Immunological and Chemotherapeutic Prevention and Control of Oncogenic Viruses

Immunological and Chemotherapeutic Prevention and Control of Oncogenic Viruses MICHAELA. CHIRIGOSAND TAXISS. PAPAS Virus and Disease Modification Section, Viral Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

I. Introduction. . . . . . . . . . . . . . . . . . . 11. Biological Systems Useful for Studying Prevention and Control of Oncogenic Viruses . . . . . . . . . . . . . . . . . . . . A. Established Procedures . . . . . . . . . . . . . . B. New Procedures . . . . . . . . . . . . . . . . C. Approaches to Prevention and Control of RNA Oncogenic Viruses. . 111. Antiviral Therapy through Biochemical Control of Oncogenic Viral Polymerase . . . . . . . . . . . . . . . . . . . . A. Classes of Inhibitors . . . . . . . . . . . . . . . B. Mechanism of Inhibition . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

89 91 91 95 102 108 109 116 118

1. Introduction Virus infection is widespread throughout the animal and plant kingdoms and produces most human illnesses (Horsfall and Tamm, 1965), including certain types of cancer (Gross, 1961). Virus-induced malignancies or benign t,umors have been demonstrated in a wide variety of warm-blooded animals and birds. Included among these are monkeys, horses, cattle, dogs, chickens, cats, ducks, guinea fowls, turkeys, rabbits, foxes, deer, gray squirrels, wood chucks, and pheasants, Virus-induced malignancies have also been reported to occur in cold-blooded animals such as frogs and salamanders. The most intensively studied virus-induced malignancies have been in monkeys, cats, cattle, mice and rats. Since man is prone to viral infections and, in view of the many virus-induced malignancies found in animals, it would be difficult to consider him to be so unique as to escape virus-induced malignancies. In recent years, RNA-containing viruses have been examined in detail as potential human oncogenic viruses (Bryan et al., 1966; Borden and Carter, 1972). The B- and C-type particles are strongly implicated in the pathogenesis of naturally occurring oncogenic infections of many animal 89

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RIICHAEL A. CHIRIGOS AND T AK I S S. PAPAS

species. Indeed, C-type viruses have been isolated from a high percentage of spontaneously occurring feline leukemia (Allen and Colc, 1972; Rickard el al., 1969). These C-type RNA viruses share many common morphological characteristics (Nowinski et al., 1970) and similarities in structural proteins and nucleic acids (Duesberg, 1970) as well as a major internal polypeptide (Parks and Scolnick, 1972). The most unique and intriguing characteristic of oncornaviruses is the presence of an RNA-dependent DNA polymerase (reverse transcriptase) (Baltimore, 1970; Temin and Rfizutani, 1970). This enzyme has been detected in every RNA tumor virus studied t o date. Of particular interest, this enzyme activity has been reported to occur in human leukemic cells. The leukemic cells of 3 acute lymphatic leukemic patients demonstrated a reverse transcriptase activity, whereas none of the normal lymphoblasts had the enzyme (Gallo et al. , 1970). This enzyme, which appears essential for tumorogenesis (Hanafusa and Hanafusa, 1971), appears uniyur to viruses with oncogenic potential. When reverse transcriptase is found intracellularly, it often is considered as a viral “footprint”; its presence in a malignant cell constitutes presumptive evidence for the viral nature of the inciting agent. If the enzyme is, indeed, needed for virus-induced transformation, the inhibition of this enzyme may reduce malignancy. It appears likely that reverse transcriptase found in all RNA leukemic and sarcoma viruses fulfills some yet undefined biological role (Baltimore, 1971). Oncogenic viral genes may themselves remain in the transformed cell and thrir presence is detectable by a techniquc of niolrcular hybridization, which indirectly measures the homology or relatcdness of grne sequenccs. Evidence from srroepidemiological studies and from ccll culture studies led Huebner and Todaro (1969) to postulatr that the cells of many and, perhaps, all vertebrates contain information for producing C-type RWA viruses. It is postulated that the viral information (the virogene) including that portion responsible for transforming a normal cell into a tumor cell (the oncogene) is most commonly transmitted from animal to progeny animal and from cell to progeny cell in a covert form. Carcinogens, irradiation, and the normal aging process all favor the partial or complcte activation of these genes. An understanding of how apparent normal cclls and apparent normal animals prevent exprrssion of endogenous viral information would appear to offcr one of the challenges for understanding the control of naturally occurring cancer. This subject is discussed in detail separately in Section II,B,2. Indeed, progress in the field of oncogenic virology in the past 3 years has been remarkable (Eckhart, 1972; Dienhardt, 1972; Orozlan et aZ., 1971; Todaro and Huebner, 1972; Temin, 1972; GiIden and Orozlan, 1972;

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91

Scolnick et al., 1972; Abrell and Gallo, 1973; Hatanaka el aZ., 1970; Teity, 1971). The objective of this review is to fullfill a nerd for summarizing the progress that has been made on the diverse approaches employed for thr prevention or control of oncogrnir viruses in light of the new information in the field. It is impossible to review comprehensively the literature pertaining to all mammalian oncogenic DSA and RNA viruses; consequently the present review is limited to the murine leukemia and sarcoma viruses. Emphasis is placed on publications that represent new or unique experimental approaches to immunological or chemotherapeutic prevention or control. Thr authors apologize for any inadvertent omission of worthwhile1publications prrtaining to the subject. Howrver, it is hoped that both the scope of the review and the items included will represent the type of research being conducted on oncogenic virus therapy throughout the world.

II. Biological Systems Useful for Studying Prevention and Control

of Oncogenic Viruses

A. ESTABLISHED PROCEDURES 1. I I L Vitro Assays The development of i n vitro (tissw culture) assay systems has facilitated the rapid testing of drugs and othcr potentially active antiviral agents. In addition, these assays have bcrn found useful in studirs designed to elucidate thc mechanism of inhibitory action of antiviral agrnts. nlurinr Irukcmia viruses are assayed by thc XC-tcst ( R o w et al., 1970; Hartlry et al., 1970) in which a quantitative number of syncytia are formed. Infective titers of murine sarcoma virus can be quantitated by the number of foci formed on mouse embryo fibroblast cultures (Hartley and Row, 1966; Fischirigcr and O’Connor, 1968). These assays havr bren shown to be useful for isolating naturally occurring viruses of the murine lcukcmic group in tissue culture (Hartley et al., 1969) and for determining the hclprr role that murinc leukrmia viruses possess for dcfectivr murine sarcoma virus. Several murinr Icukcmia and sarcoma virus-transformed cell lines havr been rstablishcd and have been rmployrd for trsting drugs (Woods et al., 1973). An alkaloid rxtract of the Narcissus bulb (Chirigos et al., 1973a) and rifampycin drrivatives (Hackrtt et al., 1972) havr been shown to exert an i n vitro inhibitory effect on murinc leukemia and sarcoma virus replication. Results of antiviral testing of srveral drugs are shown in Table I (for more dctailcd information, see rcfrrences citcd in the table).

TABLE I ANTIVIRAL ACTIVITIESOF VARIOUSINHIBITORS~. *

Inhibitor types

Enzyme source

Template primer

Site of inhibition

Antiviral (in vitro)

1. Antibiotics

a. Rifampycin b. Streptovarycin c. Distamycin

RLV(1, 34), RSV(3, 6), MSV(2, 6), AMV, FeLV(2) MLV(7) FeLv, MSV(lO), RSV(6)

e. Adriamycin

FLV, RSV, MSV(11), RLV(25), LMV(34) AMV, RLV, PK-l5(12)

f. Streptonigrin g. Actinomycin D h. Olivomycin i. Chromomycin j. Daunorubicin k. Cinerubin-A 1. Bleomycin

AMV(13) RSV(28, 33) RLV(25) RLV(25) RSV (28) RSV(28) RLV(29)

70 S(7) rA*dT,2-,8, dI*dC1Z--ls(lO), rA-dT12--18(9) dA.dT,,-is, dI.dC, rA*dT12(11), DNA(25) rA.dT12-18,d(AT), DNA, 70 S(12) rA-dT,z-18(13) 70 S(28, 33), DNA(25) DNA(25) DNA(25) 70 S(28) 70 S (28) 70 S(29)

RLV(14, 15), AMV, M-PMTV, RLV(16)

d(AT), DNA(15, 16), rA dTI2-,, (16)

d. Daunomycin

N.D.

RSV(4, 6), RLV(3), MSV(3, 5 )

N.D. IIA

MSV(8) FeLV, MSV (10)

IIA

FLV, RSV, MSV(11)

IIA

MSV(11, 12), FLV(11), RSV(12) MLV, RLV(13) N.D. N.D. N.D. N.D. N.D. N.D.

IIA IIA IIA IIA IIA IIA IIA

2. Polymers

a. Polynucleotides: PolyA, polyG, polyC, POlYU

-

IB, IIB(?) ltLV(14)

b. Modified polynucleotides: RLV(14) Poly(2’-O-methyl)A AMV(30) ePolyA FLV, MSV(17) Thiolated polyC c. Analogs of polynucleotides : MLV(18) Poly(vinyluraci1) N.I. Poly(viny1adenine) d. Other polymers : AMV, RLV, PK-15, Pyran copolymer R-C type(36) Heparin RLV(26) Histones RLV(25) Protamines RLV(2.5)

IB IB IB IB, IIA(?) IB, HA(?)

RLV, MLV(14) N.D. N.D.

MLV(18) MLV(18)

70 S, DNA, d(AT), rA-dT1&36) DNA(25) DNA(25) DNA(%)

IA

N.D.

IIA IIA IIA

N.D. N.D. N.D.

RLV(20, 21)

poly(IC), DNA(21)

IIB

N.D.

MLV, RLV(31) AMV(I9)

IINA, d(AT), rA*dT1~18(31) d(AT), 70 S(19)

RSV(22) RSV(22) N.D. FeLV, FSV, MSV(24) RLV(2Ci, 34), MLV(34)

70 S, DNA(22) 70 S, DNA(22) N.D. 70 S, rA.dTl,-,8, d(AT) (24) 70 S(34)

3. Substrate analogs

AraCTP

0 3

4. Plant extracts a. Calcium elonolate

Residual alkaloid

I IA

MSV(31) MSV(34)

5 . Miscellaneous

a. Thiosemicarbazones b. Cuz+, Hg2+ c. Cordycepin d. Natural serum inhibitor e. Ethidium bromide

N.D. IB2(?) IB1 I IIA

ltSV(22) ItSV(22) MSV(23) N.D. N.D. (Continued)

TABLE I (Continued) Inhibitor types

&. Tilorone g. Oyopropanal h. Oxohydroxypropanal i. Acridine Orange

Enzyme source

Template primer

Site of inhibition

RLV, AMV(32), MSV, FLV(35) RSV (28) RSV(28) RLV(25)

d(AT), rA.dT12-1&2, 35)

IIA

N.D.

70 S(28) 70 S(28) DNA(2.5)

N.D. N.11. IIA

N.D. N.D. N.D.

Antiviral (invitro)

a Abbreviations: AraCTP, arabinofuranosylcytosine 5'-triphosphate; RLV, Rauscher leukemia virus; MuLV, murine leukemia virus; FeLV, feline leukemia virus; FLV, Friend leukemia virus; RSV, ltous sarcoma virus; AMV, avian myeloblastosis virus; PK-1.5, pig kidney C-type particle; R-C type, reptilian C-type particle; MuSV, murine sarcoma virus; N.D., not determined; N.I., not inhibitory; rA -dTIz-,, synthetic DNA-RNA hybrid consisting of oligomer of deoxythymidylic acid (chain length 12-18) and homopolymer of riboadenylic acid; d(AT), the synthetic DNA consisting of an alternating copolymer of dA and d T ; 70 S, high molecular weight viral RNA; DNA, activated by a mild deoxyribonuclease treatment. Key to references (numbers in parenthesis indicate references): (1) Yang et al., 1972; (2) Green et al., 1972; (3) Ting et al., 1972; (4) Diggelmann and Weissmann, 1969; (5) Calvin et al., 1971; (6) Kotler and Becker, 1971; (7) Brockman et al., 1971; (8) Carter et al., 1971; (9) Arcamone et al., 1969a; (10) Chandra et al., 197%; (11) Chandra et al., 1972a; (12) T. S. Papas and M. A. Chirigos, unpublished results; (13) Chirigos et al., 1973b; (14) Tennant et al., 1972; (15) Tuominen and Kenney, 1971; (16) Abrell et al., 1972; (17) Chandra and Bardos, 1972; (18) Pitha et al., 1973; (19) Papas et al., 1973; (20) Miiller et al., 197%; (21) Tuominen and Kenney, 1972; (22) Levinson et al., 1973; (23) Wu et al., 1972; (24) Pradip et al., 1972; (25) Miiller et al., 1971; (26) Wood et al., 1968; (27) Ward et al. 1968; (25) Apple and Haskell, 1971a,b; (29) Miiller et al., 1972a; (30) T. S. Papas and M. A. Chirigos, unpublished results; (31) Hirschman, 1972; (32) T. S. Papas and M. A. Chirigos, unpublished results; (33) McDonnel et al., 1970; (34) W. A. Woods, personal communication; (35) Chandra et al., 1 9 7 2 ~(36) ; Papas et al., 1974a. See Section II1,B of text.

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2. I n Vivo Assays Several test systems employing murine leukemia arid sarcoma viruses have been shown to be useful in testing the antiviral or antitumor effect of drugs, vaccines, interferon, interferon inducers, neutralizing antibody, and other miscellaneous agents. These assays have been previously reviewed (Chirigos, 1969). Three parameters may be employed for determining the effect of the therapy applied to the virus-infected host : prevention and/or reduction of viral replication; retardation or inhibition of virus-induced neoplastic transformation (disease); and, effect on the survival time of the host. Macroscopic foci induced in the spleen by the Friend or Rauscher murine leukemia virus provide rapid quantitative assays (Axelrod and Steeves, 1964). The spleen focus assay has been used in a unique study in which helper activity has been demonstrated in tissue extracts of human leukemia patients for enhancing expression of leukemia virus in mice (Steeves et al., 1971).

B. NEW PROCEDURES From the intense study being conducted to determine whether viruses play a role as etiological agents in human cancers, several unique and interesting biochemical and virological methods have been developed. The following discussion will describe these methods and how they are adaptable to the study of preventing or controlling oncornaviruses. 1. Biochemical

The identification of the RNA-dependent DNA polymerase (reverse transcriptase) enzyme, unique only in oncogenic viruses, has allowed testing chemicals for their ability to inhibit this enzyme. This subject is discussed in detail in Section II1,B. 2. Endogenous Viruses

a. Spontaneous Occurrence or Induction of V i r u s Expression by Deoxyuridine Analogs. There is growing evidence for the presence of masked, endogenous viruses in cells of murine, avian, rodent, hamster, and feline origin. Murine leukemia virus (MuLV) can be activated in mice by certain carcinogenic stimuli or as the result of aging (Huebner et al., 1970). Evidence has also been presented that spontaneous activation of virus can occur in mouse embryo cells propagated in vitro over long periods of time (Aaronson et al., 1969).

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MICHAEL A. CHIRIGOS A N D TAKIS S. PAPAS

The most studied chemicals, capable of in vitro activation of oncogenic viruses, have been the deoxyuridine analogs, 5-bromodeoxyuridine (BUDR) and 5-iododeoxyuridine (IUDR). Cells of embryos of the high leukemic AKR mouse strain were grown in cultures as virus-negative cell lines. The cell lines, as well as clonal sublines, when exposed to IUDR or BUDR express murine leukemia virus as early as 3 days after exposure of these cells to the inducer (Lowy et al., 1971). R'lurine leukemia viruses and appearance of reverse transcriptase were also induced in well-characterized virus-free BALB/3T3 clonal cells (Aaronson et al., 1971). The implication of these studies is that the complete MuLV genome is present in the cells under investigation but in a muted nondetectable stage until activated by IUDR or BUDR. Studies of the mechanism of induction of infectious, murine leukemia virus from AKR mouse embryo cell lines by the deoxuridine analogs indicated that incorporation of IUDR or BUDR into cellular DNA plays a vital role in the activation of murine leukemia virus synthesis in the AKR cell lines (Teich et al., 1973). 5-Bromodeoxyuridine was also reported to increase production of particles, with the morphology of murine leukemia virus, in a mouse melanoma (B16) cell line (Silagi et al., 1972). This cell line did contain virus particles that were detectable, however, only by electron microscopy. Spontaneous transformation and emergence of viral information, but not of intact infectious virus, has been reported t o occur in rat cells after long-term in vitro cultivation (Rhim et al., 1972a). A rat kidney cell line was transformed when infected with the murine sarcoma virus (Kirsten), [MuSV(K)], but the cells did not produce infectious virus (nonproducing lines). The repressed virus genome could be activated by treatment of the transformed cells with BUDR. The BUDR-induced virus transformed normal rat kidney cells but did not have a similar effect on mouse embryo cells. From immunological and biochemical studies, the investigators concluded that the emergence of an infectious virus, through BUDR induction, results from activating an endogenous, rat, C-type virus that provides the helper function needed to allow the MuSV(K) viral genome to express itself in an infectious form (Klement et al., 1971). Similarly, rat C-type virus was induced in rat sarcoma cells by BUDR (Klement et al., 1972). b. Chemical Carcinogen Induction. The in vitro transformation of murine and rodent cells by chemical carcinogens has been often described and is well established. Development of tumors in animals by exposure to chemical carcinogens has also been well documented, but recent findings indicate the involvement and/or expression of a virus in chemical carcinogenesis (Chen and Heidelberger, 1969; Sanders and Burford, 1967; Sivak and

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97

Van Duuren, 1968; Berwald and Sacks, 1965; Igel et al., 1969). The mechanisms of chemical carcinogenesis are a t present unknown. Three cellular mechanisms have been proposed : (1) the chemical carcinogen selects malignant cells through clonal selection of preexisting potential malignant cells (Prehn, 1964) ; (2)the carcinogen transforms the cells by derepressing natural host cell oncogenes (Rfonal and Heidelberger, 1970); or (3) there is activation of the latent oncogenes of RNA tumor virus genomes (Irino et al., 1963; Ribacchi and Giraldo, 1966; Toth, 1963; Bergs et al., 1970). Recent findings indicate that inherited, transmitted, C-type, RNA virus genomes possess characteristics that implicate them as basic determinants of chemically induced tumors (Huebner et al., 1970, 1972). Presence of an oncogenic virus has been reported from tumors induced in vivo by chemical carcinogens. A single, neonatal injection of 7.12-dimethylbenzanthracene into mice was shown to increase the level of detectable leukemogenic activity in various tissues assayed at short intervals after treatment as well as in primary tumors (Ball and RlcCarter, 1971). Similarly, C-type RNA viruses were found prcsent in cell cultures irom transplantable and primary hepatomas induced by aromatic amine carcinogens. Virus yield was markedly enhanced by treating the hepatoma cell culture with BUDR (Weinstein et al., 1972). c. Cocarcinogenesis. The concept of cocarcinogenesis, as it is presently understood, means the eventual production of cancer resulting from many small but apparently permanent changes in the genetic material of the cell, the cellular alterations being induced by more than one, possibly numerous, carcinogenic agents. The carcinogenic agents involved may be chemical, physical, or biological. There are many reports in the literature that support, directly or indirectly, viral and chemical cocarcinogenesis. The classic example was the observation showing that benign warts induced by percutaneous applications of tar on the outer ear of rabbits rapidly turned to malignant epitheliomas when the Shope papilloma virus was injected intravenously into the same rabbits (Rfackcnzie and ROUS,1941). Shope papilloma virus alone seldom produces anything but benign papillomas, and tar painting on the rabbit ear produces epithelioma in relatively few rabbits and only after a long period of repeated applications. Together, however, the two weak carcinogenic agents precipitously induce malignant tumors when applied one after the other. A number of in vitro assay systems, useful for studying chemical carcinogenesis, exploit the observation that preinfection of cells with “nontransforming” RNA tumor viruses lead not only to accelerated transformation but also to transformation not inducible by the carcinogens alone

98

MICHAEL A. CHIRIGOS AND TAKIS S. PAPAS

(Freeman et al., 1970). I n vitro cell systems have been reported in which mouse cells chronically infected with AIiR leukemia virus were readily transformed by the chemical carcinogens : 3-methylcholanthrene (3-MC) ; 7,12-dimethylbenzanthracene(DMBA) ; and benzyopyrene (BP) (Rhim et al., 1971a,b). Such in vitro systems have been found useful in testing the carcinogenic potential of several agents. Most notable of these was the report describing the transformation of rat and hamster cells by crude extracts of city smog. Uninfected rat embryo cultures exposed to smog concentrates were not transformed, but cultures chronically infected with Rauscher leukemia virus (RLV) and exposed to smog concentrates became transformed (Freeman et al., 1971). A similar system was reported with cell lines established from normal mouse embryo cells infected with AKR leukemia virus. These continuously infected carrier cultures when exposed to concentrated airborne particulate matter (smog) became transformed. Such transformed cell lines when inoculated into mice were tumorigenic (Rhim et al., 1972b). 3. Preven,tion and Control a. Transformation by Carcinogen. A Fischer rat embryo transformation system that at a low passage level (<60) requires both the addition of exogenous, murine leukemia, type-C RNA virus and chemical carcinogen for transformation has been previously reported (Price et al., 1972). At higher passage lcvcls ( > G O ) , certain chemicals can transform the cells in the absence of virus although transformation is enhanced by either the addition of MuLV or by pretreating the cells with BUDR (Freeman et al., 1973). Employing this i n zdro system, thc ability of strrptonigrin (Sn), a n antibiotic reported to inhibit both NuSV and AIuLV expression in vitro and i n uivo, as well as inhibiting the RNA-dependent DNA polymerase of the avian myeloblastosis virus (AAZV) (Chirigos et al., 1973b), was tested for its ability to inhibit transformation by 3-MC (Price et al., 1974). Streptonigrin was found to be very effective in nanogram amounts in protecting cells from chemically induced transformation (Fig. I). The lowest dose studied (0.16 ng/ml) showed no cellular toxicity as determined by studying reduction in plating efficiency and colony size. The authors speculate that the high passage cells can be transformed by thc chemical carcinogen in the absence of added virus because low levels of the endogenous, rat, C-type virus are “turned on.” After cells undergo transformation, which is tested by their ability to grow in rats, viral antigen expressions are amplified until they reach a level that can be measured by

PREVENTION AND CONTROL OF ONCOGENIC VIRUSES

99

the crude complement fixation (CF) test. Streptonigrin was considered to act by inhibiting transformation by the chemical carcinogen. b. Induction of V i r u s by I U D R . Streptonigrin was tested for its ability to inhibit the induction of C-type virus in rat cells by IUDR. Rat cells exposed to IUDR result in expression of virus possessing several characteristics of C-type RNA viruses (i. e., presence of RNA-dependent DNA polymerase, incorporation of tritiated uridine that banded at a characteristic density in sucrose gradients, and the presence of RNA-dependent DNA polymerase that was inhibited by antiserum made against the reverse transcriptase of murine C-type virus particles). The SO% inhibition of IUDR-induced C-type virus expression by Sn (0.16 ng/ml) was of particular significance. c. Inhibition of Chemical Carcinogenesis by Viral Vaccines. In a unique investigation, vaccines prrpared from type-C RNA viruses were shown to protect against induction of sarcoma by 3-R2C mice (Whitmire and Huebner, 1973). The incidence of 3-AIC-induced subcutaneous tumors was significantly reduced by a singlc injcction of inactivated C-type RNA viral vaccine. Rauscher leukemia virus vaccine was shown to reduce the incidence of sarcomas from 78 to 50% in BALB/c mice. In addition, radiation leukemia virus vaccine and a vaccine prepared from a wild murine leukemia virus derived from a 3-MC tumor reduced more Tumorigenic

Normal FRE

(1) MuLV (2) 3-MC

- n

< 60 Passages > 60 Passages 3-MC 0.1-0.5 p l r n l

Flll

FRE

-@

> 60 Passages

Normal

3-Mc

Turnorigmic GS-1 Positive (CF)

Endogenous GS-1 Rat Leuk. "turned on"

FIG. 1. Effect of streptonigrin (Sn) o n 3-methylcholarlthrerle (%MC)-induced neoplastic transformation. MuLV, inurine leukemia virus; CF, complement fixation (test); FRE, Fischer rat embryo; F 111, Fischer rat embryo cell line 111.

100

MICHAEL A. CHIRIGOS AND TAKIS S. PAPAS

significantly the incidence of sarcoma from S6% in controls to 33 and 37%, respectively. The authors suggested that these reductions in tumor incidence, by murine C-type RNA virus vaccines, help support the concept that C-type RNA viruses serve as determinants of chemically induced cancer. d. Inhibition of Tumors with Interferon. From several studies, it has been shown that the antiviral substance, interferon, can inhibit virusinduced neoplasms in mice (DeClercq and DeSomer, 1971 ; Lieberman et al., 1971; Berman, 1970; Rhim and Huebner, 1971). The observation by Liebernian et al. (1971) that interferon treatment partially suppressed the incidence of X-radiation-induced leukemia in mice, suggested that the radiation-induced lymphoma was caused by the activation of a leukemogenic C-type RNA viral intermediate. In a recent study, interferon was found to inhibit chemical carcinogenesis in mice (Salerno et al., 1972). Mice were inoculated with 3-WIC carcinogen a t 8 days of age and treated with mouse serum interferon continuously. Treatment was shown t o inhibit the induction of subcutaneous fibrosarcomas and lung adenomas that appeared in the control, untreated, 3-MC-inoculated CF1 mice. The authors considered three possible mechanisms for the antiviral and/or cellular effects of interferon: (1) inhibition of the growth and multiplication of both tumor and normal cells; (2) nonspecific enhancement of macrophages activity and lymphocyte cytotoxicity; and (3) viral inhibition of a postulated viral intermediate, such as the endogenous, oncogenic, C-type RNA virus, which may contribute to the tumor induced by chemical treatment. The second may not bc a strong possibility since interferon and interferon inducers have bcen reported t o prolong skin grafts, indicating an inhibition of the cellular immune response (hlobraaten et al., 1973; Lindahl-RIagnusson et al., 1972; Hirsch et al., 1973a). Interferon has been reported to inhibit multiplication of both tumor and normal cells and is discussed in detail in Section II,C,2. However, the third possibility is the most provocative, since interferon has been reported effectively to prevent the expression of endogenous, oncogenic, C-type RNA virus induced by immunological activation (Hirsch et aE., 1974). 4. Immunological Induction

a. Immune Response and Oncogenic Viruses. There is compelling evidence supporting the concept that by altering one of the most common physiological mechanisms of the host-its immune mechanism-activation of a latent oncogenic virus can occur (Schwartz, 1972). I n the last few years, i t

PREVENTION AND CONTROL OF ONCOGENIC VIRUSES

101

has been demonstrated that both neonatal thymectomy and antilymphocytic treatment depress the immunological capacity of the host and simultaneously increase the incidence of chemically induced (Miller et al., 1963; Nishiauka et al., 1965; Trainin and Linker-Israel, 1970) and virus-induced tumors (Allison and Law, 1968; Law, 1969) in animals. Induction of a strong graft-versus-host reaction (GVHR) in mice with diff went I?-2 loci may lead to a significant increase in the incidence of spontaneous lymphomas in the host (Schwartz and Beldotti, 1965; Nemiransky and Trainin, 1973; Armstrong et al., 1972). The GVH reaction is an immunological response occurring when transplanted immunocytes react against histocompatibility antigens of their host. It has been reported that the GVHR leads eventually to a malignant lymphoma. This development requires an immune response by the grafted immunocytes against the recipient’s histocompatibility antigens. Latent oncogenic viruses are rapidly activated by the immune response of the graft against the host. The activated viruses can be demonstrated in cells or cell-free extracts derived from mice undergoing a GVH reaction by in vitro assay visualized by electron microscopy or by induction of lymphomas when inoculated into normal newborn mice (Armstrong et al., 1972). Reticulosarcoma and amyloid development has been reported in BALB/c mice inoculated with syngeneic cells from young and old donors (Ebbeson, 1971). The concept of immunological activation of oncogenic viruses has been elegantly described (Schwartz, 1972). A lymphocyte responding to surface antigens present on another cell transforms into a lymphoblast. During diff erentiation the virogene is derepressed, and oncogenic viruses are assembled in an environment favoring their rrplication. Virusrs shed from the plasma membranes of lymphoblasts infect cells in the microenvironment of the immunological reaction. These cells undergo malignant transformation and eventually give rise to a neoplasm. The type of lymphoma that develops is a function of the type of cell that happens to be infected by the virus. Each step in the process is determined by one or more genes. Activation of oncornaviruses by the GVH reaction has been reproduced in vitro by mixed lymphocyte culture (MLC) (Hirsch et al., 1972). Using column-purified splenic lymphocytes from parental and F1 hybrid mice, murine oncornaviruses become detectable shortly after initiating the culture. Splenic lymphocytes from parental and F1, mice in RlLC represent a “one-way” reaction because lymphocytes of the F1 hybrid do not react against parental histocompatibility antigens. b. Prevention of Immunologically Induced Viruses. Such in vivo and in vitro systems provide the investigator with excellent systems to assess

102

MICHAEL A. CHIRIGOS AND TAKIS S. PAPAS

preventive measures to abrogate the immunologically induced oncornaviruses. Employing the GVH reaction, the antiviral agent, interferon, was investigated to assess its effect on reducing or blocking virus activation (Hirsch et al., 1974). Interferon treatment of animals undergoing the GVHR not only reduced the induction of virus (from 74 to 7%) but also seemed to modify the nature of the GVH reaction itself. It is difficult to draw any conclusions as to the mechanism by which the interferon inhibited activation of endogenous virus. However, interferon shows promise as a possible block in one or more of the steps involved leading from immune responses against histocornpatability antigens to virus activation and subsequent oncogenesis. Indeed, interferon may find a role as a prophylactic agent against cancers in human transplant recipients and in patients with chronic immunological disorders. C. APPROACHES TO PREVENTION AND CONTROL OF RNA ONCOGENIC VIRUSES Current studies of the treatment of mammalian, RNA, oncogenic virus diseases is a t present directed toward four general areas: (1) testing drugs that directly block some virus-specific process; ( 2 ) evaluating the specificity of antiviral action of interferon or interferon inducers; (3) stimulating host defense mechanisms; and (4) tcsting drugs that inhibit the unique and specific reverse transcriptase enzyme of RNA oncogenic viruses. The last-mentioned approach has been intrnsively investigated and is discussed in detail in Section 111. 1. General Approaches

Advances in basic knowledge of oncogenic virus life cycles permit us to choose from several possible steps, one which is most amenable t o selective pharmacological attack. One must consider attacking the virus per se or some step in the virus-host cell interaction. There are ten preventive or control mechanisms one can consider (Fig. 2 ) . Of these, the first four have and are presently being most intensively studied. The use of a viral vaccine as a prophylactic measure has been examined and found to be effective in murine leukemia (Cohen and Fink, 1969; Friend and Rossi, 1968; Barski and Youn, 1965) and sarcoma (Schwartz et al., 1971). The use of drugs to prevent or inhibit virus infection has been discussed in several excellent reviews and recent publications (Carter, 1973; Hirschman, 1971a,b; Gallo et al., 1972; Goz and Prusoff, 1970; Pitha and Carter, 1971; Tamm and Caliquire, 1971; Chirigos, 1970).

Viral Carcinogenesis Target Cell Infection by virus (vertical or horizontal) trarismission

Host defense:

antiviral antibody anticdlular “ (Cdependent) cellular immunity

Infected, Precancerous Cell carries latent viral genome but is not ‘Ltran~formed” for uninhibited growth. Trigger-e.g., (4)

Irradiation Chemical Norioncogenic viral infection Superinfecting oncogenic viral infection Hormonal, etc.

Tumor Ce1lk“trarisformed” may or may not have overt virus particles, antigens, RNA. Carries virus-induced neoantigen(s) (including TSTA4) and/or Immune defect in host-immiinosuppression by drug or infection Initiation or stimulation of incomplete or “blocking” antibody or antibodyantigen complex

Prevention or Control

1. Vaccine-producing antiviral immunity 2. Drug-antiviral agent preventing infection

3. Interferon or Inducers 4. Ilrug-antiviral agent preventing replica-

tion or expression of viral genome (polymerase inhibitors).

5. Vaccineprevent super infection Environmental control-prevent physical or chemical insult

6

zm 4

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+-

3 0

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7. Vaccine-immune stimulators, “altered” TSTA 8. ]>rug-anticellular or antiviral t o prevent

recruitment of susceptible target cells or abrogate chronic infection 9. Viral-infection by nononcogenic lytic virus with tumor tropism 10. VacciIie-stimulator of cellular immunity: nonspecific and/or “altered” TSTA: inhibitor of “blocking” antibody or complex formation

20 8 5

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FIG.2. Possible prevention and control mechanisms of viral carcinogenesis.

$2

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MICHAEL A. CHIRIGOS AND TAKIS S. PAPAS

2. Interferon Since the time of its discovery by Isaacs and Lindenmann (1957), a vast literature has accumulated on the subject of interferon. Interferon is defined as a cellular protein produced in response to, and acting to prevent replication of, an infecting virus within the invaded cells. Interferon can be produced in cells both in tissue culture and in the intact animal. It is beyond the scope of this review to detail the knowledge accumulated on interferon over the past 16 years. Several excellent papers and pertinent reviews dealing with mechanism of induction, type of interferon inducers, physiochemical properties, and biological effects have been published (Kleinschmidt, 1972; Grossberg, 1972a,b; Rodgers and Merigan, 1972; Friedman et al., 1972; Wheelock, 1970; Gresser, 1972). Concerning mechanisms by which interferon synthesis is controlled and regulated, it is a generally held belief that interferon synthesis is induced by a derepression phenomenon involving the binding or inactivation of a hypothetical repressor molecule by the inducer, whether the inducer is viral or chemical. This control could be a t either the transcriptional, translational, or posttranslational level. Kleinschmidt el al. (1964) were the first to suggest that the stimulation of interferon synthesis in the cell came about through derepression of the gene coding for the interferon protein. Although a specific repressor has not been isolated the view that a repressor controls the synthesis of interferon is gaining general acceptance. Interferon is a host-determined protein, as evidenced by its species specificity. Interferon prepared in mouse cells was effective only against murine oncornaviruses (Rhim et aZ., 1969; Wheelock, 1970), whereas interferon that was prepared from a feline cell line was active only in feline cells (Rodgers et al., 1972). Interferon synthesis is stimulated by viruses and double-stranded RNA. Cassingeria et al. (1971) reported the site of genetic control of interferon synthesis by locating the specific genes that code for the production of interferon. Derepression of the host genome by neutralization of the repressor by an inducer, thereby permitting the coding of a messenger RNA for the interferon molecule, is in keeping with the present data concerning the mechanism of interferon induction. Burke (1966) reported that pretreatment of cells with actinomycin D, which blocks the synthesis of the messenger RNA, prevented the appearance of newly synthesized interferon, thus providing evidence that DNA-dependent RNA synthesis is a requirement for synthesis of interferon. Early studies of interferon were performed with material that had been produced by viral infection. Isaacs (1961) originally made the suggestion that the induction of interferon was a response to the presence of the

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foreign nucleic acid which had been introduced into the cell by the virus. Since then, certain bacteria and bacterial endotoxins were shown to be inducers of interferon, indicating that interferon could be stimulated by materials other than nucleic acids. In addition, other materials have been shown capable of stimulating interferon including microorganisms, bacterial, fungal, and plant extracts, and synthetic polymers. The synthetic inducers of interferon have been classified into two major groups, one of which includes compounds that are polyanionic in nature and the other being polycyclic compounds of relatively low molecular weight. Several synthetic inducers of interferon are presently known, e.g., polyriboinosinic-polyribocytidylic acid (poly IC) ; pyran copolymer (a random copolymer of maleic acid and divinyl ether); polyacytal carboxylates; poly IC-(poly-D-lysine) ; and tilorone (a diamine, namely, 2,7-bis-2-( diethylamino) fluoren-9-one). It has been well documented that interferons are active against a large number of viruses in many vertebrate species. Interferon and interferon inducers have been shown to be very effective against the murine leukemia (Rauscher, Friend, Gross) and sarcoma (Moloney) viruses (Wheelock, 1970). There is a general agreement among several investigators that interferon has no inhibitory effect on the first stages of infection: virus adsorption on the plasma membrane and penetration into the cells. The mechanism of the antiviral action of interferon on oncornaviruses has to date not been explained. Several mechanisms have been suggested but without any strong supportive evidence. Friedman el al. (1972) employing encephalomyocarditis (EMC) virus in L cells reported that antiviral activity of interferon may be directed against the translation of viral messenger RNA. Falcoff et al. (1973), from their studies on the correlation of the antiviral effect of interferon treatment with the inhibition of in vitro messenger RNA translation in noninfected cells, surmized that interferon induces a block in genetic translation in noninfected cells. There is a generally held belief that interferon inhibits transcription, but several other explanations are possible. Of considerable interest concerning the biological activity of interferon is the recent evidence indicating that interferon can exert an antitumor effect in experimental animals inoculated or infected with ancogenic viruses, inoculated with transplantable tumor cells, or inoculated with chemical carcinogens. Salerno et al. (1972) demonstrated that exogenously administered interferon prevented formation of 3-MC-induced fibrosarcomas and lung adenomas. Similarly, Kapila et al. (1971) examined the effect of pyran copolymer, an interferon inducer, on transplanted 3-MC-induced

10G

MICHAEL A. CHIRIGOS AND TAKIS S. PAPAS

tumors in mice and have shown that growth of these tumors was retarded. The antitumor effect of interferon and interferon inducers is described in two excellent reviews (Rodgers et al., 1972; Gresser, 1972). Effective antitumor activity has been reported for a variety of tumors ranging from those induced by oncornavirus to ones that are carcinogen-induced. More recently, interferon has been reported tJo act synergisticly with a tumor-cytoreductive drug (Chirigos and Pearson, 1973). The implication of this study was that interferon, when administered to mice during the period of drug-induced remission, was capable of reducing recrudescence of the leukemia. In this case it is not known whether interferon acted as an antitumor agent or whether its antitumor action was mediated by host factors stimulated by interferon. Evidence is accumulating that the interferon response may be involved in or associated with host immune recognition mechanism(s) . An enhanced interferon response by immune lymphocytes was reported by Glasgow (1966). Green et al. (1969) have reported that nonviral antigens may induce interferon production in lymphocyte cultures from sensitized human donors but not from nonsensitized donors. Recent experiments indicate that the antitumor action of interferon on murine leukemia L1210 may be mediated in part by host factors (Gresser, 1972). Moreover, there is evidence indicating that a close association exists between interferon and the function of elements of the lymphoid and reticuloendothelial system. Result,s from two studies (Gifford et al., 1971; Huang et al., 1971) suggest that the antiviral effect observed after interferon administration or production may be only a small part of its protective effects against various pathogens. Host resistance is determined by a complex series of factors including humoral antibody, cell-mediated immunity, and phagocytic action as well as interferon. All of these responses interact and, in fact, may complement one another in host recovery from infection. 3. Vaccines

Vaccination with formalin-inactivated oncogenic viruses offered excellent protection against challenge with homologous, viable infectious virus. Similarly, tissue culture cells producing noninfectious RLV or otherwise altered Friend leukemia virus (FLV) have been shown to act as excellent vaccines (Barski and Youn, 1965; Sinkovics et al., 1966; Cohen and Fink, 1969; Youn et al., 1968; Friend and Rossi, 1968). Recent findings indicate the Milcobacterium bovis BCG vaccine, when used as an adjuvant to infectious murine leukemia or sarcoma viruses,

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enhances immunity against a homologous virus challenge. Schwartz el al. (1971) demonstrated that BCG had a protective effect in the murine host injected with the murine sarcoma virus (Moloney) MSV(M). Of particular interest was the observation that exposure of mice to BCG before attempted induction of tumors with a mixture of BCG and MSV(M) completely inhibited tumor development. These mice were effectively immunized since no tumors developed when the mice were subsequently challenged with a lethal dose of MSV(M). Similar observations were reported by Larson et al. (1971, 1972) in studies with FLV. The BCG vaccine was found to be protective whether it was administered prior to or after injection of FLV. The methanol-extracted residue (MER) of attenuated tubercle bacilli of the BCG strain was found to confer marked resistance, under appropriate parameters of administration, against leukemogenesis induced by the radiation leukemia virus. The 13CG or RIIER is not known to induce the production of interferon in the host (Gresser et al., 1966), although it ran be obtained by injection of purified protein derivative (PPD) into BCG-immune mice (Stinebring and Absher, 1970). Thus the protective effect afforded hy BCG against these oncornaviruses may be clue to nonspecific cellular immunity provoked by BCG. I n an interesting study, Bliznakov (1973) demonstrated that coenzyme QlO (CoQlO), a lipid-soluble benzoquinone that increases phagocytic activity and primary hemolytic antibody formation, when administered to mice decreased splenomegaly and hepatomegaly and increased the number of surviving mice infected with FLV. In addition, CoQlO treatment was shown to reduce the percentage of mice with tumors, increased the number of survivors, and reduced the tumor size in mice with tumors induced by 3,4,9,10-dihenzopyrene. The author speculates that CoQlO stimulated the host defense system. Charney and Moore (1972) reported excellent protection against mouse mammary tumor virus (RIIMTV) in mice receiving a single dose of purified, formalin-inactivated MMTV. In a recent interesting and provocative study, Whitmire and Huebner (1972) demonstrated that the incidence of 3-MC-induced subcutaneous tumors could be significantly reduced by a single injection of inactivated C-type RNA viral vaccine. Three different viruses were shown to be effective vaccines, i.e., RLV, radiation leukemia virus, and a wild murine leukemia virus derived from a X-MC-indiiced tumor. The authors propose that the observed reduction in tumor incidence by virus vaccines help support the concept that C-type I t N L l viruses (oncornaviruses) serve as determinants of chemically induced cancer (see also Section II,B,3c).

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111. Antiviral Therapy through Biochemical Control of Oncogenic Viral Polymerase

The search for a biochemical LLhandle”that signals the onset of transformation remains a major challenge to both molecular biology of cancer and for clinical applications. The earlier discovery of reverse transcriptase in virions of RNA tumor viruses (Baltimore, 1970; Temin and Mizutani, 1970) and their presence in malignant cells (Gallo et al., 1970) stimulated new approaches to studying viral oncogenesis in human cancer. Prior to that, discovery RNA tumor viruses were known t o produce neoplasia in various species from avian to primates. However, this relationship could not be extrapolated to human cancers. The main reason for the latter has been due to the lack of a biological handle to act as a specific test in human cells. The presence of RNA-dependent DNA polymerase activity in human leukemic cells (Gallo et al., 1970) opened the possibility of using it as such a specific biological probe. A major reason is that this enzyme met the criteria required for polymerases derived from oncogenic viral sources. Specifically, it was shown to utilize natural RNA and synthetic RNADNA hybrids as templates for transcription of complementary DNA stands and transcribed 70 S AMV RNA (Sarngadharan et al., 1972). The presence of viruslike enzyme in leukemic cells stimulated the search for specific inhibitors of oncogenic viral polymerases. Reverse transcriptases have been purified from several sources including AMV, Rous sarcoma virus (RSV) RLV (Kacian et al., 1971; Hurwitz and Leis, 1972; Grandgenett et al., 1973; Leis and Hurwitz, 1972; Duesberg et al., 1971; Faras et al., 1972). Because the purification of these enzymes requires large amounts of virus, the avian (AMV) rather than a mammalian enzyme was the first to be purified and studied in some detail. The AMV enzyme was found to be composed of two subunits of total molecular weight of 170,000 (Kacian et al., 1971). By contrast, the molecular weight of RLV was determined by gel filtration to be 70,000 (Ross et al., 1971) and by glycerol gradient, 90,000 (Hurwitz and Leis, 1972). Unlike the avian enzyme (AMV) the mammalian enzyme (RLV) is unable to transcribe 70 S RNA. The latter finding could be due to the loss of a protein component in RLV essential for the transcription of 70 S. Such losses could be accounted for as artifacts of the purification process. Avian antibodies prepared against purified AMV and RSV reverse transcriptases were found to inhibit their homologous enzyme specifically and were shown not to cross-react with heterologous enzymes such as enzymes from murine, rat, hamster, and feline C-type particles (Nowinski

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et al., 1972; Watson et al., 1972; Parks et al., 1972). Antibodies against avian enzymes, on the other hand, inhibited several homologous avian polymerases that were tested, suggesting antigenic similarities of various classes of polymerases to be group specific. For detail treatment of the chemistry of reverse transcriptase, the reader is referred to recent comprehensive reviews (Temin and Baltimore, 1972; Gallo, 1972). I n order to assess the biological role of reverse transcriptase in oncogenic viruses, two experimental approaches have been pursued. One such approach is that of Hanafusa and Hanafusa (1971) involving of RSV (a) (a variant of RSV lacking the reverse transcriptase). Such particles are also shown to be defective in their ability to infect or transform indicator cells. This observation strongly implied that reverse transcriptase is an essential component of an infectious particle. The alternative approach, and possibly the method with applications in chemotherapy, is the search for synthetic or natural inhibitors that selectively inhibit reverse transcriptase and not the normal cellular polymerase. An understanding of the molecular events of inhibition and correlating such information with antiviral and antitumor activity could elucidate the importance of this enzyme in viral replication and cellular transformation. A. CLASSESOF INHIBITORS

Inhibition of viral polymerase may be achieved as a result of a variety of treatments. The significance of inhibition becomes only important when such a n inhibition elucidates enzyme function or has therapeutic merit. Thus, it could be visualized that a specific inhibitor can distinguish oncogenic viral polymerases from normal cellular polymerases (Papas et al., 1974a) or within the class of viral enzymes (although no such inhibitors have been reported as yet). If such an inhibition is coupled with antiviral and antitumor activities, then this could have clinical implications. The survey that follows describes compounds found to possess inhibitor activity against viral reverse transcriptase and to show antiviral activity. 1. Antibiotics

The first group of compounds tested and found to possess inhibitory activity against viral reverse transcriptases were antibiotics. These compounds were also found to interfere with the development of foci of transformed cells in culture. a. K i f a m p y c i n . A clinically useful, orally active antibiotic, rifampycin is synthesized from rifampycin SV (Sensi et al., 1959) which is a product of Streptomyces mediterraneous. The derivatives were initially found to inhibit

110

IIICHAEL A. CHIRIGOS AND TAKIS S. PAPAS

bacterial growth (Szilagyi and Pennington, 1971). The inhibition was shown to be produced by binding of the drug to DNA-dependent RNA polymerase and blocking the DNA-chain initiation step (Wehrli et al., 1968; Sippel and Hartmann, 1970; Bautz and Bautz, 1970; Burgess, 1971). Low concentrations of rifampycin were shown to be effective, indicating tight binding to the enzyme. Drug-resistant organisms were isolated and found, in turn, to possess resistant polymerase. The discovery that the drug could inhibit the replication of certain DNA viruses (Subak-Sharpe et al., 1969) initiated similar approaches on oncornaviruses by investigation of its effect on viral reverse transcriptases. Gurgo et al. (1971) reported that rifampycin was inactive as an inhibitor of viral polymerase. However, suitable modification by lengthening the carbon-3' position resulted in relatively eff Pctive derivatives (Gurgo et al., 1972; Yang et al., 1972; Green et al., 1972), but certain derivatives showed selective inhibition of the viral enzyme with much less effect on cellular DNA-dependent DNA polymerases (Green et al., 1972). Consistent with these observations, RNA-dependent DNA polymerase from human leukemic lymphocytes was inhibited by the derivatives shown to be effective for viral polymerases (Gallo et al., 1970). These observations imply that the latter enzyme is viruslike in origin. The fidelity of inhibition, however, was shown not to be valid when using DNA-dependent RNA polymerase from human lymphocytes (Tsai and Saunders, 1973). The latter studies indicate that the drug exerts its inhibitory action by binding to the enzyme and not interfering with the template. The mechanism of action of rifampycin has been fully investigated in the RNAdependent DNA polymerase from Escherichia coli. To date there are no published data available regarding similar studies with viral polymerases. A direct correlation has been established between inhibition of focus formation and reverse transcriptase of murine virus by rifampycin derivatives (Ting et al., 1972). Since such correlation was not observed with nononcogenic viruses, such as vesicular stomatitis virus (VSV) and vaccinia, it is suggestive that reverse transcriptase is necessary for transformation by RNA tumor viruses. b. Streptovarycin. This antibiotic is structurally similar to rifampycin: both share the ansa ring (Reinhart et al., 1966) and are shown to inhibit DNA-dependent RNA polymerase from bacterial origin but not from mammalian cells (Mizuno et al., 1968; Shmerling, 1969). The streptovarycin complex (A, B, C, D, El F, G), a mixture of several macrolides plus undetermined components, when tested against MSV polymerase was found to be a poor inhibitor (Brockman et al., 1971). Specific components such as A and C were also found to inhibit similarly. The possibility of active

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components in the complex remain to be elucidated and further work is needed for purification of various components. c. Distamycin A and Derivatives. A mixture of antibiotic substances, distamycin exhibits predominately antifungal activity. It is a basic oligopeptide isolated from cultures of Streptomyces distallious (Dimarco et al., 1962). Chemical investigations (Arcamone et al., 1964a) indicated that the structure of distamycin A is characterized by three residues of l-methyl4-aminopyrrole-2-carboxylic acid and two side chains. Some structural analogs of distamycin A have been synthesized (Arcamone et al., 1969a,b) by varying the number of pyrrole residues in the molecule. Such analogs are of great interest to the study of structure-activity relationship of the drug in various systems. Distamycin inhibits DNA polymerase activity of FLV and MSV oncogenic viruses (Chandra et al., 1972a). This inhibition was found to depend on ( a ) the number of pyrrole rings-the greater the number of pyrrole rings the greater the inhibition-and ( b ) template employed in the assay system. Templates containing thymidine and adenine are highly sensitive to the action of the drug. The inhibition of reverse transcriptases in oncogenic viruses and foci formation by distamycin derivatives indicates that both activities are dependent on the same structural component(s) of the molecule. Comparable results were obtained with vaccinia virus (Arcamone et al., 1969b). d. Daunomycin. A glycosidic anthracycline antibiotic, daunomycin is characterized by a pigmented aglycon (daunomycinone) bound by a glycoside linkage to an amino sugar (daunogamine) (Arcamone et al., 1964a,b). Daunomycin was found to inhibit reverse transcriptase activity in RNA tumor viruses (Chandra et al., 197213). Detailed studies of daunomycin and its derivatives were carried out by Chandra et al. (1972b), who showed that the inhibition against polymerases from RNA tumor viruses (MSV, FLV, RSV) selectively depends on the type of template primer used in the assay system. The inhibitory activity requires specific structural parameters. Substitution in the amino-sugar moiety, especially N-acetylation, inhibits the antitumor activity of daunomycin and influences its inhibitory action on the polymerases of RNA tumor virus. e. Adrianaycin. This antibiotic was isolated from a culture of mutant Streptomyces peucetius of the variety of caesius. The chemical structure has been elucidated and found to belong to the anthracycline group of compounds. It is chemically similar to daunomycin and differs only by hydroxylation of the fourteenth carbon. When tested with purified AMV and RLV polymerases (Papas et al., 1974b), it was found to be a potent inhibitor. The inhibition was template specific, showing greater effects

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with DNA-dependent templates d(AT), activated DNA than with the RNA-dependent synthetic poly rA -dT12-18.Similar results were obtained when the compound was tested with MSV, FLV, and RSV polymerase systems (Chandra et al., 1972b). f . Streptoniyrin. An antibiotic isolated from the broth of Streptomyces jioculus (Marsh et al., 1960), Sn was found to be active against leukemia by in vivo (RLV) and in vitro (MLV) methods. The drug was also found to inhibit purified AMV DNA polymerase. Structural modification of parent molecules resulted in compounds of varied rate of activity (Chirigos et al., 1973b) when monitored by all three test systems. The mechanism of inhibition of viral polymerase has not been studied, but there is evidence that the drug interacts physically with DNA and causes its degradation when chemically reduced in the presence of DYA (White and White, 1966). g. Actinomycin D. This drug binds to DNA but not to RNA and inhibits reactions directed by DNA but not by RNA (Reich et al., 1962; Kahan et al., 1963). Actinomycin D causes partial inhibition of virion DNA polymerase a t relatively high concentrations (McDonnel et at., 1970; Temin and Baltimore, 1972). When the endogenous product formed was examined in the absence of the drug, it contained large portions of doublestranded DNA, however in the presence of the drug it contained mostly single-stranded DNA, some DNA-RNA hybrids, and small amounts of double-stranded DNA (McDonnel et al., 1970; Temin and Baltimore, 1972). These results suggested that RNA-directed synthesis of DNA might be resistant to inhibition, whereas synthesis of DNA on the initial DNA product is inhibited. The property of actinomycin to inhibit doublestranded DNA formation has been exploited in making radioactive singlestranded DNA transcripts (probes) of viral 70 S RNA for hybridization studies (Ruprecht et al., 1973; Kacian et al., 1971; Verma et al., 1972; Ross et al., 1972). h. Olivomycin. This antibiotic selectively inhibits DNA-dependent nucleic acid synthesis in vivo and in vitro (Scholtissek, 1965; Reich et al., 1962). Inhibition of RLV polymerase is dependent on the template employed in the assay system, and there is much greater inhibition when DNA templates are used (Muller et al., 1971). i. Chromomycin. This compound complexes with DNA but not with RNA (Peacocke and Skerrett, 1956). Inhibition of RLV polymerase is observed only with DNA templates; when RNA template was used, no inhibition was seen (Muller et al., 1971). j. Bleomycin. A complex glycopeptide, bleomycin was isolated from the cultures of Streptomyces verticillus. The antibiotic possesses a significant antineoplastic activity (Kunimoto et al., 1967; Suzuki et al., 1968). Kuni-

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mot0 et al. (1967) and Suzuki et al. (1968) demonstrated that bleomycin markedly inhibits DNA synthesis in tumor cells without affecting protein synthesis. Suzuki et al. (1969) found that bleomycin produces strand scissions in DNA. Although bleomycin has not been tested against oncogenic viruses, Muller et al. (1972a) demonstrated a very strong in vitro inhibition of reverse transcriptase activity from RLV. Investigating further the mechanism of action with this enzyme, the authors found that the drug acts on the enzyme or enzyme-DNA complex but not on DNA alone. They also found that the drug did not affect the activity of two mammalian and one bacterial polymerase, In view of these experiments, they concluded that the antineoplastic activity of bleomycin is due to an inhibition of a polymerase in mammalian cell derived from RNA virus. 2. Polymers

a. Polynucleotides. Tennant et al. (1972) were the first to report that the polymerase from RLV was strongly inhibited in vitro by unprimed single-stranded polyribonucleotides (polyA, polyG, polyu). The inhibition was found to be the result of competition between the polymerase and the active template for the same enzyme-binding site (Tuominen and Kenney, 1971). The strength of inhibition depends on the particular homopolymer used: poly(U) >poly(G) >poly(A) >poly(C). Poly(U), the most active inhibitor, was not found to have absolute specificity for viral enzyme alone, it also inhibited the polymerase purified from three species of oncornaviruses as well as three out of the seven DNA polymerases purified from cells (Abrell et al., 1972). Although polyU was found to be the most effective inhibitor of RLV polymerase, polyA is a better inhibitor of replication of leukemic viruses in cultured cells (Tennant et al., 1972). More recently (Erickson et nl., 1973), it was reported that inhibition of AhlV polymerase was found to be dependent on the chain length of the polyU polymer, with a sharp drop in inhibitory activity when the polymer was reduced t o less than 200 nucleotide residues per polyU molecule. In view of this report, it is possible that lower inhibitory activity of polyU against murine leukemia virus replication in cell culture could be due to rapidly degraded noninhibitory fragments. These results strongly suggest the need for nuclease-resistant polymers. b. Modijied Polynucleotides. Poly(2’-0-methyl)A is a methylated derivative of polyA. Such modification increased the resistance of the polymer t o nuclease degradation (Rottman and Henlein, 1968). The modified derivative is an effective inhibitor of RLV polymerase in vitro with a n inhibition constant similar to that observed for polyA (Tennant et al., 1972). The modified derivative was also found to be a more potent inhibitor of leukemia virus synthesis.

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Chemically modified polycytidylic acid, prepared by partial thiolation of polycytidylic acid, significantly inhibits the polymerases of FLV and MSV (Chandra and Bardos, 1972). The modified compound is a n equally effective inhibitor of the DNA-directed DNA polymerase of both tumor viruses but shows significant selectivity in its inhibition of the RNAdirected enzyme. The magnitude of inhibition depends on the extent of thiolation. c. Analogs of Polynucleotides. Poly(vinyluraci1) and poly(viny1adenine) are vinyl analogs of polynucleotides. The structural differences between vinyl polymers and polynucleotides is the lack of sugar moieties and phosphate groups. Under suitable assay conditions, poly(vinyluraci1) was found to inhibit DNA polymerases activity of MLV, whereas poly (vinyladenine) stimulates the i n vitro reaction (Pitha et al., 1973). However, both vinyl polymers inhibit acute murine leukemia virus infection in mouse embryo cells, but they do not significantly inhibit the replication of Sindbis and vesicular stomatitis viruses. d. Other Polymers. Pyran copolymer possesses various biological activities and was found to be a potent inhibitor of purified polymerase from AMV (Papas et al., 1974a). The copolymer interacts with the polymerase a t a region other than the template site. The degree of inhibition was not template specific. The observed rate of inhibition by pyran was shown to vary with the different polymerases tested. Inhibition was shown with all oncornaviral polymerases tested, such as AMV, RLV, and pig kidney (PK-15) C-type particles, and to a lesser extent with mammalian polymerases; however, two of the three bacterial polymerases by contrast showed significant activation. Heparin is an anionic polymer found to be an effective inhibitor of DNAdependent RNA polymerase (Walter et al., 1967) a t very low concentrations. The DNA-dependent DNA polymerase activity is only affected at high concentrations. The compound also inhibits RLV polymerase (Muller et al., 1971). With such highly charged molecules, one must watch for possible artifacts of the assay. Histones are basic proteins that inhibit calf thymus, RNA-dependent DNA polymerase (Wood et al., 1968). The RLV polymerase is also blocked by the same concentration of histone (Muller et al., 1971). 3. Substrate Analogs

Arabinofuranosylcytosine 5’-triphosphate (AraCTP) is an analog of deaxcytosine 5’-triphosphate (dCTP). The compound was found not to act as a substrate of DNA and RNA mammalian polymerases (Furth and Cohen, 1968); however, it inhibits DNA polymerase by competing with dCTP (Furth and Cohen, 1968; Muller et al., 197213). Similar results

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have been obtained with reverse transcriptase from RLV (Tuominen and Kenney, 1972; Muller et al., 1972b; Schrecker et al., 1972). Since cytosine arabinoside (araC), the parent nucleoside of araCTP, interferes with both replication and transformation hy oncogenic RNA viruses, the inhibition by araCTP adds support to the generally held view that reverse transcriptase is the enzyme responsible for viral DNA synthesis in infected cells (Tuominen and Kenney, 1972). 4. Plant Extracts

Crystalline calcium elenolate was obtained from aqueous extracts of the olive plant (Olea eziropa). It is a very effective inhibitor of reverse transcriptase of murine leukemic virus and has no effect on the two Escherichia coli polymerases tested (Hirschnian, 1972). I n order t o conclude that the compound possesses relative specificity for viral enzyme, more polymerases should be tested, particularly those of mammalian origin. There is suggestive evidence that the compound interacts with the enzyme and not with the nucleic acid templates. The drug has certain advantages in that it is nontoxic t o cells in tissue culture a t high concentrations and is well tolerated by animals (Elliot et al., 1969). iln alkaloid extract of the Sacred Lily (Narcissus tarxetta I,), a medicinal plant, was found to inhibit purified D X h polymerase from AMV. The inhibitor physically combines with the polymerase, it does not affect the binding of the template to the enzyme and interferes either with the initiation or the elongation phase of the polymerization reaction (Papas et al., 1973). It has also been shown to possess antiviral activity against rarious systems. 5 . Miscellaneous Substances a. Thiosemicarbazones. N-RIethylisatin P-thiosemicarbazone (Me1RT) an antiviral agent active in variola and vaccinia infections was shown to inhibit replication of certain DYA viruses (Bauer and Apostolov, 1968; Bauer et al., 1970). The inhibition mas achieved only by treatment of cells after infection. Rous sarcoma virus, however, can he inactivated by exposure to the drug before infection (Levinson et al., 1973). The reverse

transcriptase activity of RSV was inhihited as well as the transforming ability of the virus. These experiments provide suggestive evidence for the importance of this enzyme to malignant transformation of RSV (Levinson et al., 1973). b. 1 norganic Cations. Two cations Cu?+and Hg2+can inhibits the reverse transcriptase and inactivate the transforming activity of RSV (Levinson et al., 1973). However, several cations (Asz+, CO+, Zn2+, and Ni2+) sig-

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nificantly inactivate the viral polymerase but have little effect on the transforming activity. c. Corclycepin (3-Deoxyadenosine). This drug inhibit polyA synthesis during the processing of nuclear heterogeneous RNA by terminating polynucleotide chain synthesis. Wu et al. (1972) demonstrated that the drug blocked virus production induced by IMD R from uninfected murine fibroblasts and from MuSV-transformed nonproducing cells. The inhibitory activity was shown to be specific since it acted only a t a critical time to inhibit virus production. d. Natural Serum Inhibitor. An inhibitor was detected in the sera of cats inoculated with RSV and in the sera of cats bearing spontaneous sarcomas, lymphomas, or carcinomas (Pradip et al., 1972). The inhibitor was effective against feline and murine viral polymerases but not the avian viral enzyme. The possibility that the inhibitor is a ribonuclease can be excluded because the inhibition could be overcome by excess enzyme but not by excess template. I n addition, if such an inhibitor was, indeed, a ribonuclease it will also affect the reaction given by the avian enzyme. Further characterization of this inhibitor would be of great importance since it seems to distinguish avian enzyme from mammalian enzyme. e. Ethidium Bromide. This compound forms duplexes with DNA and RNA (Ward et al., 1968) thus blocking nucleic acid synthesis in a competitive fashion (Woring, 1964). It inhibits RLV polymerase a t low concentrations (Muller et al., 1971). f . Acridine Oranye. This dyestuff binds to RNA by intercalation (Peacocke and Skerrett, 1956) and subsequently blocks DNA-dependent nucleic acid synthesis in wivo (Scholtissek, 1965) and in vitro (Sentenac et al., 1968). With the RLV polymerase system, high concentrations of the drug (Muller et al., 1971) were required when RNA was used as the template, and low concentrations when DNA was used. This reflects the relatively poor association of this compound with RNA (Peacocke and Skerrett, 1956). Table I summarizes all compounds included in the text that have been tested for activity against oncogenic viral polymerases. Most of the compounds were also tested for antiviral activity (in vitro). With only one exception, excellent correlation exists between antipolymerase activity and antiviral activity, once again emphasizing the important role of this enzyme in viral replication.

B. MECHANISJI O F INHIBITION All enzyme inhibitions are the result of an interaction of the inhibitor with some components of the enzyme system. The greater the complexity

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of the enzyme system the greater the number of inhibition sites. RNAdependent DNA polymerase is a multisubstrate enzyme utilizing template primer, four deoxynucleotriphosphates, and a metal activator. Interaction with the binding of any of these reactants results in inhibition of enzymatic activity. Inhibitors of RNAdependent DNA polymerase can generally be divided in several classes. I. A. It is relatively simple to determine by looking at the kinetics of inhibition whether the inhibitor binds at a site other than the catalytic site. Noncompetitive inhibition with respect to the substrates of the reaction indicates that the inhibitor belongs in this class of compounds. Such studies have been carried out with pyran (Papas et al., 1974a), residual alkaloid, and bleomycin (Muller et al., 1972a). B. Inhibitor inactivates by binding to catalytic site. (1) Inhibition of RNA-dependent DNA polymerase by polynucleotides could be the result of binding at the catalytic site (Tuominen and Kenney, 1971). (2) AraCTP known to inhibit both cellular and viral polymerase acts at the binding site of dCTP (Tuominen and Kenney, 1972). (3) The reported inhibition of RNA-DNA dependent polymerase of RLV by Cu2+ and Hg2+tends to implicate such an inhibition (Levinson et al., 1973). C. Adsorption onto the protein surface of the enzyme: nonspecific and weak interaction of certain substances, frequently nonpolar, that associate with protein side chain and may interfere with the binding of substrates. D. Enzyme degradation: the presence of a proteolytic enzyme could result in protein fragments that may be partially or completely inactive. E. Denaturation of the enzyme by changes in protein structure due to denaturing agents. 11. Inhibitors that inactivate substrates-substances that alter the substrate by preventing its subsequent binding to the enzyme. A. Template primer: several intercalating agents, such as actinomycin D and ethidium bromide, are involved in this class of inhibitors. Experimentally, inhibition can be overcome by increasing the concentration of the template primer. B. Deoxytriphosphates : enzymes such as phosphatases could degrade triphosphates rendering them unavailable for the reaction, thus causing inhibition. C. Metal activators: compounds acting as metal chelators could remove metals essential for the reaction. 111. Enzyme complexes-inhibitors could also interact with complexes between the enzyme and any of the substrates of the reaction. A further subclassification of the inhibitors can be made depending on

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which step of the DNA synthesis reaction is most severely affected: initiation (formation of the first phosphodiester bond) or elongation (stepwise addition of deoxyribonucleotides to the 3’-OH end group of the initiated deoxyribonucleotide chains). It is experimentally feasible to examine whether the inhibitor affects any of these steps. Cordycepin, a known chain terminator, could be used to monitor the formation of the first phosphodiester bond, and its inhibition, in turn, could lead to the study of this class of inhibitors. In very few cases has the mechanism of action of the inhibitors of viral polymerases been studied in great detail. ACKNOWLEDGMENTS The authors are greatly obliged to Dr. A. M. Schumacher for her active interest, helpful advice, and encouragement in the initiation and completion of this report. The invaluable assistance of Mim L. L. Perschau and Miss M. A. Young is gratefully acknowledged.

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