Distinct molecular species of interferons

VIROLOGY

61, 80-86

(1974)

Distinct

Molecular WILLIAM

Department

of Virology,

Rega Institute

of lnterferons

E. STEWART

for Medical Accepted

Dedicated

Species

Research, April

II University

of Leuuen, Leuuen, Belgium

30, 1974

to the memory of Professor Dallas, Texas

S. Edward

Sulkin

Interferons induced in mouse L,,, cells could be renatured from the protein-dissociating system of sodium dodecyl sulfate (SDS), mercaptoethanol, and urea after boiling at 100”. Therefore, SDS-polyacrylamide gel electrophoresis under reducing conditions was used to analyze mouse L cell interferons. Preparations, crude or partially purified, contained two well-separated molecular species of interferon: a major component (about 90% of the total activity) at 38,000 daltons and a minor component (about 10% of the total activity) at 22,000 daltons. That the smaller component was not a subunit of the larger component was evident from the finding that, while the amount of activity of the smaller species recoverable was essentially unchanged whether the preparations were reduced or not, the amount of activity of the larger species recoverable was greatly increased (about 10.fold), rather than decreased, under reducing conditions. Also, when the interferon eluted from unreduced gels at 38,000 daltons was boiled in SDS, mercaptoethanol, and urea, latent activity was “unmasked” giving an increase of about lo-fold in activity. The data indicate that mouse L cell interferon preparations contain two molecular species of interferon: a smaller species renaturable with or without reduction and a larger and predominant species requiring disruption of disulfide bonds for efficient renaturation.

INTRODUCTION A vast and contradictory body of literature has accumulated on the molecular sizes of interferons (Ng and Vilcek, 1972; Weil and Dorner, 1973), reporting an array of molecular sizes from a given animal species; the size of interferon seemingly depending on the types of cells induced, on the type of inducer, or on several ill-defined variables. To date, however, it has not been resolved whether the various molecular sizes of interferons from a given animal species are, in fact, different molecular species, that is, differing in their primary structures. One interpretation, prompted by the near-multiple relationships in sizes of some interferons of the same animal species, is that basic subunits of the interferons aggregate to form oligomers. Indeed, Carter and his associates (Carter, 1970; Carter and Pitha, 1971; Marshall, Pitha, and 80 Copyright All rights

0 1974 by Academic Press, of reproduction in any form

Inc. reserved.

Carter, 1972; Pitha, Marshall, and Carter, 1973) have persisted in reporting that mouse and human interferons with molecular weights of 38,000 and 24,000, respectively, could be dissociated, albeit only partially, to “monomers” of 19,000 and 12,000 molecular weight, respectively, merely by dialysis against low ionicstrength buffer; they have, therefore, espoused the idea that the apparently different molecular weights of interferons are simply different oligomeric species of a basic interferon subunit. However, as has been succinctly pointed out by Weil and Dorner (1973), several data appear to be at variance with this unifying explanation of size polymorphism, and considering the physicochemical variabilities encountered in even homologous interferons (Ke and Ho, 1967, 1968; Falcoff, 1972; Edy, Billiau, and De Somer, 1974), it is more tempting to speculate that primary structural differences exist.

MOLECULAR

SPECIES

Recently, it was found that mouse L cell interferons could be renatured after boiling in a solution of sodium dodecyl sulfate (SDS), mercaptoethanol, and urea (Stewart, De Clercq, and De Somer, 1974; Stewart, De Somer, and De Clercq, 1974). Since there are no known protein aggregates linked by noncovalent bonds that survive treatment with SDS at 100” for 1 min (Maizel, 1970), the present studies were undertaken, using the technique of electrophoresis in SDS-polyacrylamide gels, to determine the basic molecular unit(s) of dissociated and reduced interferons. The data reported here are incompatible with the interpretation that there is a single basic subunit for mouse interferon. MATERIALS

AND

METHODS

Interferon preparation and assay. Mouse interferons were induced in L,,, cell monolayer cultures with Newcastle disease virus at a multiplicity of 10 plaque-forming units/cell in serum-free Eagle’s minimal essential medium (MEM). Medium harvested at 18 hr postinoculation was adjusted to pH 2 at 4” for several days, and clarified supernatant fluids obtained after centrifugation at 1000 g were adjusted to pH 7.2 by dialysis against 0.01 A4 phosphate buffer and were stored at -70” as crude interferon stock with a specific activity of about lo4 NIH mouse reference interferon units/mg of protein. Partially purified interferon preparations were prepared from crude preparations by precipitation of extraneous proteins by addition of ammonium sulfate to 20% saturation at pH 2 and room temperature. Clarified supernatant fluids obtained after centrifugation at 1000 g were adjusted to pH 7.2 by dialysis against 0.01 M phosphate buffer and were stored at -70” as partially purified interferon stock with a specific activity of about lo6 NIH mouse reference interferon units/mg of protein. Interferons were characterized as being sensitive to trypsin, active against several viruses on mouse cells but relatively inactive on human and rabbit cells, and unable to induce antiviral activity in actinomycin D-treated L cells. Interferons were assayed by plaque-reduction assays against vesicular stomatitis

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OF INTERFERONS

virus on L,,, cell monolayer cultures as described elsewhere (Stewart, De Clercq, and De Somer, 1972b); 1 unit is equal to approximately 2 NIH mouse reference units. Polyacrylamide gel electrophoresis (PAGE) of interferons. Samples in glass

tubes were constituted to contain 1% SDS or 1% SDS, 1% mercaptoethanol and 5 M urea, heated to 100” for 1 min by immersion in boiling water, and O.l-ml aliquants were subjected to electrophoresis on SDS-polyacrylamide gels by the technique of Summers, Maizel, and Darnell (1965), using 6 mA per 20 cm of gel for 24-28 hr. Bromphenol blue in sucrose was added to make samples 10% sucrose prior to electrophoresis; electrophoresis was stopped when bromphenol blue had migrated about 15 cm into the gels, and gels were scanned by ultraviolet light (280 nm) for location of marker proteins. Gels (10% polyacrylamide) were preelectrophoresed for at least 2 hr to remove excess persulfate. Electrophoresis buffer was 0.1 M phosphate, pH 7.2, containing 0.1% SDS (and in some experiments also 0.1% mercaptoethanol). Protein markers used to calibrate protein mobility were bovine serum albumin, ovalbumin, chymotrypsinogen, and cytochrome c (BioRad Laboratories, Richmond, CA) and human -y-globulin (Calbiochem, San Diego, CA). Reduced samples were calibrated against gels simultaneously electrophoresed with reduced markers, and unreduced samples were calibrated against gels simultaneously electrophoresed with unreduced markers. Gels, extruded from electrophoresis tubes by pushing from the bottom with a tight-fitting glass rod, were sliced into O.&cm segments which were pulverized in 2 ml of MEM containing 10% calf serum. After elution at room temperature overnight, samples were assayed for interferon. RESULTS

Indirect evidence for molecular heterogeneity: differential requirements for partial and complete renaturation of interferon preparations. Mouse L cell interferons were

previously reported (Stewart, De Clercq, and De Somer, 1974) to renature only

82

WILLIAM

E. STEWART

partially after being denatured in SDS alone, but to renature completely after being denatured in SDS, mercaptoethanol, and urea. As shown in Table 1, this relationship applied equally to the crude and partially purified preparations used here. Interferon samples were dialyzed overnight against sodium phosphate buffer, buffer containing SDS, or buffer containing SDS, mercaptoethanol, and urea; in other experiments, interferon samples were adjusted to 1% SDS, 1% mercaptoethanol, and 5 M urea by direct addition of the reagents to the samples in glass tubes. Aliquots (1 ml) in glass tubes were then immersed in boiling water for 1 min. Samples were then diluted 1: 10 with MEM containing 10% calf serum and were assayed for interferon activity. While the preparations exhibit only partial activity after treatment with SDS, they exhibit full activity after treatTABLE

1

DIFFERENTIALEFFECTS OF SDS ON MOUSE L-CELL INTERFERONPREPARATIONSUNDER REDUCINGAND NONREDUCINGCONDITIONS Treatment

r

Interferon

titer

Crude interferon

4.8 :2.5 4.0 4.3 5.0

units/ml)

Partially purified interferon I

II

5.0 <2.5 4.0 4.0 4.7

5.3 <2.5 4.5 4.8 5.1

5.0 <2.5 4.3 4.5 5.0

5.0

5.3

5.0

P None 100” SDS SDS + 100” SDS + mercaptoethanol + urea SDS + mercaptoethanol + urea + 100”

(log,,

0 Indicated interferon preparations were dialyzed overnight at room temperature against more than 100 vol of 0.01 M phosphate buffer,‘pH 7.2, containing indicated reagents; SDS, 1%; mercaptoethanol, 1%; urea, 5 M. Samples heated at 100” for 1 min by immersion in boiling water. b Indicated interferon preparations were constituted to contain the indicated reagents by addition of 10% SDS solution, 10% mercaptoethanol solution, and crystalline urea to final concentrations of SDS I%, mercaptoethanol 196, and urea 5 M, indicated samples were then heated by immersion in boiling water.

II

ment with SDS, mercaptoethanol, and urea. In the presence of SDS, or SDS, mercaptoethanol, and urea, the interferons are stabilized against inactivation by heat; however, upon addition of protein-rich medium (which presumably allows interferon to renature by removing the SDS from the interferon protein by an exchange process) the interferon activity again becomes heat labile. These data suggest either: (A) that the interferon preparations are homogeneous and contain a single molecular species that can renature to a certain degree from cross-linked peptide chain, but that can renature more efficiently from a reduced conformation; or (B) that there are two distinct molecular species of interferons in the preparations, one species containing disulfide bonds that must be disrupted for efficient renaturation to occur, and one species renaturable from detergent alone (perhaps lacking essential cross links). SDS-PAGE of unreduced interferon preparations. These experiments showed

that interferon activity was recoverable after boiling in SDS. Since no protein aggregates linked by noncovalent bonds are known that will remain aggregated after such treatment (Maizel, 1970), this afforded the possibility for determining the basic molecular unit(s) of dissociated interferons. Both crude and partially purified interferon preparations were constituted to contain 1% SDS, were boiled for 1 min, and were subjected to electrophoresis on SDS-polyacrylamide gels. Absence of reduction of disulfide bonds was monitored by the use of human y-globulin marker. Eluates were assayed directly for interferon activity, as the SDS presumably is removed from the eluted SDS-interferon by the protein in the eluting MEM containing 10% calf serum. Evidence that the SDS-interferon complex is dissociated, is that while the SDS-interferon complex is heat stable (Table l), the interferon activity becomes heat labile after addition to a protein-containing medium. As shown in Fig. 1, two bands of approximately the same amount of activity were eluted from gels of both crude and partially purified preparations, one with a peak of activity at

MOLECULAR

SPECIES

about 38,000 daltons and one with a peak of activity at about 22,000 daltons. The total amount of recovery in each gel was only about 10% of the total activity of the interferon preparation prior to boiling in SDS, but was about 100% of the renaturable activity of the SDS-boiled samples applied to the gels. SDS-PAGE of reduced interferon preparations. The results with unreduced samples suggested that the activity at the 22,000-MW range might represent a monomer of the activity at 38,000; the latter, if unreduced and containing cross links, might be expected to migrate somewhat faster than predicted for a dimeric molecular weight of 44,000 (Dunker and Rueckert, 1969). This would imply that the smaller unit was more active than the inefficiently renatured “dimeric” form, presumably composed of intermolecularly bonded “monomeric” units. Samples were, therefore, constituted to contain 1% SDS, 1% mercaptoethanol, and 5 M urea prior to heating at 100” for 1 min and were then subjected to electrophoreses on SDSpolyacrylamide gels. Efficiency of disulfide reduction was monitored by the disappearance of y-globulin marker and appearance of H- and L-chains generated from the y-globulin. As shown in Fig. 2, the activity again

io FRACTION

83

OF INTERFERONS

eluted from gels in two bands, at 38,000 and at 22,000. The amount of activity of the smaller species was not increased compared to that recovered from unreduced samples; in fact, it was slightly but (in several experiments) consistently diminished in reduced gels. However, the amount of activity recovered at 38,000 was increased about lo-fold over that recoverable from unreduced samples. Total recovery from reduced gels was about 100% of the total activity of the interferon preparations prior to boiling in SDS, mercaptoethanol, and urea, which was also about 100% of the renaturable activity of the reduced, SDS-boiled samples applied to the gels. In other experiments, identical results were obtained when mercaptoethanol was included in the buffer tanks during electrophoresis; this is in agreement with the finding of Fish, Reynolds, and Tanford (1970) that reduced samples in SDS stay dissociated after removal of the reducing agent. “Unmasking” of inactive interferon conformations eluted from unreduced gels. These data show that there are two molecular populations of interferon in the preparations: one of 38,000 and one at 22,000. The finding that reduction of the samples increased the amount of activity of the larger species suggested that either: (A) the

2’0 NUMBER

3’0

FIG. 1. SDS-polyacrylamide gel electrophoresis of unreduced mouse L-cell interferon preparations. Samples of crude interferon (-0-) or partially purified interferon (-0-l preparations containing approximately IO” units/ml were constituted to contain 1% SDS and were heated at 100” for 1 min, and 0.1-ml aliquots were electrophoresed as described in Materials and Methods. Molecular weight standards (-x-): y-glob, human y-globulin; BSA, bovine serum albumin; oval, ovalbumin; chymo, chymotrypsinogen; cyto, cytochrome c. Migration left to right.

84

WILLIAM

E. STEWART

10 FRACTION

20 NUMBER

II

30

FIG. 2. SDS-polyacrylamide gel electrophoresis of reduced mouse L-cell interferon preparations. Samples of crude interferon (-0-l or partially purified interferon (-0-l preparations containing approximately lo5 units/ml were constituted to contain 1% SDS, 1% mercaptoethanol, and 5 M urea and were heated at 100” for 1 min, and O.l-ml aliquots were electrophoresed as described in Materials and Methods.

larger interferon cannot efficiently renature from an unreduced conformation; or (B) the majority of this species is inactively bound to other molecules, from which it is liberated by disruption of disulfide bonds. If the former were the case, material eluted from unreduced gels at 38,000 should consist of predominantly inactive conformations of interferon. This being the case, it should be possible to “unmask” the latent activity of these denatured molecules by renaturing them from SDS under reducing conditions. Therefore, samples eluted from unreduced gels were constituted to contain 1% SDS, 1% mercaptoethanol, and 5 M urea and were immersed in boiling water for 1 min; samples were then dialyzed overnight against 0.01 M sodium phosphate buffer, pH 7.2, to remove excess reagents and were diluted in MEM containing 10% calf serum and assayed for interferon activity. As shown in Fig. 3, the activity recoverable from unreduced gels at 38,000 MW was significantly increased by such treatment, whereas activity levels at other positions were essentially unchanged.

the larger molecule is a dimer of the smaller “subunit.” However, it becomes untenable to argue that a dimer so labile as to dissociate into monomers merely by exposure to low ionic-strength buffer (Carter, 1970; Carter and Pitha, 1971; Marshall, Pitha, and Carter, 1972; Pitha, Marshall, and Carter, 1973) would not dissociate in boiling SDS solution (which treatment should disrupt all noncovalently bonded molecules; Maizel, 1970). Interchain disulfide-bonded subunits would remain associated in boiling SDS; however, the finding that the amount of the smaller interferon species did not increase upon reduction in SDS, while the activity of the larger molecule greatly increased under these conditions (and the activity stayed at 38,000), shows that the disulfide bond is intramolecular, rather than intermolecular, and must be reduced for efficient renaturation. The results, thus, show that mouse L cells produce two distinct molecular populations of interferons. While the total activity of the preparations is seemingly accountable in these two populations, the possibility cannot be ruled out that there DISCUSSION are other molecular species which do not The near-multiple relationships of the renature after treatment with boiling SDS sizes of the two interferons in mouse L cell or boiling SDS under reducing conditions. interferon preparations (-20,000 and Indeed, the smaller species of interferon is -40,000) might tempt the speculation that somewhat less efficiently recovered from

MOLECULAR

SPECIES

10 FRACT

20 I ON

85

OF INTERFERONS

30 NUMBER

FIG. 3. Effect of reduction on activity eluted from SDS-polyacrylamide gels electrophoresed with unreduced mouse L-cell interferon. Samples of partially purified interferon containing approximately lo5 units/ml were constituted to contain 1% SDS and were heated at 100” for 1 min, and O.l-ml aliquots were electrophoresed as described in Materials and Methods. Eluted samples were assayed (-0-) or were constituted to contain 1% SDS, 1% mercaptoethanol, and 5 M urea and heated at 100” for 1 min prior to dialysis and assay (-•-):

SDS under reducing conditions than from SDS without reduction. Also, Yamazaki and Wagner (1970) have reported that rabbit interferon is destroyed by SDS (a finding I have been unable to confirm); further, Mogensen and Cantell (1974) have reported that, while human leukocyte interferon is stable in SDS alone, it is largely inactivated by treatment with SDS under reducing conditions (a finding I have been able to confirm). To the contrary; human fibroblast interferon, while completely renaturable from SDS under reducing conditions, is only partially renaturable from SDS alone (Stewart, De Clercq, and De Somer, 1974). Studies similar to those described here will likely help clarify the molecular compositions of interferon preparations from different species, different cells of given species, different inducers, and a number of other variables. Indeed, studies to be reported elsewhere (Stewart, Bodo, Paucker, and Tovey, manuscript in preparation), have revealed that while some other mouse interferon preparations are identical to the preparations reported here, some are monospecific. For practical purposes of production and purificat.ion studies, it becomes increasingly important to know the molecular composition of interferon preparations, for

the different molecular species can have differing requirements for renaturation (Stewart, De Somer, and De Clercq, 1974). Studies are currently underway to determine: the relative spectrum of antiviral activities induced in cells (Stewart, Scott, and Sulkin, 1969) by each of the isolated molecular species of interferon; the nonantiviral activities (priming and blocking; Stewart, Gosser, and Lockart, 1971a, b; double-stranded RNA toxicity enhancement; Stewart, De Clercq, Billiau, Desmyter, and De Somer, 1972; Stewart, De Clercq, De Somer, Berg, Ogburn, and Paucker, 1973; cell-growth inhibition; Gresser, Bandu, Tovey, Bodo, Paucker, and Stewart, 1973) resident in each molecular species; and the cell-binding capacities (Stewart, De Clercq, and De Somer, 1972a) of each of the molecular species. ACKNOWLEDGMENTS I thank Frieda De Meyer and Willy Zeegers for excellent technical assistance. This work was aided by Grant DRG-1PlOA from the Damon Runyon-Walter Winchell Memorial Fund for Cancer Research and Grant 310 from the Jane Coffin Childs Memorial Fund for Medical Research. REFERENCES

CARTER,W. A. (1970). structure.

Proc.

Nat.

Interferon: evidence for subunit Acad. Sci. USA 67, 620-628.

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E. STEWART

W. A., and PITHA, P. M. (1971). Structural requirements of ribopolymers for induction of human interferon: evidence for interferon subunits. In “Biological Effects of polynucleotides” (R. F. Beers, Jr. and W. Braun, eds.), pp. 89-105. Springer-Verlag, New York. DUNKER, A. K., and RUECKERT, R. R. (1969). Observations on molecular weight determinations on polyacrylamide gel. J. Biol. Chem. 244, 5074-5080. EDY, V., BILLIAU, A., and DE SOMER, P. (1974). Stabilization of mouse and human interferons by acid pH against inactivation due to shaking and guanidine hydrochloride. Proc. Sot. Exp. Biol. Med. (in press). FALCOFF, R. (1972). Some properties of virus and immune-induced human lymphocyte interferons. J. CARTER,

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16, 251-253.

FISH, W. W., REYNOLDS,J. A., and TATFORD, C. (1970). Gel chromatography of proteins in denaturing solvents. J. Biol. Chem. 245, 5166-5168. GRESSER, I., BANDU, M.-T., TOVEY, M., BODO, G., PAUCKER, K., and STEWART, W. E. II (1973). Interferon and cell division. VII. Inhibitory effect of highly purified interferon preparations on the multiplication of leukemia L 1210 cells. Proc. Sot. Exp. Biol.

Med.

142, 7-10.

KE, Y. H., and Ho, M. (1967). Characterization of virus- and endotoxin-induced interferons obtained from the serum and urine of rabbits. J. Viral. 1, 883-890. KE, Y. H., and Ho, M. (1968). Studies on physicochemical inactivation of rabbit interferons. Proc. Sot.

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MAIZEL, J. V., JR. (1970). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), pp. 179-246. Academic Press, New York. MARSHALL, L. W., PITHA, P. M., and CARTER, W. A. (1972). Inactivation of interferon: the effect of protonation. Virology 48, 607-611. MOGENSEN, K. E., and CANTELL, K. (1974). Human leukocyte interferon: role for disulfide bond. J. Gen. Virol.

22, 95-103.

NC, M. H., and VILCEK, J. (1972). Interferons: physicochemical properties and control of cellular synthesis. Aduan. Protein Chem. 26, 173-241. PITHA, P. M., MARSHALL, L. W., and CARTER, W. A. (1973). Interferon: the dissociation of rIn rCn-

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induced proteins by protonation. J. Gen. Virol. 21, 169-174. STEWART, W. E. II, DE CLERCQ, E., BILLIAU, A., DESMYTER, J., and DE SOMER, P. (1972). Increased susceptibility of cells treated with interferon to the toxicity of polyriboinosinic.polyribocytidylic acid. Proc. Nat. Acad. Sci. USA 69, 1851-1854. STEWART, W. E. II, DE CLERCQ,E., and DE SOMER, P. (1972a). Recovery of cell-bound interferon. J. Virol. 10, 707-712. STEWART, W. E. II, DE CLERCQ,E., and DE SOMER, P. (1972b). Cellular alternation by interferon. A virusfree system for assaying interferons. J. Virol. 10, 896901. STEWART, W. E. II, DE CLERCQ,E., and DE SOMER, P. (1974). Stabilization of interferons by “defensive” reversible denaturation. Nature (London) (in press). STEWART, W. E. II, DE CLERCQ, E., DE SOMER, P., BERG, K., O~BURN, C. A., and PAUCKER,K. (1973). Antiviral and non-antiviral activities of highly purified interferon. Nature (London) New Biol. 246, 141-143. STEWART, W. E. II, DE SOMER, P., and DE CLERCQ,E. (1974). Protective effects of anionic detergents on interferons: reversible denaturation. Biochim. Biophys. Acta (in press). STEWART, W. E. II, GOSSER,L. B., and LOCKART,R. Z., JR. (1971a). Priming: a non-antiviral function of interferon. J. Viral. 7, 792-801. STEWART,W. E. II, GOSSER,L. B., and LOCKART, R. Z., JR. (1971b). Distinguishing characteristics of the interferon responses of primary and continuous mouse cell cultures. J. Gen. Virol. 13, 35-50. STEWART, W. E. II, SCOTT, W. D., and SULKIN, S. E. (1969). Relative sensitivities of viruses to different species of interferon. J. Viral. 4, 147-153. SUMMERS, D. F., MAIZEL, J. V., and DARNELL, J. E. (1965). Evidence for virus specific non-capsid proteins in poliovirus infected HeLa cells. Proc. Nat. Acad. Sci. USA 54, 505-513. WEIL, R., and DORNER,F. (1973). Interferon structure: facts and speculation. In “Selective Inhibitors of Viral Functions” (W. A. Carter, ed.), pp. 107-122. CRC Press, Cleveland. YAMAZAKI, S., and WAGNER, R. R. (1970). Purified rabbit interferon: attempts to demonstrate interferon-specific 3H-protein. J. Virol. 5, 270-273.