Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 182-186, January 1975

Interfel-on Action 1I. Membrane-Bound Alkaline Ribonuclease Activity in Chick Embryo Cells Manifesting Interferon-Mediated Interference* (viral interference/vesicular stomatitis virus)

PHILIP I. MARCUSt, THOMAS M. TERRYt, AND SEYMOUR LEVINET t Microbiology Section, U-44, University of Connecticut, Storrs, Conn. 06268; and $ Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Mich. 48201

Communicated by Theodore T. Puck, October 21, 1974

virus (VSV)-HR, Indiana serotype, were prepared as reported previously. (19). Interferon and Interferon-Mediated Viral Interference. Chick interferon was prepared as has been reported (20), and produced no adverse effects on cell macromolecule synthesis or growth. Mock-interferon preparations represented growth medium from plates of chick cells that were not exposed to inducers of interferon but otherwise weke treated identically. All preparations produced comparable results when equated to interferon activity as measured by a 50% reduction assay of VSV plaque-forming particles [PRso (VSV) uhits]. Viral resistance was induced by interferon preparations over a 24hr period at 370, at which time mock-treated cells also were processed. Mouse interferob (50,000 U/ml) was obtained by infecting mouse L-cells with ultraviolet-irradiated Newcastle disease virus, and processing through the zinc acetate stage of purification (20). No reduction in the yield of VSV plaqueforming particles was observed on chick embryo cell monolayers treated with 5000 PR50 (VSV) units/ml of mouse cellderived interferon. Viral interference induced by poly(I)poly(C) (P-L Biochemicals) was accomplished by exposing aged chick cell monolayers for 24 hr at 370 to fresh medium that contained DEAE-dextran (10 Mug/ml) and various concentrations of the polyribohucleotide. All experiments included bioassays for viral interference. Membrane Fractionation. After treatment with interferon, mock-interferon, or poly(I) * poly(C), cell monolayers were removed virtually intact by incubating for about 5 min at 370 in saline D (21) containing 5 mM EDTA. The sheets of cells were chilled to 40, washed twice with cold saline 1), swelled in RSB-K (22), and Dounce homogenized to disrupt the cells. Subsequent steps to secure membrane fractions followed the procedure of Caliguiri and Tamm (22). Visible membrane bands were collected from the 25 to 30% sucrose interface (fraction 3) and from the 30 to 40% sucrose interface (fraction 4), diluted with RSB-K buffer and collected by centrifugation at 40,000 X g for 4 hr and resuspension in RSB-K. VSV In Vitro Transcription. Preparation of transcribing nucleoprotein (TNP) from VSV followed the procedure of activity was also Szil~gyi and Urvayev (23). Transcriptase assayed by their procedure. Reaction mixtures (50 ul) contained: 5 ,.d of TNP; 3.5 mM dithiothreitol; 20 mM Tris- HC1 (pH 8.0); 0.1 M NaCl; actinomycin D (0.8 ,ug); 0.64 mM each of ATP, CTP, and GTP; 6.4 MAM UTP and about 25,000 cpm of [5'-3H]UTP (specific activity 14 Ci/mmol, Schwarz/

Membrane fractions from thick embryo ABSTRACT cells manifesting,vifai interference mediated by interferon or poly(I). poly(C) contain high levels. of an alkaline ribonuclease. Enhanced RN se activity is not observed when inhibitors of cell protein or RNA synthesis are present during interferon treatment, or when heterologous interreron is used. The RNase associated with comparable membrane fractions from cells treated with mock-interferon is about 1/10 as active, and shows qualitative differences. In principle, divergent views of interferon action may be reconciled to a common mode of action by Postulating that viral interference results from a newly induced or activated RNase of cellular origin and proper specificity that acts to reduce the accumulation and functional capacity of newly synthesized viral RNAs, particularly niRNA. Previous data in support of interferon's acting to inhibit virion-derived transcription in vivo are now interpretedas demonstrating enhanced degradation of viral transcripts (mRNA).

Interferon-mediated interference with viral replication has been reported to act at the level of translation (1-5) and transcription (6-11). These seemingly divergent views of interferon action may be reconciled to a single mechanism by postulating that a cellular ribonuclease with proper specificity acts to reduce the intracellular accumulation of newly synthesized viral mRNA, or to alter its capacity for translation (6, 12, 13). Modulation of such an RNase by cellular (14-16) or viral inhibitors, or shifts in the location or orientation of the enzyhie in relation to the sites of transcription or translation might provide still a higher order of restriction on the accumulation or function of viral RNAs, and perhaps some kinds of cellular RNA critical for viral mRNA translation (17), thereby achieving precise control over messenger expression. This communication presents preliminary evidence to support such a concept by demonstrating that a membrane-associated alkaline RNase appears in chick embryo cells that manifest viral interference induced either by homologous interferon or by poly(I) * poly(C). MATERIALS AND METHODS

Cells and Virus. Primary chick embryo cell monolayers aged in vitro for 5-7 days as described previously were used throughout this study (18). These cells produce high levels of interferon upon induction, and are more responsive to the action of interferon (18). Stock cultures of vesicular stomatitis Abbreviations: VSV; vesicular stomatitis virus; PR,o, 50% reduction in viral plaques; TNP, transcribing nuclteoprotein. * Paper I in this series is ref. 6.

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FIG. 1. Effect of fraction 4 membranes from variously treated chick embryo cells on the accumulation of acid-precipitable viral RNA in the standard reaction mixture for VSV TNP transcription in vitro. "cpm" represent product labeled with [3H] UTP as precursor, and are cumulative to the time of sampling (abscissa). All reaction mixtures except the control (TNP) received membrane protein at a final concentration of 6 g/i100 Al, from cells previously treated as noted in the text. Interferon treatment produced a 104-fold decrease in virus yield and full protection against cell killing (19).

FIG. 2. Effect of interferon concentration on the activity of fraction 4 membranes in the VSV TNP in vitro transcription reaction. Conditions were as described in the text. Membrane protein (6 lug/100 Al) added to the in vitro reaction mixture was from chick embryo cell monolayers previously treated with 2000, 200, 20, or 2 PRjo (VSV) units/ml of chick interferon, or mock treated. Yields of VSV plaque-forming particles from these monolayers were 4 X 104, 6 X 104, 10 X 104, 3 X 107, and 1 X 108/ml, respectively. The three highest doses of interferon protected against cell killing (19). 3H cpm as in Fig. 1 legend.

Mann), and included up to 15 Al of membrane fractions in RSB-K (200 PR5o (VSV) units] initially produce a measurable decline in the rate of VSV RNA accumulation, and an earlier plateauing and solubilization of previously synthesized RNA, compared to all controls. For lower doses the effect is less. The curves gen-

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feron, thus defining a requirement for homologous interferon to produce the effects recorded in Figs. 1 and 2. Perhaps the chick-derived interferon preparation itself contained a nonspecific factor with a high affinity for membranes. To test this possibility we prepared membrane fractions from the Vero line of green monkey kidney cells treated with 5000 PR5o (VSV) units/ml of chick interferon. None of these fractions reduced in vitro transcription by VSV TNP. Effect of Membrane Fractions from Poly(I) -Poly(C)-treated Cells on In Vitro Transcription. Cell monolayers were exposed to poly(I) poly(C) in the presence of DEAE-dextran (10 ,ug/ ml) to induce high levels of interference with VSV replication. Fig. 3 demonstrates that all three concentrations of poly(I)poly(C) used in this experiment (0.02, 0.2, and 2.0 ,Ag/ml) produced membrane fractions that were highly active in preventing the accumulation of VSV transcripts. Diluting poly(I) * poly(C) beyond 0.02 ug/ml revealed a dose-response effect similar to that illustrated in Fig. 2 for interferon (data not shown). When cycloheximide was added to cells simultaneously with poly(J) - poly(C) and DEAE-dextran, the membrane fractions were inactive as tested in the in vitro transcribing system and behaved much like the preparations from mock (not shown) or mock + cycloheximide-treated cells -

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FIG. 3. Effect of poly(I)-poly(C)-induced interference on the activity of fraction 3 membranes in the VSV TNP in vitro transcription reaction. Membrane protein (6 ,Ag/100 Ml) added to the in vitro reaction mixture was from cells previously treated with DEAE-dextran (10 ,g/ml) and poly(I) - poly(C) at concentrations of 2, 0.2, or 0.02 ,g/ml. All three doses of inducer produced background levels of virus upon challenge with VSV, and protected against cell killing (19). Controls consisted of membranes from mock-treated cells and poly(I) poly(C)-treated cells ex-

posed simultaneously to cycloheximide (50 ,sg/ml). 3H cpm as in Fig. 1 legend.

erated in Figs. 1-3 represent cumulative radioactivity in RNA.

Homologous Interferon Is Required to Produce Membrane Fractions Effective Against Transcript Accumulation In Vitro. Chick-cell derived interferon was used to induce the membrane changes responsible for the effects on in vitro transcription recorded in Figs. 1 and 2. In contrast, membrane fractions prepared from chick embryo cells treated with 5000 PR5o (VSV) units of mouse interferon derived from L cells behaved like membranes from cells treated with mock inter-

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FIG. 4. Production of VSV mRNA as substrate through heat inactivation (500, 5 min) of virion transcriptase. The time course of VSV TNP RNA synthesis was followed at 310 by measuring the incorporation of 3H ([3H1UTP as precursor) into acid-insoluble material in the standard reaction mixture (0). After 2 hr the mixture was held at 500 for 5 min, cooled, and returned to 310 for subsequent measurements of acid-insoluble product (0). The stable product represents single-stranded primary transcripts of VSV, i.e., mRNA (23, 32).

(Fig. 3). Inactivation of Transcriptase and Stabilization of VSV RNA by Heat Treatment (500, 5 min). Adding active membranes from cells treated with interferon or poly(J) -poly(C) to the reaction mixture after the in vitro transcription product had accumulated for 1 or 2 hr produced a relatively rapid loss of previously synthesized VSV RNA, suggesting the presence of an active RNase. Curves generated by this experimental manipulation represent the vector resulting from at least two reactions, the synthetic process of transcription, and the concomitant or subsequent degradation of its product. To simplify analysis, we generated isotopically labeled VSV transcripts over a linear portion of the reaction, usually for 90 min, and then stopped further transcription by inactivating the heat-labile virion transcriptase by subjecting the reaction mixture to 500 for 5 min (24, 26). Results typical of this treatment, shown in Fig. 4, demonstrate that heating stops transcription immediately, and that the resultant transcripts remain stable. Preparations of heat-treated VSV RNA were stable after freezing and storage at -70°. All subsequent experiments were performed with these 8H-labeled VSV transcripts as substrate. Enhanced Ribonuclease-Like Activity Associated with Membranes from Cells Treated with Interferon or Poly(I) * Poly(C). Membrane fractions 3 or 4 from cells treated with interferon, interferon + actinomycin D, or mock-interferon were adjusted to equal concentrations of protein, and measured amounts were added to a heat-stabilized reaction mixture containing 2500 cpm of 3H-labeled VSV primary transcripts. Samples were removed at different times and assayed for acid-insoluble radioactive material. Fig. 5a represents a typical assay. All the membrane preparations produced a loss of acid-insoluble VSV RNA at linear rates, indicating that they had some RNase-like activity. However, membrane fractions from cells treated with interferon solubilized the viral RNA at a significantly faster rate than that produced by membranes from the various control cells. In the experiment illustrated in Fig. 5a, 6 ug of membranes from interferon-treated cells per

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100 M1 of reaction mixture solubilized 50% of the VSV RNA in 16 mim at 310, whereas an equivalent amount of membrane from cells treated with mock interferon required 76 min, or about five times longer. A similar experiment with membranes from poly(J) -poly(C)-treated cells (Fig. 5b) revealed a solubilization half-time of 7.5 min, with mock-treated membranes requiring 66 min to reach that same level, i.e., nine times longer. As shown in Fig. 5, treatment with actinomycin D or cycloheximide during induction with either interferon or the polyribonucleotide reduced the RNase-like activity to less than that of the mock preparations. In a series of such assays, membrane fractions 3 or 4 from cells showing high levels of viral interference consistently solubilized VSV RNA at 5 to 20 times the rate of comparable mock preparations. Mixing mock- and interferon-membranes had no adverse effect on the nuclease activity of the latter.

Preliminary Characterization of the Ribonuclease Activity Associated with Membranes from Interferon or Poly(I) Poly(C)Treated Cells. Detailed characterization of the membraneassociated RNase is not complete; however, we can report that the nuclease activity from cells showing viral interference is optimal at about pH 8.2 and 600, and requires Mg++. While EDTA at 3 mM, in the presence of 0.75 mM MgCI2, inhibits the activity completely (it can be recovered upon addition of excess Mg++), the chelating agent has little effect on an equivalent activity of crystalline bovine pancreatic RNase assayed under similar conditions. The nuclease activity associated with 1 ,ug of membrane protein per 100 Ml of assay mixture at 310 is equivalent to 0.01 Mg/100 Mil of pancreatic RNase, and is derived from about 106 chick embryo cells. Free sulfhydryl groups may not be required for the activity of the membrane-bound RNase, since it is not affected by 1 mM pchloromercuribenzene sulfonic acid. Sonication, or the addition of the neutral detergent Triton N-101 (0.1%) had no deleterious effect, nor did it enhance the nuclease activity, but it was inactivated at 700 after 10 min. We also observed that heat-stable (700, 5 min), activity in standard and commercial (Searle Co.) preparations of an alkaline RNase inhibitor from mammalian cells (14-16) inactivates essentially all the nuclease activity of interferon- or poly(I). poly(C)-membranes while leaving unaffected about one-half of the activity on mock-membranes. For example, 1 unit of nuclease inhibitor (14) added to 1 ug of interferon- or mockmembrane protein per 100 MuI of assay mixture produced a 24and 1.6-fold reduction, respectively, in the rate of viral RNA solubilization. The RNase activity can be eluted and recovered from membranes by exposing them to 1 M KCl for 30 min. We have not yet tested the membrane preparations for DNase activity, or on other RNAs as substrates.

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DISCUSSION Our data demonstrate that the induction of interferon-mediated interference in chick embryo cells by interferon or poly(I) -poly(C) is accompanied by a marked increase in the activity of a membrane-associated alkaline RNase. This RNase is active against the RNA transcripts synthesized in vitro from the genome strand of VSV by virion-bound transcriptase, is expressed at 5 to 20 times the ribonuclease activity per weight of protein of comparable fractions from cells treated with mock interferon, and is qualitatively different from that nuclease activity. The nuclease associated with

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FIG. 5. Degradation of VSV primary transcripts (mRNA) by membrane-bound ribonuclease from cells manifesting viral interference induced by chick interferon (a) or poly(I)-poly(C) (b). Membrane protein (6 ,g/100 ,1 of reaction mixture) was derived from cells treated with interferon (500 U/ml), poly(I)-poly(C) (0.2 Mg/ml), or mock treated in the presence or absence of actinomycin D (ACD) (1 Mg/ml) (a), or cycloheximide (50 ,g/ml) (b). Each reaction mixture contained 2500 cpm of VSV mRNA prepared as shown in Fig. 4. Samples were removed as a function of time after addition of membrane and assayed for acid-insoluble radioactivity.

interferon-membranes is totally sensitive to a heat-stable, Pronase-resistant inhibitory factor found in standard preparations of an alkaline RNase inhibitor from mammalian cells, and has optimal activity at about pH 8.2, with a significant decrease at pH 7.2. The comparable activity on membranes from the mock-treated cells declines little if at all from 8.2 to 7.2, and is relatively resistant to this same inhibitory factor. Thus, cytoplasmic membranes, the cell organelles most likely to become involved with viral synthesizing processes (22, 27), acquire a highly enhanced capacity to degrade mRNA. Increased RNase activity in topologically propitious points in the cell may profoundly affect viral replication, or, for that matter, cellular events reflecting nonantiviral effects of interferon (28). Syntheses of cellular RNA and protein are required for the expression of enhanced RNase activity, although it is not yet clear whether new molecules are synthesized or membrane changes resulting from interferon action provide new or more favorable sites for binding preexisting or activated nuclease. Degradation of viral nucleic acid by the release or activation of cellular nucleases is usually attributed to lysosomal enzymes (29). Our demonstration of an alkaline RNase tends to eliminate lysosomes and their acid nucleases as a source of this enzyme. The membrane-bound RNase may represent an endonucleolytic activity similar to that present in highly purified mouse interferon (12). However, our preliminary studies suggest that, for chick cells, the membrane-bound nuclease found upon interferon treatment and that found free in both

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interferon and mock-interferon preparations represent different molecular entities. For example, the heat-stable component of the inhibitor from mammalian cells does not inhibit the free nuclease-in contrast to its action on the membrane-bound enzyme. Although definition of the exact relationship between the interferon [or poly(I) -poly(C)] dose and membrane-bound RNase activity is incomplete, our preliminary results already show two effects characteristic of the interferon system: first, increasing the dose of interferon [or poly(I) -poly(C) ] results in an increase in response, i.e., in membrane-bound RNase activity, and second, high doses of either inducer tend to approach saturation in their effect-producing almost equal amounts of membrane-RNase activity. These results are strikingly similar to the dose-response curves reported earlier for the effects of these two inducers of interferon action on the accumulation in chick embryo cells of VSV RNA from virionderived transcription (ref. 6; Figs. 3 and 4). The present data provide evidence that *a membranebound alkaline RNase may constitute one facet of interferon action. From this vantage, we can interpret more accurately our previous report of inhibition of virion-derived primary transcription in chick embryo cells as reflecting an increase in the rate of degradation of transcription product; Enhanced nuclease activity associated with membranes might also account for some recent results (30), which provide evidence for primary transcription in interferon-treated cells-seemingly in conflict with other reports (6, 8). Thus, selective nucleolytic action might permit detection of primary transcripts through molecular hybridization (31, 11), the technique used, even though the transcripts were nonfunctional as mRNA. Continued exposure of transcripts to nuclease action would lead eventually to a net decrease in the rate of viral mRNA accumulation, as we and others have observed (6, 8, 11). Presumably, the efficiency at which primary transcripts function would depend upon the extent to which the transcripts interact, during the initial stages of viral replication with cytomembranes laden with proper nuclease. As we noted earlier (6), there is no a priori reason for supposing that the interferon system consists of a single molecular species with a single mode of action. In fact, the myriad nonantiviral effects attributed to interferon treatment (28) cast doubt on such thinking. Nonetheless, it is tempting to consider that properly selective and situated RNase(s) could act to effect many of the intracellular changes attributed to interferon action, as astutely pointed out by Graziadei and coworkers (12, 13). Add to this the possibility of inducing inhibitors of RNase action, and there emerges a finely tuned regulatory system, aimed at a key molecule in the synthetic capacity of the virus, or cell-mRNA. Certainly these experiments do not preclude translation or transcription processes per se as sites of interferon action, but raise the possibility that increased levels of membrane-bound cellular RNase may control the extent to which viral mRNA reaches the translation system intact. In principle, then, viral mRNA, as both the product of transcription and the messenger for translation, provides a common target for properly selective and situated RNase molecules. Further experimentation is required to determine whether the membrane-bound alkaline RNase described here constitutes such a molecule.

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This research was supported by USPHS Grant AI-09312 from the National Institute of Allergy and Infectious Diseases, and benefited from the use of the Cell Culture Facility supported by Grant CA-14733 from the National Cancer Institute. We thank Margaret J. Sekellick for a critical reading and discussionof the manuscript, and John J. Papale for excellent assistance in some recent experiments. 1. Sonnabend, J. A. & Friedman, R. M. (1973) In Interferons and Interferon Inducers, ed. Finter, N. B. (North-Holland Publ., Amsterdam-London), pp. 201-239. 2. Friedman, R. M., Metz, D. H., Esteban, R. M., Tovell, D. R., Ball, L. A. & Kerr, I. M. (1972) J. Virol. 10, 11841198. 3. Falcoff, E., Falcoff, R., Lebleu, B. & Revel, M. (1973) J. Virol. 12, 421-430. 4. Gupta, S. L., Graziadei, W. D., III, Weideli, H., Sopori, M. L. & Lengyel, P. (1974) Virology 57, 49-63. ;). Samuel, C. E. & Joklik, W. K. (1974) Virology 58, 476491. 6. Marcus, P. I., Engelhardt, D. L., Hunt, J. M. & Sekellick, M. J. (1971) Science 174, 593-598. 7. Oxman, M. N. & Levin, M. J. (1971) Proc. Nat. Acad. Sci. USA 68, 299-302. 8. Manders, E. K., Tilles, J. G. & Huang, A. S. (1972) Virology 49, 573-581. 9. Bialy, H. S. & Colby, C. (1972) J. Virol. 9, 286-289. 10. Gravell, M. & Cromeans, T. L. (1972) Virology 50, 916919. 11. Bean, W. J., Jr. & Simpson, R. W. (1973) Virology 56, 646651. 12. Graziadei, W. D., III, Weideli, H. & Lengyel, P. (1973) Biochem. Biophys. Res. Commun. 54, 40-46. 13. Taylor-Papadimitriou, J., Spandidos, D. & Georgatsos, J. G. (1971) Biochem. Biophys. Res. Communz. 43, 149-155. 14. Roth, J. S. (1956) Biochim. Biophys. Acta 21, 34-43. 15. Roth, J. S. (1962) Biochim. Biophys. Acta 61, 903-915. 16. Kraft, N. & Shortman, K. (1970) Aust. J. Biol. Sci. 23, 175184. 17. Gupta, S. L., Sopori, M. L. & Lengyel, P. (1974) Biochem. Biophys. Res. Communi. 57, 763-770. 18. Carver, I). H. & Marcus, P. I. (1967) Virology 32, 247-

257. 19. Marcus, P. I. & Sekellick, M. J. (1974) Virology 57, 321338. 20. Marcus, P. I. & Salb, J. M. (1966) Virology 30, 502-516. 21. Ham, R. G. & Puck, T. T. (1962) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. O. (Academic Press, New York), Vol. ), p. 90. 22. Caliguiri, L. A. & Tamm, I. (1970) Virology 42, 100-111. 23. Sziligyi, J. F. & Urvayev, L. (1973) J. Virol. 11, 279286. 24. SzilAgyi, J. F. & Pringle, C. R. (1972) J. Mol. Biol. 71, 281291. 25. Finter, N. B. (1973) in Iuterferons and IWterferon, Inducers, ed. Finter, N. B. (North-Holland Publ., AmsterdamLondon), p. 137. 26. Marcus, P. I. & Sekellick, M. J. (1975) Virology, in press. 27. Friedman, It. M. & Sreevalsan, T. (1970) J. Virol. 6, 169-

175. 28. Nebert, D. W. & Friedman, It. M. (1973) J. Virol. 11, 193-197. 29. Fiszman, M. Y., Bucchini, D., Girard, M. & Lwoff, A. (1970) J. Geni. Virol. 6, 293-304. 30. Repik, P., Flamand, A. & Bishop, D. H. L. (1974) J. Virol., in press. 31. Flamand, A. & Bishop, D. H. L. (1973) J. Virol. 12, 1238-

1252. 32. Bishop, D. H. L. & Roy, P. (1971) J. Mol. Biol. 58, 799814.