The Induction of Interferon by Natural and Synthetic Polynucleotides

The Induction of Interferon by Natural and Synthetic Polynucleotides CLARENCE COLBY,JR. Department of Biology, University of California, Sun Diego, La Jolla, California

I. Introduction . . . . . . . . . . . A. Discovery of Interferon . . . . . . . . B. Properties of Interferon . . . . . . . . . . . . 11. The Induction of Interferon by Viruses . 111. The Induction of Interferon by Nonviral Agents . . . . A. Induction versus Release of Preformed Interferon . . . B. Induction of Interferon by Microorganisms . . . . . . . . C. Induction of Interferon by Polyanions . D. Induction of Interferon by Double-Stranded RNA . . E. Induction of Interferon by Synthetic Polynucleotides . . IV. Double-Stranded RNA in Cells Infected with DNA-Containing Viruses . . . . . . . . . . . . . . . . A. Vaccinia Virus Double-Stranded RNA . . B. Double-Stranded RNA in Other DNA-Virus Systems . . V. Discussion of the Mechanism of Induction of Interferon . A. The Nature of the Intracellular Receptor Site . . . B. Intracellular Processes Occurring after the Inducer-Receptor . . . . . . . Site Complex Is Formed . C. The Nature of the Inducer Molecule . . . . . References . . . . . . . . . . .

1 1 2

4 5 5 6 G 8

10 21 21 22

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1. Introduction

A. Discovery of Interferon In 1957 Isaacs and Lindenmann ( 1 ) treated portions of the chorioallantoic membrane of embryonated chicken eggs with heat-inactivated influenza virus for 3 hours, then incubated the membrane fragments in fresh solutions of buffered saline for an additional 24 hours. When the fragments were removed, the solutions contained a substance that interfered with viral replication. They named this antiviral substance “interferon,’’ It could be quantitated by measuring the reduction in yield of infectious virus particles produced from membrane fragments pretreated with various dilutions of interferon prior to infection. The antiviral activity was nondialyzable, sensitive to proteolytic digestion with trypsin, 1

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CLARENCE COLBY, JR.

stable at high and low pH, heat-stable, and insensitive to antibodies against the strain of influenza virus used to induce its formation (1-3).

B. Properties of interferon 1. PHYSICAL AND CHEMICAL PROPERTIES OF INTERFERON

The observations cited above suggested that interferon is a protein, and this has been confirmed in many laboratories. Indeed, the purification of interferon involves standard techniques of protein chemistry such as : ( a ) precipitation with ammonium sulfate or by acidification; ( b ) solution of interferon or impurities in organic solvents; ( c ) selective adsorption of interferon to a solid absorbent, followed by its elution; ( d ) separation of interferon from contaminants on ion exchangers; ( e ) purification by gel filtration; and ( f ) purification by electrophoresis ( 4 ) . The first work in this area was done by Burke ( 5 , 6) who obtained a limited purification of chick interferon. Lampson et a2. (7) obtained a 4500-fold puri6cation of chick interferon. These investigators also demonstrated that the physicochemical properties of chick interferon induced by herpes simplex virus are identical with those of the interferon induced by influenza virus (8). The best purification of interferon was achieved by Merigan et al. ( 9 ) . The interferon was purified 20,000-fold and contained more than lo6 unitslmg protein. The physical and chemical properties of interferon may be summarized as follows ( 4 ) . Interferon is a protein containing most of the common amino acids and some carbohydrate, including glucosamine. Disulfide groups, amino groups, and the methyl group of methionine are required for antiviral activity. Substitution on sulfhydryl or hydroxyl groups does not affect biological activity. Interferon is isoelectric at pH 6.5-7.0 but is stable from pH 2 to 10. The protein appears to have a molecular weight between 25,000 and 35,000,and hence is probably a single polypeptide chain. However, there are numerous reports of interferons with molecular weights as large as 150,OOO. These might be aggregates of interferons with other proteins, or they might be self-aggregates. Furthermore, the monomeric interferons of different species differ in molecular weights. Thus, one should refer to “the interferons” when discussing the field in general and reserve the singular form for those discussions that refer to a particular cell system and a particular inducer.

PROPERTIES OF INTERFERON 2. BIOLOGICAL The greatest dilemma facing anyone attempting to draw definite conclusions from the vast literature about the biological properties of inter-

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ferons is the fact that the majority of experiments used crude preparations. Many investigators have found a variety of specific and nonspecific viral inhibitors in interferon preparations. Lockart ( 10) has suggested the following criteria for the acceptance of a viral inhibitor as an interferon: ( i ) The inhibitor must be a protein and be formed as a result of the addition of an inducing substance to cells or animals. (ii) The antiviral effect must not result from nonspecific toxic effects on the cells. (iii) The proposed interferon must inhibit the growth of viruses in cells through some intracelhlar action involving both RNA and protein synthesis on the part of the cells. (iv) The inhibitor must be active against a range of unrelated viruses. If an interferon shows marked specificity for homologous cells, this is highly suggestive that it is in fact an interferon. a. Species Specificity. Interferons appear to be synthesized from messenger RNA transcribed from a host gene rather than from viral genetic information. The evidence for this statement is overwhelming. Inhibition of host RNA synthesis, either with a virus (11) or with actinomycin D ( 12, 13), reduces or eliminates interferon production. Secondly, a variety of nonviral substances can induce interferon (see Section 111). Finally, the interferon produced by one cell type is biologically active only on cells of the same species but against a variety of viruses. To demonstrate this last point, the use of p u d e d interferon is particularly important. Buckler and Baron ( 1 4 ) obtained reduced yields of vaccinia virus from chick cells treated with chick interferon, normal allantoic %uid, mouse serum interferon, and normal mouse serum. However, if the cultures were carefully washed after interferon treatment prior to infection with vaccinia virus, then only the chick interferon exhibited antiviral activity. Using interferons purified 6000-fold from chick and mouse cells, Merigan (IS) clearly demonstrated the species specficity of both preparations. b. Lack of Virus Specificity. The sensitivity of animal viruses to a given preparation of interferon varies remarkably ( 1 6 ) . Of the RNA-viruses, the arboviruses appear to be among the most susceptible, the myxoviruses running a close second; Newcastle disease virus is the most resistant. A definitive study was recently done by Stewart et al. ( 1 6 ) . They determined the relative sensitivities of Sindbis, vesicular stomatitis, Semliki Forest, and vaccinia viruses to interferon-treated mouse embryo, hamster kidney, rabbit kidney, human embryonic lung, and bat embryo cells. They found large cell-dependent variations of sensitivity of the viruses. For example, vaccinia was the least sensitive virus in human, rabbit, and bat cells but the most sensitive in mouse and hamster cells. c. Mechanism of Action of Interferon. Interferons do not directly inactivate virus particles (17, 1 8 ) , nor do they inhibit virus adsorption,

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CLARENCE COLBY, JR.

penetration, or uncoating (19). Synthesis of both viral RNA and protein is inhibited in cells pretreated with interferon, but this inhibition is not seen if host RNA and protein synthesis are eliminated during the pretreatment ( 2 0 ) . Thus, it appears that interferon causes a stimulation of synthesis of an antiviral protein. This antiviral protein may act by inhibiting the translation of viral messenger RNA ( 2 6 2 2 ) .

II. The Induction of Interferon by Viruses

Since the initial experiments by Isaacs and Lindemann ( 1 ) with heatinactivated influenza virus, virtually all classes of animal viruses have been shown to elicit an interferon response in an appropriate host (23, 2 4 ) . Not only are the arboviruses and the myxoviruses among the most sensitive to the antiviral action of interferons, but they are also good inducers. These groups contain the largest number of interferon inducers. However, the amount of interferon induced by the various members of these groups varies markedly. Thus, it seems likely that the level of interferon response is a genetic characteristic of the virus, even though the interferon itself is a host-determined protein. The enteroviruses appear to be poor inducers of interferon. However, it is important to remember that infection with enteroviruses, such as Mengo virus (25) and poliovirus ( 2 6 ) , is accompanied by a rapid inhibition of cellular RNA synthesis. Thus, these viruses may be blocking the synthesis of the messenger RNA for interferon. In support of this idea, Johnson and McLaren (27) reported good induction of interferon b y the RMC strain of poliovirus, which does not appear to shut off cellular RNA synthesis. Similarly, four virulent strains of poliovirus do not induce interferon whereas five strains of low virulence do ( 2 3 ) . The reports on the induction of interferon by DNA-containing viruses are sparse. Vaccinia virus is the DNA virus most studied with respect to its ability to induce interferon (28-30) and the effect of interferon on its replication ( 2 1 , 3 1 , 3 2 ) .Fruitstone et al. ( 3 3 ) found that UV-irradiated herpes simplex virus induces interferon in chick embryos. Human adenovirus induces interferon in vivo in chicks ( 3 4 ) and in vitro in chick embryo fibroblasts ( 35). A particularly interesting virus with respect to the interferon system is Newcastle disease virus. As mentioned above (Section I, B, 2, b ) , this virus is quite insensitive to the antiviral activity of interferon. However, the induction of interferon by this virus depends on the state of the virus and of the host system employed. It grows well in chick embryo cells, yet it induces little or no interferon in this system ( 3 6 ) . On the other hand, infection of mouse L-cells with Newcastle disease virus results in abortive replication (37) and the production of high titers of interferon

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(38). A complete study of this phenomenon by Youngner et al. (39) showed that both infective and UV-treated Newcastle disease virus stimulated interferon production in mice and in mouse L-cells. Continued exposure of the virus to ultraviolet irradiation did not depress the interferon-inducing capacity of the virus. They also found that the infective virus did not elicit an interferon response in chick embryo fibroblast cultures. However, as the infectivity of the virus preparation was eliminated by UV irradiation, the induction of interferon became maximal. Furthermore, continued irradiation of the virus was accompanied by a rapid loss of its capacity to induce interferon. Finally, heat-inactivated virus was ineffective in stimulating interferon production from either cell type in uitro, but it was able to cause the release of serum interferon in mice. In another interesting series of experiments, De Clercq and De Somer ( 4 0 ) found that the exposure of Newcastle disease virus to an acid environment caused a complete loss of infectivity, and when the virus was mixed with 500 pg of yeast RNA per milliliter prior to acid treatment, the infectivity was reduced to 0.1-0.01%of original levels. However, the addition of RNA at pH 2 to the virus resulted in a much earlier appearance of interferon when compared with virus that had not been treated with RNA. The cell system used was a continuous rabbit kidney cell line, RK13. Thus, it appears that in some permissive cell culture systems, such as chick embryo and rabbit kidney cells, the lytic infective cycle of Newcastle disease virus is accompanied by the production of a viral function that interferes with the induction of interferon. When this virus function is eliminated by ultraviolet irradiation or infection in a nonpermissive host, such as mouse L-cells, the interferon-stimulating system becomes unencumbered and the antiviral protein is made and released.

111. The Induction of Interferon by Nonviral Agents Investigators in the interferon field between 1957 and 1963 concentrated on the induction of these antiviral proteins by infectious or inactivated animal viruses. Because of the obvious potential therapeutic value of protecting an animal against a variety of viral infections by the animal's own interferon system, many workers began searching for nontoxic, nonviral inducers of interferon. The results of these efforts have been recently reviewed by Ho and his colleagues (41, 4 2 ) .

A. induction versus Release of Preformed interferon Many of the nonviral substances are active only in intact animals, only if injected intravenously or intraperitoneally, and often require large

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CLARENCE COLBY, JR.

dosages. It was therefore suspected that the appearance of interferon in the bloosd of animals treated with these substances might be the result of a mechanism quite different from that in virus-infected animals. Specifically, it was suspected that viruses inducc the synthesis of interferon while the nonviral materials cause the release of interferon from a preformed pool. The induction of interferon by Sindbis virus and by bacterial endotoxin is a model system for considering this point. The requirement for RNA and protein synthesis for the induction of interferon was studied in the rabbit using actinomycin D and puromycin, respectively (43, 44). The production of interferon by Sindbis virus was sensitive to both drugs whereas neither affected the production of endotoxin-induced interferon. Furthermore, virus-induced interferon was sensitive to body temperature and insensitive to the elimination of corticosteroids by adrenalectomy. In contrast, the induction of interferon by endotoxin was unaffected by changes in body temperature and was increased 10-fold after the animal had undergone adrenalectomy (45, 46).

B. Induction of Interferon by Microorganisms Many microorganisms other than animal viruses elicit the appearance of interferon (41, 42). These include rickettsia, live and killed bacteria, trachoma-inclusion conjunctivitis agent, mycoplasma, and protozoa. Various products of microorganisms such as bacterial endotoxins and fungal preparations such as statolon and helenine are also active. It is likely that the appearance of interferon in animals following the injection of microorganisms is due to the release of preformed interferon. In the case of the Gram-negative bacteria, endotoxin is probably responsible for interferon production. In the cases of the other microorganisms, the active principle has not been discovered. Intracellular growth of bacteria is not required for interferon production in animals, whereas the growth of Rickettsia tsutsugamuchi is essential for the production of interferon (47, 48).

C. Induction of Interferon by Polyanions 1. FUNGAL EXTRACIS

A filtrate of Penicillium stoloniferum was shown, in 1952, to have antiviral activity ( 4 9 ) . Ten years later, a second active filtrate was obtained and was called “statolon” (50). Kleinschmidt et al. ( 5 1 ) showed that preparations of statolon, a complex anionic polysaccharide, induces

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interferon in chick embryo cells and that the statolon-induced interferon has the same properties as those of virus-induced chick embryo interferon. Youngner and Stinebring ( 52) compared the interferon production in mice by statolon and bacterial endotoxin. Since cycloheximide did not reduce the level of circulating interferon in mice injected with statolon, these workers concluded that statolon stimulates the release of preformed interferon in mice. However, they also reported differences in the kinetics of appearance and in the failure to induce cross tolerance. These results indicated that endotoxin and statolon induce preformed interferon either from different cell populations or by different mechanisms. There are other fungal extracts that have interferon-inducing or releasing properties. Helenine, an exbact of Peniciltium funiculosum, is an active inducer of interferon (53,5 4 ) . Interferon may be induced by extracts of P . chrysogenum, P . cyaneo-fulvum ( 5 5 ) and from extracts of the mushroom Cortinellus shiitake ( 56). The active principle responsible for interferon induction by all of these extracts is known and is discussed below in Section 111, D. 2. PYRANCOPOLYMERS Regelson (57) discovered that synthetic copolymers of maleic acid anhydride induce interferon in mice while De Somer and his colleagues ( 58) have thoroughly studied the antiviral properties of polyacrylic and polymethacrylic acids. Merigan ( 59) investigated the release of circulating interferon in mice injected with a variety of synthetic polyanions of known chemical composition. Polymers containing maleic anhydride, divinyl ether, vinyl methyl ether, vinyl acetate, and styrene were examined together with similar polymers modified by amidation or methyl esterification. The structural characteristics of these polymers required for interferon-releasing activity have been discussed by Merigan ( 60). 3. NUCLEIC ACIDS

Isaacs (61) offered the suggestion that viruses might elicit the induction of interferon by presenting a foreign nucleic acid to the cell. This suggestion was tested in two ways. Preparations of chick liver and mouse liver RNA were incubated with chick and mouse cells in culture at the time of infection with vaccinia virus ( 6 1 ) .The RNA from heterologous species was more effective in inhibiting vaccinia virus growth than homologous RNA. The second series of experiments involved treating chick, mouse, and rabbit cells with homologous and heterologous ribosomal RNA (62). Homologous RNA induced interferon only if it was first treated with nitrous acid, a deaminating agent, whereas heterologous RNA induced interferon without chemical modification. However,

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“foreign” nucleic acid did not appear to be the whole story. That is, other foreign nucleic acids, such as rat liver DNA and RNA from encephalomyocarditis virus, turnip yellow mosaic virus and E. coli, did not induce interferon production ( 6 2 ) .

D. Induction of Interferon by Double-Stranded RNA By 1966, nine years after the discovery of interferon ( I ) , the antiviral substance had been partly characterized physically and chemically, it had been shown that both viral and nonviral agents could elicit the production of interferon, and it was known that there were two fundamentally different mechanisms for its production, viz., release of interferon from preformed pools (usually by nonviral agents in intact animals) and induction, a process involving the requirement for de m o o RNA and protein synthesis. Yet, the molecular basis for both the induction of interferon and the action of interferon remained a mystery. The series of events leading to the experimentation in the last few years with respect to the induction of interferon by natural and synthetic polynucleotides is intriguing. Braun and Nakano reported that oligodeoxynucleotides ( 6 3 ) and complexes of poly(A) and poly(U), or of poly(C) and methylated bovine serum albumin ( 6 4 ) , stimulate the formation of antibodies in mice. In the latter report, single-stranded polynucleotides had no adjuvant activity. These observations prompted Field et al. (65) to examine the capacity of polynucleotides to induce interferon in rabbits, in rabbit spleen cell suspensions and in primary cultures of rabbit kidney cells. Poly( I) -poly(C) was the most active complex, but poly( A ) .poly( U ) and poly( I ) an( CpC) were also active. The interferon was characterized by its species specificity, trypsin sensitivity, isoelectric point, and molecular weight. The single-stranded polymers, ( I ),,, ( C) n, ( A ) n, and ( U ) were inactive. Lampson et al. (66) made the important observation that naturally occurring double-stranded polynucleotides induce interferon. Helenine, the mycelial extract of P . funicuEosum which is an active interferon inducer), was deproteinized with phenol and purified by chromatography on ECTEOLA-cellulose. The purified RNA (free of detectable polysaccharide, protein, and DNA), in microgram quantities, induced interferon in rabbits and resistance to viral infection in mice. The RNA was characterized as double-stranded RNA by its thermal stability, its reduced thermal stability at low ionic strength or in the presence of formaldehyde, and its resistance to ribonuclease. When the RNA was “melted” it was

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sensitive to ribonuclease and was no longer an active interferon inducer. Various single-stranded RNA's and double-stranded DNA at 100-1000 times higher concentrations elicited no interferon response. Lanipson et al. (66) postulated that the double-stranded RNA in helenine might reflect the presence of a fungal virus with double-stranded RNA either as its genetic information or as its replicative form. Kleinschmidt and Ellis ( 67, 68) centrifuged preparations of statolon, the interferon-inducing preparation from P . stoloniferurn, on sucrose gradients and found two bands, both of which were active. When the heavier band was examined by electron microscopy, hexagonal particles measuring 30 nm were found. No particles were found in the inactive fractions from the sucrose gradients. The lighter visible band contained interferon-inducing activity but did not contain viruslike particles. Banks et al. (69) confirmed the presence of virus particles in statolon and found virus particles in helenine as well. The purified viruses were shown to be serologically different. In a subsequent study, Kleinschmidt et a2. ( 7 0 ) demonstrated that the statolon virus particles, which he called mycophage PSI, contained complementary RNA and that the active material in the lighter band in the sucrose gradients was double-stranded RNA. The interferon-inducing activity in extracts of P . cyaneo-fuluurn is associated with the double-stranded RNA of a polyhedral virus (55) and the activity in extracts of the mushroom Cortinellus shiitake is also associated with double-stranded RNA (56). The double-stranded RNA of fungal viruses is not unique in its capacity to induce interferon. Tytell et al. ( 7 1 ) purified noninfectious double-stranded RNA from reovirus type 3, and Field et al. ( 7 2 ) isolated the double-stranded replicative form of MS2 coliphage. Both preparations were extremely active in inducing interferon and resistance to viral infection (71, 7 2 ) . In both of these studies the antiviral activity induced by the double-stranded RNA's was subjected to the appropriate experimental tests and identified as interferon. RecentIy the list has been expanded to include such RNA isolated from rice dwarf virus virions, cytoplasmic polyhydrosis virions ( 7 3 ) , and from cells infected with mengo virus ( 7 4 ) ,influenza vims (751, and vaccinia virus ( 7 5 ) . Thus, it appears that double-stranded RNA from a variety of sources is capable of inducing interferon in vivo and in vitro. Because of the high specific activity for interferon induction by double-stranded RNA, as compared with the large doses required of the other nonviral inducers, Field et al. ( 7 2 ) postulated that the double-stranded replicative form of RNA present in cells infected with RNA-containing viruses is responsible for starting the interferon response.

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E. Induction of Interferon by Synthetic Polynucleotides 1. INDUCTION OF INTERFERON BY POLY ( I ) ‘POLY( C ) IN VIVO a. Animal-Vim Systems That Respond to Poly(Z). P o l y ( C ) .The first use of poly(I).poly(C) as an inducer of interferon in intact animals was by Field et al. (65). These workers found that ( I),. (C ) ,induces interferon in rabbits and resistance to viral infection in mice. Table I lists some of the other animal-virus systems that respond to ( I ) n * (C), treatment. In some cases, serum interferon titers were measured. In others, ( I ) * ( C ) was shown to prolong survival of animals infected with lethal doses of virus, to cause the regression of tumors, or to prevent tumor growth. Park and Baron ( 7 7 ) made the important discovery that 11

TABLE I In Viuo R.ESPONSETO I’OLY(I).PoLY(C) Animal

Virus, t,umor, or disease

Assay

Vesicular stomat.itis virus Mouse pneumonia vinrs Columbia SK Plasmodium bwghei FIerpes simplex virus

Interferon Titer Survival Survival Survival Recovery from herpatic kerator conjunctivitis Decreased growth rate Reticulum cell sarcoma, Mouse lymphoma, fibrosarcoma, of tumor or sirrvival leukemia, adenovinis 12 tumor Mouse Decreased growth rate Mouse sarcoma vinis of tumor Mouse Interferon tit,ers and Mengo virus, vesicular stomatitis virus survival Mouse Japanese B encephalitis Survival virus Hamster Adenovirus 12 Number of tumors Hamster Simian virus 40 Number of tumors Hamster Friend leukemia virus Survival Mouse Survival RClp tumor

Rabbit Mouse Mouse Mouse Rabbit

Mouse

Chick

Vaccinia virus Sendai virus Influenza virus Yellow fever virris Rabies vinrs Brain Ilous sarcoma vinis

Tail lesions Survival Survival Survival Survival Tumor growth

Reference Field el al. (66) Jahiel et d. (76) Park and Baron (77), Park el al. (78), Pollikoff et al. (79) Levy et al. (80)

Sarma el al. (81) Merigan el al. (88) Postic and Sather (83) Larson el aE. (8.4) Larson et al. (86) Larson et al. (86) Greaser and Bourali (87) Nemes et al. (88)

Nenies el al. (88)

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( I )n. ( C ) treatment allowed rabbits to recover from herpetic keratoconjunctivitis. Youngner and Hallum (80) have concluded that poly( I ) -poly(C ) is similar to endotoxin rather than viruses in its mechanism of interferon stimulation. That is, the induction of interferon in intact animals by poly( I ) -poly(C ) is not sensitive to cycloheximide, while that induced by Newcastle disease virus is sensitive, suggesting that the former does not require de nouo protein synthesis. However, this could be due to the inhibition of a cycloheximide-sensitive step in the viral infection cycle. b. Toxic Effects of Poly( I ) * Poly ( C ) . The possibility of using nonviral interferon inducers as prophylactic agents against viral diseases in man has intrigued virologists for several years. Since poly( I ) *poly(C ) is active at such low doses ( 6 5 ) , it was immediately considered to be a promising agent for clinical studies. There have been several recent reports concerning side effects of poly( I ) -poly(C ) treatment. Absher and Stinebring (90) found that 500 pg of ( I ) n - ( C ) nwas lethal to one-half of the mice that were injected. The symptoms of the affected animals suggested an involvement of the central nervous system. In addition, ( I ) n -( C ) , enhanced the lethality of lead acetate in these mice. The authors postulated that ( I ) (C), may act in the intact animal as a stimulator of interferon precisely because of its toxic effects. Lindsay et al. (91) found that very small doses of poly( I ) *poly(C ) cause fever in rabbits. Adamson and Fabro (92) found that at much higher doses, poly( I ) .poly( C ) exerts an embryotoxic effect in rabbits. Treatment of females with 2 mg per kilogram body weight on the eighth and ninth days of pregnancy resulted in 80%resorptions. The incidence of malformed fetuses was only slightly increased by ( I ) n .( C ),treatment. Poly( I ) .poly( C ) at very high doses causes cerebellar symptoms and death within an hour in young chickens but not in rabbits (93). Of all the animal species tested, the dog was the most sensitive to repeated administration of ( I ) * ( C ) (94). The effects, including retching, emesis, diarrhea, tremors, and convulsions, appear to have a vascular, hepatic, or hematologic basis. Necrotic changes appear in the liver, bone marrow, bone, spleen, and other organs. In contrast, the monkey showed no severe toxic effects, particularly when the helical polynucleotides were given intranasally ( 94). Diploid human cell strains in culture treated continuously with poly( I ) .poly( C ) show no change in the growth rate, plating efficiency, or morphology. There is no neoplastic transformation in these cells, nor any change in the karyotype (94). Poly(I).poly(C) has been tested on a limited scale in man. TWO patients with advanced cancer (one with reticulum cell sarcoma and one ,1

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wid1 transitional cell sarcoma of the urinary bladder) were given ( I ) n *( C ),. Both showed positive interferon titers in their sera. Two other patients failed to respond. One of the four exhibited transitory fever, and there were no other clinical symptoms of toxic effects (94). Poly(1) poly(C) has also been found to protect human volunteers against rhinovirus infection (95).

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2. THE IN VITROINDUCTIONOF INTERFERON BY POLY( I) -POLY(C )

a. Cell-Virus Systems That Respond to Poly( I ) .Poly(C). Since Field et al. ( 6 5 ) discovered that poly( I ) *poly(C ) induces interferon in rabbit cells in culture, there have been confirmatory reports from many laboratories. Some of the systems studied are listed in Table 11. The cell systems differ from each other with respect to the amount of poly( I ) *poly(C ) required to induce detectable levels of interferon. This is probably due to at least two factors: first, the capacity of the cells to take up the inducer may differ, and second, the capacity of the cells to recognize and respond to the inducer may also differ. The first point was emphasized by the discovery by Dianzani et al. (96) that the interferon-inducing capacity of poly( I ) .poly( C ) in mouse L-cells is enhanced 100-fold by the presence of DEAE-dextran. An enhancement by DEAE-dextran of the antiviral activity of ( I ) * ( C ) has also been found in rabbit kidney cells (98, 102), human leukocytes, and human amniotic membrane cells (79), and chick embryo fibroblasts (10). The polycation might exert its stimulatory action either by protecting the polynucleotide from enzymatic degradation or by facilitating its uptake to an intracellular site. The enhancement of interferon-stimulating activity of ( I ) n ( C ) ,, by a number of polycations, such as neomycin, streptomycin, DEAE-dextran, methylated albumin, protamine, histone, and colistin, was studied by Billiau et al. (102), who were unable to choose between these two mechanisms. However, the results of subsequent experiments have led Dianzani et al. (106) to conclude that the activity of DEAE-dextran in L-cells is exerted mainly by increasing the cell permeability to the inducer rather than by protecting it against RNase. A similar conclusion was reached for the action of DEAE-dextran on the uptake of labeled ( I ),,.( C )II by chick embryo fibroblasts (100, 107). b. Znduction versus Release by PoZy(Z) -Poly(C) in Vitro. As pointed out above (see Section 111, A), interferon can arise by two presumably different mechanisms, release from a preformed pool or de nova induction. Finkelstein et al. ( 9 7 ) studied the induction of interferon by Newcastle

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TABLE I1 In Vitro RESPONSETO PoLY(I).PoLY(C) ~

~~~

Virus

Assay

Vesicular stomat,itis virus Vesicular stomat.it>isvirus Semliki Forest virus Vesicular stomatit,is virus

Interferon titer Interferon titer Irit,erferon titer Interferon titer

Field et al. (66)

Vesicular stomatitis virus

Interferon titer

Finkelstein el al.

Vesicular stomatitis virus Vesicular stomatitis virus

Interferon titer Interferon titer

Human amniotic membrane Chick embryo fibroblasts

Vesicular stomatitis virus

Interferon titer

VilEek et al. (98) Falcoff and PerezBercoff (99)

Sindbis virus

Colby and Chamberlin (100)

Chick embryo fibroblasts Chick embryo fibroblasts Rabbit kidney Mouse embryo Mouse embryo Mouse embryo Human fibroblasts Rabbit kidney Rabbit kidney Dog kidney Calf kidney Hamster kidney Mouse embryo Human fetal lung Human amniotic membrane Hiinian kidney Human kidney

Vesicular stomatitis virus

Interferon titer and resistance Resistance

Vaccinia virus

Resistance

Colby (101)

Semliki Forest virus Vesicular stomatitis virus Mouse sarcoma virus Friend leukemia virus Human cytomegalovirus Vaccinia virus Herpes simplex virus Vesicular stomatitis virus Vesicular stomatitis virus Vesicular stomatitis virus Vesicular stomati tis virus Vesicular stomatitis virus Vesicular stomatitis virus

Resistance Resistance Resistance Resistance Resistance Resistance Resistance Resistance Resistance Resistance Resistance Resistance Resistance

Billiau et al. (10.2)

Vesicular stomatit,is virus Rhinovims

Resistance Resistance

Cells Rabbit spleen Rabbit kidney Mouse L-cells Human skin fibroblasts Mouse peritoneal macrophage Rabbit kidney Human leukocytes

Reference

Dianzani et al. (96) Finkelstein et al. (97)

(97)

Colby (101)

Rhim et al. (103) Rabson et al. (104) Field et al. (106)

disease virus, statolon and ( I ) n * ( C ) nin human skin fibroblasts. Virusinduced interferon appeared later and was more sensitive to actinomycin D and puromycin than ( I)n- (C).-induced interferon. Finkelstein et al. concluded that either two different mechanisms of induction are operative or both inducers result in de novo synthesis of interferon, but that an extra process more sensitive to antimetabolites, such as uncoating, is involved in the case of virus-induced interferon. Pretreatment of rabbit

CLARENCE COLBY, JR.

14

kidney cells (98, IOS),chick embryo fibroblasts (100), and human leukocytes and amniotic membrane cells (99) with actinomycin D results in the reduction or elimination of ( I ) ( C ) .-induced interferon. These results were interpreted to indicate that ( I ) n * ( C ) ninduces the de not10 synthesis of interferon. Recent results from VilEek's laboratory (108,109) provide new insight into this problem. In one set of experiments, rabbit kidney cells were pretreated with either actinomycin or puromycin, and then interferon was induced with ( I ) ( C )n. Interferon production was blocked with actinomycin but not with puromycin. However, when the antimetabolites were added 3.5hours after the inducer, the opposite result was obtained. That is, puromycin inhibited interferon production while there was a 4-fold increase in interferon in the actinomycin-treated cells (108). The authors concluded that poly( I ) -poly(C ) causes the release of preformed interferon, and they postulated an inhibitor of this release ( 1 0 8 ) .VilEek has extended these observations to include the effects of treatment at various times with cycloheximide (109). When poly( I ) -poly( C) alone is given to the cells, maximum interferon release is seen at 4 hours. In the presence of cycloheximide, interferon release continues and is maximal at 22 hours. Mouse L-cells did not show this effect with cycloheximide. VilEek's current views are that poly( I ) .poly( C) probably induces the de novo synthesis of interferon, this synthesis being sensitive to actinomycin but resistant to cycloheximide, and that there is an interferon inhibitor in rabbit kidney cells that normally depresses the release of interferon after 4 hours but that is sensitive to cycloheximide (109). Bausek and Merigan (110) infected human fibroblasts with Newcastle disease virus and after 1 hour treated the infected cells with poly( I ) poly( C ) . The induction of interferon by ( I).. ( C ) occurred early and appeared to be terminated shortly before the appearance of the virusinduced interferon. Since full yields of viral interferon (interferon released at late times) were found immediately after the repression of ( I ) * ( C ) .-induced interferon, it appeared that the repressor does not directly inactivate interferon itself. Rather, the authors suggest that a repressor substance blocked the continued formation of nonviral interferon, without significantly affecting the induction of viral interferon (110).

-

-

.

-

.

3. THE SPECIFICITY OF INTERFERON INDUCTION BY VARIOUSPOLYNUCLEOTIDES

.

a. Is the Polynucleotide-Induced Resistance to Viral Infection Due to Interferon? The in vitro experiments using ( I ) ( C ) described above (Section 111, E, 2, b ) indicate that the polynucleotide can induce cells

15

THE INDUCTION OF INTERFERON

to synthesize interferon. Many workers have also found that concentrations of (I)..( C ) . far too low to induce detectable interferon in the culture medium confer a state of viral resistance on cells in culture (100105). They have assumed that the resistant state of ( I ) ( C ).-treated cultures is mediated by interferon. This view has recently received strong support from a series of experiments carried out by Schafer and Lockart ( 1 1 1 ) . Two stable cell lines derived from African green monkey kidney cells were used: Vero cells, which are able to respond to monkey interferon but which cannot be stimulated to make interferon ( 1 1 2 ) ; and LLC-MK, cells, which can both make and respond to interferon. Monolayers of both types were incubated with either monkey interferon or poly( I ) .poly( C ) for 18 hours. The cultures were then infected, and after 18-24 hours the virus yield was determined. Monkey interferon treatment resulted in a 99.9% reduction in the virus yield from both cell lines. Virus replication in LLC-MK, cells was reduced by 99% after treatment with ( I ) n . ( C ) n .In contrast, viral replication was unaffected in ( I).. (C).-treated Vero cells. Schafer and Lockart ( 1 1 1 ) ruled out the possibility that Vero cells failed to respond to poly( I ) .poly( C ) because of a permeability barrier and therefore concluded that poly( I ) -poly(C ) specifically derepresses only the interferon operon. Thus, it appears that the use of very low concentrations of polynucleotides to induce a state of viral resistance in animal cells in culture is a valid method for studying the specificity of the interferon induction mechanism. b. N o Requirement for Specific Base Sequences. It may be suggested that a peculiar sequence of nucleotides must be present in order for a polynucleotide to serve as an inducer of interferon, but the initial findings that double-stranded RNA’s from a variety of sources are very efficient inducers (66, 71, 72) makes the suggestion unlikely. The fact that the associated synthetic polynucleotides poly( I ) .poly( C ) are also an efficient inducer ( 6 5 ) , does not support the suggestion that either a sequence of inosinic acid residues or a sequence of cytidylic acid residues is required. This possibility was directly tested and excluded by Colby and Chamberlin (100). The associated homopolymers poly( I) .poly( C ) are equal in efficiency as an interferon inducer to the alternating polymer p l y ( I-C ) . In addition, synthetic double-stranded polynucleotides containing sequences of A, U, G, and X have been found to be active inducers (65, 100, 113). C. Requirement for Secondary Structure. In their initial report of the use of ( I ) * ( C ) as an inducer of interferon, Field et al. (65) emphasized that the single-stranded polynucleotides poly( I ) , poly( C ) , poly( A), and poly( U ) , are inactive at concentrations more than 10,000 times greater

.

. .

16

CLARENCE COLBY, JR.

than those allowing detectable viral interference with the double-stranded homopolymer pair poly( I ) *poly(C). These results have been confirmed in other laboratories (98, 100). A similar requirement for the doublestranded helical conformation has been reported for naturally occurring RNA’s (see Section 111, D). Recently Baron et al. ( 1 1 4 ) reported that certain batches of commercially available synthetic “single-stranded polynucleotides are active as inducers of interferon in cells in culture and in the intact rabbit. Control experiments excluded the possibility that the interferon-inducing activity was due to small amounts of contaminating poly( I ) .poly( C ) . These included base analyses of degraded samples of the polynucleotides, chromatography on benzoylated DEAE-cellulose, and determination of the spectrum of activity on various cell types. It is interesting that mammalian cells, such as those from rabbit, mouse, and man, were much more responsive to the “single-stranded polynucleotides than were chick embryo cells. If one considers that the significance of the yield reduction assay begins at 0.5 log,, inhibition of virus yield, then chick embryo cells did not respond to 300 pg/ml of the most active preparations tested. Baron et al. (114)pointed out that polynucleotide preparations from some commercial sources were totally inactive and that there was wide variation in the activities of high concentrations of different preparations from the same supplier. They suggest that the difference in activity between double-stranded RNA and single-stranded RNA may be simply related to the greater resistance of the former to ribonuclease. This implies that a few of the samples of poly( I ) and poly( C ) were much more resistant to ribonuclease than others. Since the active samples contained no detectable helical RNA, several explanations are possible. One is that the active samples might be contaminated with polycations rendering the polynucleotide more resistant to RNase. Another is that the active polynuceotides were in a conformation intermediate between that of a hydrogen-bonded double helix and a random coil. The possible significance of the latter suggestion may best be considered in the light of some of the recent work done in Merigan’s laboratory. De Clercq and Merigan ( 1 1 3 ) compared the physicochemical properties and the virus resistance-inducing properties of several synthetic polynucleotides at different magnesium and hydrogen ion concentrations. The homopolymer pairs (U),-(X),, ( A ) n * ( I ) n(,I ) n . ( X ) n ,and (A),. ( X ) ,induced cellular resistance to virus infection as measured by a reduction in plaques on treated cultures. The activity of these polynucleotides increased with increasing magnesium ion concentration and with decreasing pH. However, the activity of these polynucleotides was much lower than that of poly( I ) .poly( C ) or poly( A ) .poly( U ) .

THE INDUCTION OF INTERFERON

17

When De Clercq and Merigan (113) tested single-stranded polynucleotides at 40 pglml they found that ( U),, ( C)n,and ( A ) , were inactive and that ( G)”, ( I),, and X, gave some antiviral activity. The antiviral activity of the latter three did not increase in excess Mg2+whereas ( A ) , and ( C ) , were slightly active with higher Mg2+ concentrations. The authors found that the single-stranded polynucleotides were active at concentrations over 10,000-fold higher than the equivalent activity of p l y ( I ) .poly( C ) and 100-fold higher than that of poly(A) .poly( U). De Clercq and Merigan (113) suggested that there may be a causal relation between the increase in thermal stability and viral-resistance inducing capacity of the single-stranded polynucleotides in their altered environments. That is, they suggested that because of the greater degree of secondary structure induced by Mg2+or H+,the single stranded polymers may be more resistant to RNase degradation and thereby be more effective inducers. However, they also point out that the different polynucleotide types could have a different affinity for the cellular site of initiation of interferon production and that some specific structural elements might be extremely favorable in triggering the interferon production sites within the cell (113). I strongly favor this second suggestion, but several other experiments must be considered before the case can be made. The situation at this point is essentially that we are attempting to solve a problem of three unknowns with only two equations. The variables we have been considering are ( i ) the efficiency of interferon induction, (ii) the secondary structure, and (iii) the thermal and RNase stability of the polynucleotides. We can now consider the relationship between interferon induction and RNase susceptibility of a series of polynucleotides having the same secondary structure, viz., a stable hydrogen-bonded double helix. d . E f e c t s of Chemical Modification of the Polynucleotides. De Clercq et al. (115) compared two helical alternating copolymers with respect to their ability to induce interferon and their thermal stability and RNase susceptibility. One, poly ( A-U ), has alternating riboadenosyl and ribouridylyl units. The other, poly(A 5 U ) , is identical except that one of the oxygens of the phosphate group not involved in the phosphodiester linkage is replaced by sulfur. This replacement had no effect on the “melting” temperature (both have T,,, = 48OC at 0.01 ionic strength). However, there was a remarkable enhancement of the interferon-inducing capacity of the thio-substituted polymer and a concomitant increase in RNase resistance. The authors stated, “it is tempting to causally relate the parallel increase of antiviral activity and resistance to ribonuclease degradation in poly( A 2 U ) ” (115). I prefer to relate the two phenomena to the same cause rather than

CLARENCE COLBY, JR.

18

to relate causally the two phenomena. The essential clue is the unchanged T , , which indicates that the stability of the helix is unchanged. Thus, the increased resistance to RNase of poly( A 9 U ) is more likely due to an inhibitory action of the sulfur atom at that portion of the active site of RNase responsible for the recognition of the phosphodiester linkage. Similarly, poly(A 5 U ) may have a higher affinity than poly(A - U ) for the intracellular site that triggers the production of interferon. These ideas are supported by the following experimental results obtained by Colby and Chamberlin ( I O O ) , who studied a number of synthetic polynucleotides with respect to their ability to induce interferon in chick embryo cells as measured by viral resistance. The yield reduction assay was used, since it had been found to be quantitatively superior to the plaque reduction assay. Primary cultures of chick embryo cells were treated with 10 pglml of the polynucleotide to be tested and 10 pg/ml of DEAE dextran for 2,4 hours. The role of the DEAE dextran was the facilitation of uptake of the polynucleotides (100, 107). The treated cells were then challenged with lo7 plaque-forming units of Sindbis virus, and after 22 hours the culture medium was assayed for infective virus. Table I11 lists the polynucleotides tested. They are divided into two groups; inducing polynucleotides and noninducing polynucleotides. The synthetic single-stranded polynucleotides ( I )n, ( C )n, (G) n, ( A)n, and ( U ) , were inactive as inducers at 10 pglnil. These results are in agreement with those of De Clercq and Merigan (113) and of Baron et d. ( 1 1 4 ) ,who found that concentrations greater than 10 pg/ml are required to elicit antiviral activity in mammalian cells with these single-stranded polynucleotides. Indeed, Baron et al. ( 1 1 4 ) were unable to detect significant viral resistance with 300 pg/ml of the most active preparations of both poly ( I ) and poly ( C ) in chick embryo cells. Seven of the 20 polynucIeotides tested were active as interferon inducers. These shared the common properties of being helical polyribonucleotides. However, the yield reduction assay revealed that the efficiency of the inducing polynucleotides varied considerably. The effiTABLE 111 POLYNYCLBOTIDES

TESTED AS

Inducing polynucleot.ides

INTERFERON I N D V C E R S IN C H I C K

EMBRYO CELLS

Noninducing polynitcleot,ides

19

THE INDUCTION OF INTERFERON

TABLE I V EFFICIENCY O F I N D U C T I O N A N D RIBONUCLEASE SENSITIVITY OP HELICALPOLYRIBONUCLEOTIDES

Polynucleotide

Minimum concentration (dmC

Titer reduction index

POlY (I).POlY(C) POlY(1-C) p M G ) . p o b (C) poly(I).poly(I-BrC) poly ( A - V poly (A-BrU) P O ~ Y ( A ) . P(U) ~~Y

0.001 0.003 0.001 0.01 0.01 0.01 2.0

6.3 5 .9 5.1 4.0 2.5 2.4 0

Riboriuclease sensitivity (pmoles/hr/mg RNase) 3.8 17 <0.0% I .4 110 41 13

ciency of induction may be expressed either by determining the minimum concentration of the polynucleotide allowing detectable viral resistance or by expressing the efficiency of induction by the titer reduction index, defined as the log of the virus titer from control cultures divided by the log of the virus titer from cultures treated with 0.1 pg/ml polynucleotide. Table IV gives the efficiencies of induction of the active polynucleotides and also presents their sensitivity to degradation by pancreatic RNase. Poly( G ) .poly( C ) is very resistant to RNase, yet it is not any better than ( I ) ( C ) or ( I-C ) as an inducer. Bromination of the cytosine residues in ( I-BrC) increases the RNase resistance of the polynucleotides 10-fold as compared with (I-C)n, yet ( I-C)n is a better inducer than (I-BrC),,. Finally, poly( A ) .poly( U ) is degraded by RNase at a rate 1/10 that of poly(A-U), yet the latter is by far the superior inducer. It is quite clear from the work of De Clercq and Merigan ( 1 1 3 ) and Colby and Chamberlin (100) that the secondary structure of the polynucleotide is a very important element in the induction of interferon. Obviously, the polynucleotide must remain intact long enough to reach the intracellular site. In the case of induction by high concentrations of single-stranded polynucleotides ( 113, 114),their altered secondary structure may play such a role. However, in the case of induction by very low concentrations of helical polyribonucleotides, the results do not support the idea that the sole requirement is that the polynucleotide should survive RNase degradation. e. Requirement for 2’-Hydroxyl Groups on the Sugar Moieties. If an appropriate secondary structure were all that is required of a polynucleotide in order that it might serve as an inducer of interferon, one might expect DNA to be an effective inducer. Lampson et al. (66) reported that calf thymus DNA is inactive as an interferon inducer in rabbits, and

-

20

CLARENCE COLBY, JR.

Colby and Chamberlin (100) found that DNA from coliphage X does not induce interferon in chick embryo cells. In addition, Colby and Chamberlin (100) found that three DNA-like polynucleotides, poly( dA-dT), poly( dG) .poly( dC) and poly( d I ) *poly(dC), are inactive as interferon inducers in chick embryo cells (see Table IV). The inactivity of poly( dG) .poly( dC) has recently been confirmed by Gresser (116). In contrast, De Clercq et al. ( 1 1 7 ) have reported that high concentrations of poly( dG) *poly(d C ) , poly( dA) -poly(d T ) , poly( dA-dT), and coliphage DNA cause a reduction in virus plaque formation in human skin fibroblasts. Field et al. ( 7 2 ) postulated that a special replicative form of the DNA or a viral DNA.RNA complex might be responsible for induction of interferon in cells infected with DNA viruses. Also, the “RNaseresistance theory” would predict that single-stranded polyribonucleotides should be very strong inducers if protected from RNase digestion by hydrogen-bonding to an appropriate polydeoxyribonucleotide. VilEek et al. (98) found that poly( I ) -poly(dC) gave resistance to vesicular stomatitis virus in rabbit kidney cell cultures. However, the concentration required was lo4 times that for poly(I).poly(C) (65). Colby and Chamberlin (100) tested the hybrid polynucleotide pairs poly( 1.1).poly( dC) and poly(d1)-poly(rC) and found them to be inactive at 10 pglml (100). The alternating helical copolymer, poly( rA-dU) was also inactive ( 100). It is possible that the hybrid polymers might be excluded from, or taken up more slowly by, the chick embryo cells in culture. Alternatively, one might postulate that the hybrid polymers are broken down much more rapidly than the helical polyribonucleotides by intracellular nucleases. In order to test these possibilities, Colby and Chamberlin (100) prepared 3zP-labeled poly( I-C) and 32P-labeledpoly( I ) *poly(dC) and measured their rates of uptake and intracellular breakdown. The rates of uptake were found to be identical and poly( I-C) was broken down slightly faster than the inactive homopolymer pair, poly( I ) .poly( dC) ( 100). Thus, permeability and intracellular stability differences cannot account for the difference in the interferon-inducing activity of these polynucleotides. It therefore appears that, at low concentrations, the intracellular receptor site can recognize particular functional groups on the inducer molecule as well as its molecular conformation. Specifically, the receptor site appears to have a strong affinity for the double-stranded helix and a strong requirement for the presence of 2‘-hydroxyls on the sugar moieties. We suggest that the 10,000-fold difference in activity between polyf I ) *poly(C ) ( 6 5 ) and poly( I) -poly(dC) (98) is a reflection of the latter requirement ( 100).

THE INDUCTION OF INTERFERON

21

The issue has recently been complicated by the finding of Nemes et al. ( 7 3 ) that a preparation of a DNAmRNA hybrid synthesized from f l phage DNA is active as an interferon inducer in both rabbits and rabbit kidney cells in culture. The smallest amount of the f l hybrid required to induce resistance to vesicular stomatitis virus was 300 times that for poly( I ) * poly( C ) . Thus, this hybrid appears to be significantly more active than the purely synthetic hybrid, poly ( I )-poly ( dC) ( 98). One possible explanation is that the secondary structures of the synthetic and natural hybrids are quite different. Another possibility is that there may be contaminating double-stranded RNA in the preparation. The hybrid sample that Nemes et al. ( 7 3 ) tested was prepared by transcribing f l phage single-stranded DNA with E. coli RNA polymerase in a reaction mixture that allowed approximately one equivalent of RNA to be synthesized (118). Robertson and Zinder (119, 120) have found that some of the excess RNA from a reaction mixture greater than 1:1 is double-stranded. Colby et al. (121) have confirmed this finding using antibodies specifk for double-stranded RNA and DNAaRNA hybrids (122, 123). We are currently testing purified components of such a reaction mixture for their individual interferon-inducing properties.

IV. Double-Stranded RNA in Cells Infected with DNA-Containing Viruses A. Vaccinia Virus Double-Stranded RNA The work discussed in Section I11 indicates that double-stranded polyribonucleotides have a far greater specific activity as interferon inducers than double-stranded polynucleotides that have deoxyribose moieties in one or both chains. Double-stranded RNA is expected to be present in cells infected with RNA viruses, but not in cells infected with DNA viruses. Nevertheless, Colby and Duesberg (75) have found such an RNA in vaccinia virus-infected chick embryo cells. The infected cells were labeled with [3H]uridine and the total nucleic acids were extracted by the dodecyl sulfate phenol method. Pancreatic DNase was used to degrade the DNA, and the single-stranded RNA's were degraded at high salt concentration with pancreatic RNase. Finally, the double-stranded RNA was purified by gel filtration on an agarose column. The [3H]RNA was characterized as double-stranded RNA by the following criteria (75, 124) : 1. It is resistant to digestion with a mixture of pancreatic RNase, T1RNase and pancreatic-DNase.

22

CLARENCE COLBY, JR.

2. Its resistance to RNase is lost when it is thermally denatured and rapidly cooled. 3. When infected cells are labeled with [3H]thymidine and the same purification procedures are used, no labeled material appears in the exclusion volume of the agarose column. 4. It has a T , of 77°C in 0.01 M NaC1. 5. Its buoyant density in Cs,SO, is 1.64 gm/ml (DNA = 1.43 and single-stranded RNA = 1.68). 6. Its buoyant density in Cs,SO, increases 0.04 gmlml after thermal denaturation. 7 . It self-anneals after thermal denaturation. 8. The same efficiency of self-annealing occurs when the thermally denatured material is treated with DNase or is banded in Cs2S0,. 9. Very low concentrations of it induce interferon in chick embryo cells as measured by resistance to Sindbis virus. The double-stranded RNA appears to be virus-specific. After thermal denaturation the [3H]RNA hydridized with vaccinia DNA but not with calf thymus DNA or E. coli DNA. Approximately 1%of the input RNA hybridizes with chick DNA. This is probably due to minor contamination of the vaccinia virus double-stranded RNA with the RNase-resistant RNA of normal cells (75, 125). Another indication that the double-stranded RNA is virus-specific is that complementary RNA, which can be converted to double-stranded RNA, is made in vitro using vaccinia virus "cores" ( 1 2 6 ) . Finally, vaccinia virus double-stranded RNA can be purified from vaccinia-infected HeLa and mouse L-cells ( 1 2 6 ) . The double-stranded RNA appears to be made by transcribing overlapping regions of vaccinia DNA, thereby producing complementary RNA ( 1 2 6 ) . Some of these complimentary sequences then anneal in the cytoplasm of the infected cell to give helical RNA in the region of overlap with single-stranded segments on each end ( 1 2 6 ) . At least 70% of the double-stranded RNA isolated by the above technique is in an intracellular form that is resistant to extensive RNase degradation prior to phenol extraction ( 1 2 6 ) . Thus it is quite possible that this population of molecules is responsible for the induction of interferon in cells infected with vaccinia virus.

B. Double-Stranded RNA in Other DNA-Virus Systems Cells infected with other poxviruses, such as chick embryo cells infected with fowl-pox virus and rabbit kidney cells (RK13) infected with myxoma virus, also contain double-stranded RNA ( 1 0 1 ) .There have been no reports of double-stranded RNA in animal cells infected with DNA viruses that replicate in the nucleus and that induce interferon.

THE INDUCTION OF INTERFERON

23

The phenomenon of double-stranded RNA in DNA virus-infected cells is not restricted to eukaryotic cells. Bprvre and Szybalski (127) have found that complementary RNA is transcribed from the b2 region of coliphage A. Double-stranded RNA that is virus-spec& has also been isolated from E . coli infected with phage T4 (128). The role of all of these virus-specific double-stranded RNA’s in the replicative cycle of the DNA viruses is unknown.

V. Discussion of the Mechanism of Induction of Interferon

A. The Nature of the lntracellular Receptor Site

From the experimental evidence presented in Section 111, it may be concluded that the mechanism of interferon induction involves a high degree of specificity and that wide variations may exist in the intracellular concentrations of polyanions required for the induction of interferon. Thus, there are definite physical and chemical requirements the inducer molecule must satisfy. The common structural requirements are (1) a sufficiently high molecular weight, ( 2 ) a regular and dense sequence of negative charges on a long-chain backbone, and (3) a stable primary or secondary structure (116). For the specificity involving low concentrations of polynucleotides, the polymers are most effective if they are in a helical configuration. In addition, there is a strong requirement for 2’-hydroxyl groups on the sugar moieties. If the induction mechanism is sensitive to the fine physical and chemical structure of the inducer molecule, there must be some medium by which this specificity is imposed. Colby and Chamberlin (100) postulated the existence of a specific intracellular receptor site as an integral part of the interferon induction mechanism. The extent of interaction between the intracellular receptor site and the inducer molecule would determine the amount of interferon induced. Two classes of macromolecules are known to be able to interact with nucleic acids in a very specific fashion: other nucleic acids and proteins. Nucleic acids recognize and interact with other nucleic acids with specificity via a base-pairing mechanism. If the receptor site is a nucleic acid, then there are two immediate predictions concerning its interaction with the inducer molecule. Specific base sequences in the inducer would be required, and the nature of the sugar residue would be inconsequential. The experimental evidence is contrary to both of these predictions. On the other hand, many proteins, such as polymerases, nucleases,

24

CLARENCE COLBY, JR.

and aminoacyl-tRNA synthetases, are known to be able to recognize a particular secondary structure. Furthermore, only proteins have the ability to recognize particular functional groups, i.e., to distinguish ribose from deoxyribose. We have therefore postulated that the intracellular receptor site recognizing the interferon-inducing molecule is a protein (100).

I should like to interpret some of the experimental observations mentioned above within the framework of our current knowledge of proteinsubstrate interactions. Specihally, the discussion will be based on our understanding of competitive inhibition at the submolecular level. The normal substrate of an enzyme can be modified physically or chemically in such a way that its interaction with the active site of the enzyme is greatly enhanced or reduced. If helical polyribonucleotides interact with the receptor site most efficiently (and are therefore the best interferon inducers) and if single-stranded polyribonucleotides with the secondary structure of a totally random coil do not interact at all with the receptor, then there should be a series of intermediate secondary structures that would interact with the receptor to an intermediate extent. One would expect that much higher concentrations of such molecules as compared with helical molecules would be required for activity. The results of De Clercq and Merigan (113) and of Baron et al. (114) fit this expectation quite well (see Section 11, E, 3 c). Similarly, one would expect the specificity with respect to the presence of 2'-hydroxyl groups to be overcome by high concentrations of polynucleotides containing deoxyribose. Such results have been reported by VilEek et al. ( 9 8 ) and De Clercq et al. ( 1 1 5 ) . Finally, the discovery that the thiophosphate analog of poly(A-U) is a more potent inducer than poIy( A-U) itself (115) may be interpreted as an example of an enhancing effect on the interaction between an inducer molecule and the receptor site. If so, then the approach of De Clercq et al. (115) may be the best for finding very potent interferon inducers that could be used therapeutically at concentrations far below the level of toxicity.

B.

lntracellular Processes Occurring after the InducerReceptor Site Complex Is Formed,

In Section 111, E, 2, b above, the experiments of VilEek (109) and Bausek and Merigan (110) are described, They suggest that there is an interferon-regulating system, the synthesis of which is more sensitive to cycloheximide than is the synthesis of interferon itself. Recently, Tan et al. (129) have confirmed these results and have provided evidence that the regulating substance requires messenger RNA synthesis. Furthermore, the control mechanism for interferon production may operate differently in different tissues or in different cell populations ( 1 3 0 ) .

THE INDUCTION OF INTERFERON

25

Ho et al. ( 1 3 1 ) have suggested that the production of interferon is a two-step reaction. They postulate that the product of the interferon gene is a protein called “preinterferon,” which may act as interferon in the cell, but which is not released as detectable interferon. The second postulated step is the conversion of preinterferon to interferon, a reaction that would be inhibited by the product of a control gene. The action of cycloheximide would be to stop the synthesis of the control protein and thereby allow the continued conversion of preinterferon to interferon. These ideas are presented schematically in Fig. 1 ( 1 3 1 ) . This scheme requires at least one and possibly two new proteins, the control protein that blocks the conversion of preinterferon to interferon and possibly a protein which catalyzes this conversion. The experimental evidence may be fitted into a somewhat simplified scheme requiring two assumptions: ( i ) that the synthesis of the receptorsite protein is more sensitive to cycloheximide than the synthesis of interferon, and (ii) that the inducer-receptor site complex is relatively stable. By this model, the formation of the complex would allow interferon synthesis to occur, but after a few hours more receptor site protein would be synthesized and the interferon gene would be turned off again. This process would be sensitive to cycloheximide and interferon production would continue in its presence (109,110,129).In the case of simultaneous administration of poly( I ) .poly( C ) and Newcastle disease virus infection, the second burst of interferon synthesis ( 1 1 0 ) would reflect the interaction of the newly synthesized receptor site protein with the replicative form of the viral RNA (the synthesis of which would be sensitive to antimetabolites ) .

’

Interferon

gene

L

rnRNA-

//

Preinterferon-Interferon

Control protein-mRNA-

Facilitation

@

Control

gene

Inhibition @

FIG. 1. The stimulation and regulation of interferon production by p o l y ( 1 ) . poly( C).

e,

facilitation;

8, inhibition.

26

CLARENCE COLBY, JR.

There is no direct evidence for either of these models. However, they offer predictions that may be tested experimentally and that may ultimately lead to an understanding of the induction of interferon on a molecular basis.

C. The Nature of the Inducer Molecule The value of studying the stimulation of interferon production by nonviral inducers is 2-fold. From the practical standpoint, nonviral inducers of interferon would be superior to viral inducers if the interferon mechanisms are to be used in the treatment and prevention of viral diseases in man. For the basic research scientist, nonviral inducers offer the advantage of eliminating the many virus-directed processes found in virus-infected cells, such as the shutting off of host cell synthetic functions, etc. However, in the last analysis, we must always relate the information gained from the study of other systems to the virus-infected cell. What must happen in cells infected with a virus in order that they may produce interferon? Most of the work covered by this review is consistent with the original postulate of Field et al. (65) that the doublestranded replicative form of the RNA-viruses may be the inducer molecule. However, there is experimental evidence that suggests that this may not be the case. Lockart et al. (132) studied the ability to induce interferon of wild-type Sindbis virus and temperature-sensitive mutants of Sindbis that do not make significant amounts of viral RNA. The latter were found not to induce interferon at the nonpermissive temperature. However, cells infected and incubated for 2 hours at the permissive temperature before being shifted to the higher temperature produced interferon. This result could be eliminated by inhibiting protein synthesis with cycloheximide. Thus, it appeared that early viral protein synthesis is required for interferon production, in agreement with experiments by Skehel and Burke (133). One might postulate that the early protein needed is the viral RNA polymerase required to synthesize the replicative form. However, when Lockhart et al. (132) tested several RNA+temperature-sensitive (ts) mutants they again found no interferon production and concluded that viral RNA replication, or its accumulation in the cell is insufficient to account for interferon production. These experiments have been repeated by Marcus (134) with conflicting results. Using an interferon assay system that is at least 100 times more sensitive, Marcus found excellent induction of interferon with all three types of Sindhis; wild type, ts-RNA+ mutants and ts-RNAmutants. It appears that stock preparations of all of the viruses (which are produced at the permissive condition) contain an inducer of inter-

THE INDUCTION OF INTERFERON

27

feron exclusive of the virion. Purified preparations of the viruses gave results compatible with the idea that the replicative form is the interferon inducer molecule. That is, purified preparations of the RNA- mutants do not induce interferon at the nonpermissive temperature, whereas comparable preparations of RNA+ mutants do. The situation is camplicated further by the fact that the RNA' mutants appear to turn off cellular macromolecular synthesis with different efficiencies ( 134). In Mengo virus infection, viral functions do not appear to be required. Both the double-stranded replicative form ( R F ) and the multistranded replicative intermediate of Mengo virus induce interferon in mice (74, 135). The double-stranded RF is infectious in cells growing in tissue culture (136). Falcoff and Falcoff (136) found that the infectivity of the doube-stranded RF could be completely destroyed by UV-irradiation without altering its ability to induce interferon at all. As mentioned above (Section 11) the lytic infective cycle of Newcastle disease virus appears to be accompanied by the production of a viral function that interferes with interferon induction. Elimination of this viral function by UV-irradiation or infection of a nonpermissive host cell is accompanied by interferon production. Dianzani et at. (137) infected mouse L-cells with Newcastle disease virus in the presence of cycloheximide. At 4 hours actinomycin D was added, at 5 hours the cycloheximide was washed away, and at 8 hours interferon production and viral resistance was measured. Thus, in the first 4-hour period, cell mRNA could be transcribed but not translated. In the last 3-hour period no new cell RNA could be transcribed, so translation was limited to those messages transcribed in the first period. The authors (137) found that interferon was produced during the period of translation but no antiviral resistance occurred. This result indicated that the mRNA for interferon had been transcribed during the period of protein synthesis inhibition. Dianzani et al. (137) could detect no RNase-resistant RNA in cells infected with Newcastle disease virus in the presence or absence of inhibitors of protein synthesis. Unfortunately, this control experiment was done 5 hours after infection, i.e., after the 1-hour treatment with actinomycin D, so they were unable to report on the absence or presence of viral RNA synthesis during the first 4 hours when only the protein synthesis inhibitors were present. Nevertheless, it appears that the stimulus for induction of interferon in this system may be provided by a component of the input virus or an associated physical event ( 1 3 4 ) . Since the RNA in the virion is singlestranded, Dianzani et al. (137) concluded that certain single-stranded viral RNA's may induce interferon. Thus, we are faced with the same dilemma we encountered in the case of certain batches of commercially

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prepared “single-stranded polynucleotides ( 114). High input concentration could be part of the answer, since these workers used a very high multiplicity of virus infection (137), but the explanation may be more complicated. If the input virions were uncoated by a cellular process and the viral RNA was neither replicated nor transcribed, then it is possible that the RNA retained a conformation similar to that in the densely packed virion particle, that is, one intermediate between a totally random coil and a rigid helix. Such a structure might interact with the intracellular site for interferon induction in a positive manner. This postulated explanation assumes that one of the input RNA molecules can be responsible for interferon induction. Another alternative is that two of the input RNA molecules interact to form a stable helical structure which would then trigger the interferon response. This possibility is reasonable in light of Robinson’s (138) recent finding that 1020%of the RNA isolated from Newcastle disease virions may be converted to an RNase-resistant structure by annealing conditions. Another clue relating to the mystery of the Newcastle-interferon system has recently been supplied b y Huppert et al. (139). These workers studied viral RNA synthesis in chick embryo cells infected with UVirradiated virus. They found UV-irradiated virus totally unable to produce infectious virus is still able to induce viral RNA synthesis. They reasoned that while a single hit may destroy the possibility for infectious progeny, a specific hit in the cistron coding for viral RNA replicase would be required to eliminate viral RNA synthesis. In addition to showing that viral RNA was synthesized, Huppert et al. (139) also found RNaseresistant RNA in cells infected with unirradiated or irradiated Newcastle disease virus. While these results do not prove that double-stranded RNA is the interferon inducer in these cells, they do eliminate the major objection to this hypothesis (139). In summary, the following ideas seem consistent with the experimental results. Under normal conditions of infection of animal cells with an RNA-containing virus it is likely that the replicative form and/or the replicative intermediate acts as an interferon-inducing molecule. In the case of infection with the poxviruses, the virus-specific double-stranded RNA is a likely candidate. In cells infected with nonreplicating viruses at a high multiplicity, the viral RNA may contain portions with sufficient secondary structure to allow interferon induction. Finally, there may be animal viruses that direct the synthesis of messenger RNA molecules with primary structures that permit or demand secondary structures similar to those recently reported for bacteriophage mRNA’s (140-142). These might be interferon-inducing molecules, providing that the portions with secondary structure are of sufficient size.

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