Oklahoma Agricultural Experi-ment Station projects OK 1438, 1725, and 1990.

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