[1] DEFINITION AND CLASSIFICATION OF INTERFERONS 3

[1] D e f i n i t i o n a n d C l a s s i f i c a t i o n o f t h e I n t e r f e r o n s

By SIDNEY PESTKA a n d SAMUEL BARON

Interferon, the body's most rapidly produced defense against viruses, is a protein secreted by body cells when they are stimulated by viruses, bacteria, foreign cells, foreign macromolecules, or numerous other com- pounds. The secreted interferon then stimulates surrounding cells to produce other proteins, which, in turn, may regulate virus multiplication, the immune response, cell growth, and other cell functions. At least three distinct types of interferon may be produced, depending on the type of stimulus and the type of cell stimulated. Moreover, recent medical studies indicate that interferon is promising as a treatment for virus infections and, perhaps, cancer and autoimmune diseases, so that great interest in the interferons has been generated.

Since antiviral activity has been a hallmark of detecting and measuring interferon, it is ordinarily considered as part of the definition of inter- feron. However, since the ratios of antiviral activity to antiproliferative ~ and natural killer-cell z activity vary markedly among the natural purified human leukocyte interferon species, it should not be surprising to find a homologous molecule with antiproliferative and other activities, but es- sentially devoid of antiviral activity. If such a putative molecule shows extensive homology to interferon, it should be considered an interferon. Now that we know the amino acid sequences of many human leukocyte interferons,a-s classification of the interferons should be based on primary structure and biological activity rather than on other criteria. Such con- siderations are even more pertinent because we do not fully know the nat- ural physiological role of the interferons. Although antigenic properties have been useful in classifying and identifying interferons, as has been

M. Evinger, M. Rubinstein, and S. Pestka, Arch. Biochem. Biophys., in press (1981). z j. R. Ortaldo, A. Mantovani, D. S. Hobbs, M. Rubinstein, S. Pestka, and R. B. Herberman,

in preparation. a W. P. Levy, J. Shively, M. Rubinstein, U. Del Valle, and S. Pestka, Proc. Natl. Acad.

Sci. U.S.A. 77, 5102 (1980). 4 W. P. Levy, M. Rubinstein, J. Shively, U. Del Valle, C.-Y. Lai, J. Moschera, L. Brink,

L. Gerber, S. Stein, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A., 78, in press. K. C. Zoon, M. E. Smith, P. J. Bddgen, C. B, Anfinsen, M. W. Hunkapiller, and L. E. Hood, Science 207, 527 (1980).

6 G. Allen and K. H. Fantes, Nature (London) 287, 408 (1980). 7 M. Streuli, S. Nagata, and C. Weissmann, Science 209, 1343 (1980). 8 D. V. Goeddel, E. Yelverton, A. Ullrich, H. L. Heyneker, G. Miozzari, W. Holmes,

P. H. Seeburg, T. Dull, L. May, N. Stebbing, R. Crea, S. Maeda, R. McCandliss, A. Sloma, J. M. Tabor, M. Gross, P. C. Familletti, and S. Pestka, Nature (London) 287, 411 (1980).

Copyright © 1981 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 78 All rights of reproduction in any form reserved.

ISBN 0-12-181978~7

4 DEFINITION [1]

noted, ",1° identification of components by their antigenic properties with- out concomitant structural and functional information is prone to serious errors. Nevertheless, standard antibodies and monoclonal antibodies to human leukocyte interferon 11'12 may provide an important practical means of defining a molecule as an interferon even though several species of leu- kocyte interferon are not recognized by some of the monoclonal anti- bodies) 2 Thus, as noted above, although the definition of the interferons must reside in protein structure homologous with the known interferons, for practical purposes this does not provide a rapid or convenient means of determining whether a molecule is an interferon. For these purposes, antigenicity as well as the general characteristics of mode of production, species activity profile, mechanism of action, and cell surface receptors provide useful operational methods to assist in defining the interferons. However, except perhaps for antigenicity of the major classes of inter- feron, none of these procedures alone provides definitive identification. The sections below and throughout this volume and Vol. 79 provide de- tails of these adjunct definitions.

The variety of abbreviations used for the three types and various spe- cies of interferons are given in Table I. Types I human interferon are rep- resented by the leukocyte and fibroblast types (generally acid stable); type II is the immune interferon (usually acid labile). Type I and type II designations are less useful because they overlap other functional and structural criteria for classification. A number of abbreviations used are shown in Table I. The most common abbreviation for interferon in the lit- erature has been IF. Combined with L, F, and I, the abbreivation pro- vides a pneumonic for recalling which is which. A nomenclature recently proposed is (IFNo~,/3, and 7 for leukocyte, fibroblast and immune types, respectively) may be confused with prior designations of the purified spe- cies. Many purified natural human IFL species have been isolated (al, az, 131, f12,/33, 71, 7z, 73, 74, 7s, and 8).~4 It is proposed that recombinant interferon be differentiated from natural interferon by use of a lower case " r " prior to the designation. So far, two recombinant species can proba- bly be matched to the natural species (IFLa2 and/31 are probably equiva- lent to IFLrA in primary sequence; and IFLTa probably is equivalent to

9 S. Udenfriend, personal communication. ~9 j . H. Julliard, T. Shibasaki, N. Ling, and R. Guillemin, Science 208, 183 (1980). 1~ D. S. Secher and D. C. Burke, Nature (London) 285, 446 (1980). 12 T. Staehelin, B. Durrer, J. Schmidt, B. Takacs, J. Stocker, V. Miggiano, C. St~ihli,

M. Rubinstein, W. P. Levy, R. Hershberg, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 78, 1848 (1981).

~3 Interferon Nomenclature, Nature (London) 286, 110 (1980). 14 M. Rubinstein, W. P. Levy, J. A. Moschera, C.-Y. Lai, R. D, Hershberg, R. T. Bartlett,

and S. Pestka, Arch. Biochem. Biophys., in press (1981).

[1] DEFINITION AND CLASSIFICATION OF INTERFERONS 5

TABLE I ABBREVIATIONS USED AND DESIGNATIONS OF HUMAN INTERFERONS

Subspecies designations Interferon Type

class number Abbreviations used Natural Recombinant

Leukocyte I IFL, LelF, LIF, ifnLe, IFNa

Fibroblast I IFF, FIF, INF, ifnF, IFN/3 Immune II IFI, ImIF, IFN T

IFLal IFLrB IFLrC

/3~ IFLrA

/32 IFLrF /33 IFLrG Tt IFLrH T2 T3 IFLrD, IFLrl

T4 T5 3

o Abbreviations used for interferon have included IF, IFN, INT. In the table, ab- breviations and designations that have been employed for the various interferon types and species are given. The natural species have been isolated from human leuko- cytes? 4 No correspondence between the natural and the recombinant interferons is im- plied by their position in the table except for IFI_~z and #t, which are probably equiv- alent to IFLrA; and IFLT3, which appears to be equivalent to IFLrD and IFLrl.

IFLrl or IFLrD). 4a4az However, many distinct recombinant cDNAs and genes for leukocyte interferon have been identified and sequenced, r,8,~6-as It will be possible eventually to match all the natural species with each of the recombinant species when additional structural data are available. Ac- cordingly, more information about the amino acid sequences of the natu- ral products is necessary. It will also probably be necessary to have addi- tional recombinants.

Although human leukocyte interferon does not appear to be glycosyl-

~s C. Weissmann, personal communication. 16 D. V. Goeddel, D. W. Leung, T. J. Dull, M. Gross, R. M. Lawn, R. McCandliss, P. H.

Seeburg, A, Ullrich, E. Yelverton, and P. W. Gray, Nature (London) 290, 20 (1981). ~7 S. Maeda, R. McCandliss, T.-R. Chiang, L. Costello, W. P. Levy, N. T. Chang, and

S. Pestka, in "Developmental Biology Using Purified Genes" (D. Brown and C. F. Fox, eds.) I C N - U C L A Syrup. Mol. Cell. Biol. 23 in press. Academic Press, New York, 1981.

~s S. Pestka, S. Maeda, D. S. Hobbs, W. P. Levy, R. McCandliss, S. Stein, J. A. Moschera, and T. Staehelin, in "Cellular Responses to Molecular Modulators" (W. A. Scott, R. Werner, and J. Schuitz, eds.) Miami Winter Symp. 18, in press. Academic Press, New York, 1981.

6 DEFINITION [1]

ated T M to a significant extent, natural human fibroblast interferon appears to be a glycoprotein. Nevertheless, whatever the ratio of the glycosylated to the nonglycosylated species in native preparations, the nonglycosyl- ated species are active. 7"s'19-~4

A number of recombinant hybrid leukocyte interferon molecules have been constructed, 25-27 and many more will surely be made. These will also include molecules with additions, deletions, substitutions, overlaps, and the like. To provide some systematic framework for keeping track of this very extensive array, we suggest that these be described with sub- scripts designating the amino acids in the corresponding interferon (from NH~-terminal to COOH-terminal end). For example, hybrids A1-91/D~-16e and D1-9~/Aa~-~05 represent recombinants at the PvulI re- striction endonuclease site. Position 1 is considered to be the amino ter- minus of the predicted corresponding natural interferon species and is cysteine in both IFLrA and IFLrD. A simple deletion of ten amino acids at the COOH-terminus would be simply designated IFLrA~_Iss; an inter- nal deletion, for example, as IFLrA~-es.rl-~es. Such designations would specify hybrid and modified proteins with precision.

Cellular Production of Interferon. Interferon is produced by cells that are stimulated by viruses or foreign materials (e.g., bacteria, foreign ani- mal cells, macromolecules). These stimuli activate cellular genes for in- terferon production. Activation of the structural gene leads to production of interferon, which is synthesized and then secreted from the cell. These events, which can occur during a virus infection, are illustrated in the first panel of Fig. 1. Interferon is not ordinarily produced in large amounts until the foreign stimulus that induces interferon is present in the body.

lg S. Pestka, M. Evinger, R. McCandliss, A. Sloma, and M. Rubinstein, in "Polypeptide Hormones" (E. F. Beers, Jr., and E. G. Bassett, eds.), p. 33. Raven, New York, 1980.

20 S. Bose, D. Gurari-Rotman, U. T. Ruegg, L. Corley, and C. B. Anfinsen, J. Biol. Chem. 251, 1659 (1976).

21 T. Taniguchi, M. Sakai, Y. Fujii-Kuriyama, M. Muramatsu, S. Kobayashi, and T. Sudo, Proc. Jpn. Acad. Ser. B 55, 464 (1979).

32 M. Houghton, A. G. Stewart, S. M. Doel, J. S. Emtage, M. A. W. Eaton, J. C. Smith, T. P. Patel, H. M. Lewis, A. G. Porter, J. R. Birch, T. Cartwright, and N. H. Carey, Nucleic Acids Res. 8, 1913 (1980).

23 D. V. Goeddel, H. M. Shepard, E. Yelverton, D. Leung, R. Crea, A. Sloma, and S. Pestka, Nucleic Acids Res. 8, 4057 (1980).

24 R. Derynck, J. Content, E. De Clercq. G. Volckaert, J. Tavernier, R. Devos, and W. Fiers, Nature (London) 285, 542 (1980).

3~ C. Weissman, Communication at First International Congress of Interferon Research, Washington, D.C., November 1980.

26 D. V. Goeddel, Communication at First International Congress of Interferon Research, Washington, D.C., November 1980.

37 E. Rehberg and S. Pestka, in preparation.

[1] DEFINITION AND CLASSIFICATION OF INTERFERONS 7

2 ® 0

\ @ ~ O~,,O

L . . . .

t_.__j ?___

°

I !

. . . . ~ J

Flo. 1. Cellular events during virus stimulation of interferon (IF). In the first panel, virus comes in contact with a body cell (1) and penetrates the cell membrane. The virus then re- leases its genetic material and multiplies (2). Released from the cell (3) into the surrounding fluid, some of the new virus infects adjacent cells (4) and releases the virus genetic material (5).

During early stages of infection of the first cell, some event (viral nucleic acid?) stimu- lates a gene in the cellular DNA, which contains the stored genetic information for interferon (A). This leads to the production of messenger RNA for interferon; the messenger RNA leaves (B) the cell nucleus and then is translated by the cell's ribosomes (C) into interferon protein. The newly produced interferon is secreted by the cell (D) into the surrounding fluid, where it contacts adjacent cells (E). The adjacent cell (second panel) is activated by the in- terferon to produce (F) new messenger RNAs, which are translated (G) into new proteins, the antiviral proteins (AVP). These proteins in turn can modify the cell's protein-synthesiz- ing machinery and other biochemical events so that viral nucleic acid functions poorly or is degraded (while cell nucleic acid functions normally), thereby inhibiting virus multiplication (see Section VII of Vol. 79 of this series for details of the biochemical events).

If the initial interferon-producing cell survives the virus infection, processes E, F, and G may also operate in that cell to form AVP and, thereby, reduce any continuing multiplication of virus. Evidence exists that the antiviral state may be directly transferred between adja- cent cells (second panel to third panel) by the passage of an undefined inducer of the AVP.

Continued high-level production of interferon requires the continuous presence of a stimulus. Once the stimulus is eliminated, interferon pro- duction ceases.

Types of Interferon. As considered above, three different classes of human interferons have been identified: leukocyte interferon, fibroblast interferon, and immune interferon. ~s These are coded by different struc- tural genes as determined by distinct amino acid sequences 3-6,14,29-al and distinct antigenicities. In fact, the human leukocyte interferons represent

~s S. Baron and F. Dianzarti, eds., Tex. Rep. Biol. Med. 35, 1-573 (1977). 2a E. Knight, Jr., M. W. Hunkapiller, B. D. Korant, R. W. F. Hardy, and L. E. Hood, Sci-

ence 2tl7, 525 (1980).

8 DEFINITION [1]

TABLE II THREE CLASSES OF INTERFERON

Interferon Major producer class Stimulus for production cell types

Leukocyte Viruses Null lymphocytes Bacteria B lymphocytes Foreign cells Macrophages Mitogens for B lymphocytes

Fibroblast Viruses Fibroblasts Polynucleotides Epithelial Inhibitors of RNA and protein synthesis Myeloblasts

Lymphoblasts

T lymphocytes Immune Foreign antigens Mitogens for T lymphocytes Galactose oxidase Calcium ionophores

multiple individual species determined by distinct structural genes, a,6,7,14, ,6-1,,3z Multiple messages and genes for the human fibroblast interferons have also been reported. 33"34 The three classes of interferon have impor- tant differences in the stimulus required for induction, in the cell produc- ing them, and probably in their physiological functions (Tables II and III). In the murine system, three classes of interferon analogous to the human types appear to exist (see later chapters). Many other animal phyla, such as fish 35'36 and reptiles, 37,3s can produce their own interferons. Plants also may have an interferon-related defense against viral infection. 3a

Action of lnterferon. The binding of interferon to a cellular surface re- ceptor initiates the induction of some intracellular proteins. These pro-

30 H. Okamura, W. Berthold, L. Hood, M. Hunkapiller, M. Inoue, H. Smith-Johannsen, and Y. H. Tan, Biochemistry 19, 3831 (1980).

3~ S. Stein, C. Kenny, H.-J. Friesen, J. Shively, U. Del Valle, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 77, 5716 (1980).

32 S. Pestka, S. Maeda, R. McCandliss, W. P. Levy, P. C. Familletti, A. Sloma, and D. S. Hobbs, Proceedings of Conference on Clinical Potentials of Interferons in Viral Diseases and Malignant Tumors, Japan, December 2-4, 1980.

3a p. B. Sehgal and A. D. Sagar, Nature (London) 288, 95 (1980). 34 j . Weissenbach, Y. Chernajovsky, M. Zeevi, L. Shulman, H. Soreq, U. Nir, D. Wallach,

M. Perricaudet, P. Tiollais, and M. Revel, Proc. Natl. Acad. Sci. U.S.A. 77, 7152 (1980). 35 H. K. Oie and P. C. Loh, Proc. Soc. Exp. Biol. Med. 136, 369 (1971). 36 j . DeSena and G. J. Rio, Infect. lmmun. 11, 815 (1975). 3r E. Falcoff and B. Fauconnier, Proc. Soc. Exp. Biol. Med. 118, 609 (1975). 3s A. S. Galabov and E. H. Velichkova, J. Gen. Virol. 28, 259 (1975). ag R. Mozes, Y. Antignus, I. Sela, and I. Harpaz, J. Gen. Virol. 38, 241 (1978).

I l l DEFINITION AND CLASSIFICATION OF INTERFERONS

TABLE III PROPERTIES OF THE INTERFERON SYSTEM

Distinguishing properties Experimental test

Production 1. Normally an unexpressed genetic function

of cells 2. Requires both cellular RNA and protein

synthesis

Molecule 1. Amino acid sequence specific for interferon

species 2. Activity blocked by interferon type-specific

antibody 3. Specific recognition by monoclonal anti-

bodies 4. Intact polypeptides required for activity

Action 1. Antiviral action is through cell activation to

inhibit intracellular virus replication with- out direct action against virions

2. Induces broad biochemical alterations of cells

3. Broad antiviral action 4. Certain viruses more sensitive than others

to antiviral action

5. Action requires both cellular RNA and pro- tein synthesis

6. Generally is most effective on cells from animal species related to the producing cell species

Normally undetectable

Production blocked by metabolic in- hibitors of RNA and protein syn- thesis

Amino acid sequencing

Neutralization by type-specific anti- body

Radioimmunoassay or enzyme im- munoassay

Inactivation by proteases

Treatment of cells with interferon fol- lowed by removal of interferon leaves the cells temporarily resistant to viruses

See Table IV

Inhibits replication of diverse viruses Interferon-sensitive viruses (e.g., ve-

sicular stomatitis and Sindbis vi- ruses) are inhibited more than inter- feron-resistant viruses (e.g., herpes simplex and vaccinia viruses).

Action blocked by metabolic inhibitors of RNA and protein synthesis

Often little activity on cells from heter- ologous species; characteristic spe- cies activity profile

t e ins , e i t he r d i r e c t l y o r i n d i r e c t l y , t h e n inh ib i t mu l t i p l i c a t i on o f d i v e r s e v i ru se s , a f fec t the i m m u n e r e s p o n s e , d e c r e a s e cel l g r o w t h , and p r o v i d e the m e c h a n i s m for t he mul t ip l i c i t y o f e f fec t s o f i n t e r f e ron . Inh ib i t i on o f v i ru s mu l t i p l i c a t i on , the b e s t - u n d e r s t o o d ac t i on o f i n t e r f e ron , is i l lus- t r a t e d in the s e c o n d p a n e l o f F ig . 1. T r a n s f e r o f the i n t e r f e r o n - i n d u c e d an- t iv i ra l s ta te to a d j a c e n t , u n t r e a t e d ce l l s c a n o c c u r w i t h o u t the c o n t i n u e d p r e s e n c e o f i n t e r f e r o n ( th i rd p a n e l , F ig . 1). T h e m e c h a n i s m o f m a n y o f t h e s e e v e n t s is d e t a i l e d in c h a p t e r s o f th is v o l u m e a n d Vol . 79.

R e g u l a t i o n o f the i m m u n e r e s p o n s e t h roug h i n t e r f e r o n ac t ion on the s p e c i a l i z e d ce l l s t ha t a re r e s p o n s i b l e fo r i m m u n i t y is a ro le o f i n t e r f e ron

l 0 DEFINITION [1]

that is being studied. Although interferon inhibits many individual im- mune functions, occasionally its final effect may be to enhance the im- mune response, perhaps by inhibiting suppressor immune functions at critical times. Natural killer-cell activity and other cytolytic activities ap- pear to be enhanced by interferon.

Interferon inhibits growth of many tumor cells more effectively than it inhibits growth of normal cells. Tumor inhibition by interferon may occur directly by inhibiting the tumor cells, or indirectly by activating the body's immune cells (macrophages and lymphocytes).

Properties. The properties of the interferon system definitively distin- guish it from other defense mechanisms (Table III). Normally, the inter- ferons appear to be unexpressed genetic functions of mature cells. After induction, synthesis of interferon requires cellular RNA and protein syn- thesis. The resulting secreted interferon molecules are hydrophobic. Some are glycosylated (fibroblast interferon; probably immune inter- feron), whereas other species are largely nonglycosylated (leukocyte in- terferons). Molecular weights of interferons have been reported from 15,000 to 70,000.

Distinctive properties of the action of interferon include inhibition of virus replication through alteration of host cells, not by direct action against virions; induction of multiple biochemical alterations of cells (see below); inhibition of diverse viruses to different degrees; induction of al- teration of cells by processes requiring cellular RNA and protein synthe- sis; and a characteristic species profile so that interferon produced in one animal species is usually most active on cells from the same or related animal species. However, two human leukocyte interferons (IFLya and IFLy~) have high antiviral activity on bovine cells, but relatively low ac- tivity on human cells. 14 Other striking exceptions are likely to be found as well.

Various Effects oflnterferons. The interferons exhibit a wide range of biological and biochemical effects on cells and the whole animal (Table IV). As considered above, interferons can exert antiviral, immunoregula- tory and antitumor actions. In addition, interferons may inhibit the growth of some normal cells, alter cell membranes, activate macro- phages, increase cytotoxicity of lymphocytes, influence subsequent pro- duction of interferon, and exert hormone-like activation of cells. Avail- able information indicates that the different interferons may induce the various effects of interferon to different degrees.

Of particular interest are the hormone-like properties of the interferon system. Interferon resembles certain hormones. 4° Interferon and many polypeptide hormones activate a number of different body cells. Further

4o j . E. Blalock and E. M. Smith, Proc. Natl. Acad. Sci, U.S.A. 77, 5972 (1980).

[1] DEFINITION AND CLASSIFICATION OF INTERFERONS 1 l

TABLE IV VARIOUS EFFECTS OF INTERFERON

Biological effects Antiviral action Immunoregulatory action Antitumor action Cell growth inhibition Alteration of cell membranes Macrophage activation Enhancement of cytotoxicity of lymphocytes Influence on subsequent production of interferon Hormone-like activation of cells

Biochemical effects Induction of new cellular proteins Alteration of initiation factor elF-2 Induction of 2',5'-oligoadenylic acid synthetase Activation and possibly also induction of endonuclease activated by 2' ,5'-oligoadenylic

acid Alteration of tRNA concentrations Induction of dsRNA-activatable protein kinase that phosphorylates ribosome-associated

proteins and elF-2 Induction of protein phosphatase Induction of 2'-phosphodiesterase Changes in glycosyl transferase Membrane transport and binding alterations

evidence of the similarity is the observation that thyrotropic hormone, gonadotropic hormone, and interferon appear to bind to the same cell re- ceptor. 41 Also, as cited above, interferon has been reported to cause ef- fects in cells similar to the effects normally caused by norepinephrine; conversely, norepinephrine causes antiviral effects in cells similar to the effects caused by interferon. 4° These findings emphasize that the simple demonstration of interferon-like activity by a substance does not indicate that it is interferon.

The variety of functional cellular effects of interferon suggest a corre- sponding variety of biochemical effects (Table IV). Thus, as anticipated, interferon produces a large number of cellular biochemical and physiolog- ical changes. These are summarized in the chapter"Enzyrnology of Inter- feron Action" by P. Lengyel in this series, Vol. 79 [19]. Sections III, IV, and V of Vol. 79 provide numerous chapters describing assays for the many biochemical alterations induced by interferon. Nevertheless, al-

41 L. D. Kohn, R. M. Friedman, J. M. Holmes, and G. Lee, Proc. Natl. Acad. Sci. U.S.A. 73, 3695 (1976).

12 DEFINITION [1]

though much is known about interferon and interferon action, many of the physiological roles of the interferons are a matter for speculation at this time.

Principles of Production of Interferon for Laboratory and Clinical Use. For production of a particular type of interferon, it is necessary to select an appropriate producer cell and an appropriate stimulus (Table II). Increased production of fibroblast interferon occurs when cellular protein synthesis is altered at specific times. Fibroblast and leukocyte interferon production can be enhanced by treatment of cells with a small concentra- tion of interferon (priming). Optimum cell and nutrient concentrations must be used as well as an appropriate suspension system or surface for the cells. The time of harvest of interferon is important so that its yield can be maximized and unwanted cell products, such as cellular inhibitors of interferon production and action, can be minimized. Stability during storage is another important consideration. Specific details of high-level interferon production by various cells are described in several chapters.

Endogenous interferon may be induced within a cell system or the whole animal or man by administering any one of a number of interferon inducers. The levels and duration of endogenous interferon often exceed those achieved by administration of exogenous interferon. However, in- duction of endogenous interferon has the dual disadvantages of side ef- fects of the inducer and diminished interferon production after repeated and frequent administration of inducers. Methods for production and in- duction of endogenous interferon are considered in some chapters.

Principles of Purification. Purification of interferon has been carried out by many different methods. Binding of interferons to specific ligands attached to solid supports (concanavalin A-Sepharose, Blue Sepharose) has proved to be a useful step for the concentration and purification of interferons. Controlled-pore glass has also been used to concentrate the fibroblast and immune interferons. For the relatively stable human leuko- cyte interferons, trichloroacetic acid precipitation can be used for concen- tration of the bulk material. High-performance liquid chromatography (HPLC) can be used to purify both human leukocyte 14 and fibroblast 31'42 interferons to homogeneity. The use of HPLC for purifying proteins was applied successfully for the first time with the human interferons. 4s The special methods and equipment used in these procedures as well as the procedures themselves are outlined in the appropriate chapters dealing with the purification of the interferons. Since the interferons are generally

4e H.-J. Friesen, S. Stein, M. Evinger, P. C. Familletti, J. Moschera, J. Meienhofer, J. Shiv- ely, and S. Pestka, Arch. Biochem. Biophys. 206, 432 0981).

43 M. Rubinstein, S. Rubinstein, P. C. Familletti, M. S. Gross, R. S. Miller, A. A. Waldman, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 76, 640 (1979).

[1] DEFINITION AND CLASSIFICATION OF INTERFERONS 13

hydrophobic proteins, reverse-phase HPLC on octyl silica 14"31 and other supports 4~ has been used effectively to purify interferons. In addition, it is necessary to use polypropylene tubes or siliconized glassware wherever possible to avoid losses due to binding of interferon to surfaces. Rapid assay of the interferons by procedures described in these volumes are useful in monitoring the purification procedures.

With the availability of monoclonal antibodies to the interferons, puri- fication has been simplified and the overall yield of purification increased. Monoclonal antibodies have provided rapid purification of the interferons from relatively crude media. 11,1z,44 For example, interferon produced by bacterial recombinants has been purified to homogeneity with monoclonal antibodies bound to solid supports. 44

Interferon as a Natural Defense against Viruses. Interferon is the most rapidly produced of the known body defenses against viruses. Pro- duced within hours of virus infection, interferon continues to be produced throughout the infection. A causal relationship between interferon and natural recovery from most virus infections is strongly supported by evi- dence developed since the discovery of interferon. This evidence has been reviewed elsewhere. 4s

Medical Application to Virus Diseases and Cancer. A number of virus diseases appear to be amenable to experimental treatment with inter- feron. The virus diseases include rabies, hepatitis, respiratory virus infec- tions, encephalitis, and eye infections. In addition, interferon may have a role in the therapy of cancer. So far, initial clinical trials with interferon to treat certain viral diseases 4e and cancer are encouraging. 47-4a Substantial supplies of interferon are required to perform the clinical trials to evaluate this properly. Alternatively, endogenous interferon production may be ar- tificially stimulated within the body.

Interferon for therapeutic use may be produced by stimulating human cells in culture. Many chapters in this volume describe these procedures. In addition, production of interferon in bacteria containing recombinant plasmids with interferon sequences, 7,s,21-24 and purification of this inter- feron 44 provides an economical and large source of the human interferons

44 T. Staehelin, D. S. Hobbs, H.-F. Kung, and S. Pestka, this volume [7i]. 4s S. Baron, ASM News 45, 358 (1979). 48 R. B. Pollard and T. C. Merigan, Pharmacol. Ther. Part A. 2, 783 (1978). 47 H. Strander, K. CanteU, G. CarlstrOm, and P. A. Jakobsson, J. Natl. Cancer Inst. 51,733

(1973). 4s T. C. Merigan, K. Sikora, J. H. Breeden, R. Levy, and S. A. Rosenberg, N. Engl. J. Med.

299, 1449 (1978). 49 j. U. Gutterman, G. R. Blumenschein, R. Alexanian, H.-Y. Yap, A. U. Buzdar, F. Ca-

baniUas, G. N. Hortobagyi, E. M. Hersh, S. L. Rasmussen, M. Harmon, M. Kramer, and S. Pestka, Ann. Intern. Med. 93, 399 (1980).

1 4 D E F I N I T I O N [ 2 ]

for clinical as well as basic research. In fact, clinical trials with a recom- binant interferon, IFLrA, were initiated in January 1981. With the avail- ability of sufficient amounts of human interferon for appropriate clinical studies, information regarding the safety and efficacy of human interferon will soon be forthcoming. Nevertheless, because numerous human leuko- cyte interferon species as well as fibroblast and immune interferons exist and because a large number of synthetic combinations and varieties are possible, defining specific ones with optimal activity against a given dis- ease will be a continual process of refinement that will occupy scientists for many years.

[2] S t a n d a r d i z a t i o n o f A s s a y o f I n t e r f e r o n s

By NORMAN B. FINTER

There is a need in almost all aspects of interferon (IF) research to be able to measure the amounts of IF involved. Until now, such measure- ments have usually made use of one important property of IF, namely the ability to render cells resistant to infection with a virus. Thus in a typical antiviral bioassay a series of dilutions of the IF are made, for example, in twofold steps, and each is added to one or more replicate tissue cultures of an appropriate cell. The cultures are incubated, usually overnight, and in those treated with a sufficient amount of the interferon, an antiviral state develops in the cells. The cultures are then all challenged with-a con- venient virus; after further incubation for an appropriate time, the extent of virus growth in each culture is determined in some way. The relation between the amount of virus growth and the amount of IF (usually the logarithm of the reciprocal of the dilution) is established graphically or by calculation. Arbitrarily, some particular degree of inhibition of virus growth, such as 50%, is taken to define the end point; the reciprocal of the corresponding dilution of interferon gives the potency of the preparation in "units ." An appropriate reference IF preparation is included in the assay, and from the result obtained with this, the triers for the other prepa- rations are adjusted to standardized "laboratory units," or, when possi- ble, to international units (IU) (see below). Titers are usually expressed in units per milliliter; in most assay systems, quite big changes in the ac- tual volume of diluted IF used in the assay make little or no difference to the end point obtained.

Although such bioassays are clumsy, tedious, and relatively inaccu- rate, they are still necessarily used in most routine work for two reasons:

Copyright ~ 1981 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL 78 All rights of reproduction in any form reserved.

ISBN 0-12-181978-7