Effect of interferon on exogenous murine leukemia virus infection

VIROLOGY

84, 134-141 (1978)

Effect of interferon

on Exogenous

Murine

MORDECHAI ABOUD, RUTH SHOOR, Department

of Life Sciences, Bar-Ilnn

Leukemia

Virus Infection

SAMUEL SALZBERG’

AND

University,

Ramat-Gan,

Israel

Accepted August a,1977

When interferon (IF)-treated NIH/3T3 cells were exogenously infected with the Moloney strain of murine leukemia virus (M-MLV), no viral progeny release was detected as long as IF remained in the medium. This was evidently an inhibition of the virus replication and not a consequence of interfering with the establishment of the infection since, when IF was removed either before infection or later after infection, virus release gradually approached the rate of untreated control after a temporary delay. Furthermore, a direct examination revealed that IF treatment had no effect on the formation of infectious centers. IF treatment led to a reduced viral RNA synthesis. A similar inhibition of viral RNA synthesis was observed when the potent protein synthesis inhibitor cycloheximide (CH) was added early after infection. However, when added at a late stage, the drug had no effect on viral RNA synthesis. It appears, therefore, that IF-induced inhibition of viral RNA synthesis is not a feedback consequence of an inhibition of a later step but, rather, an inhibition of some early step. This conclusion was substantiated by the finding that when IF was eliminated from pretreated cultures up to 10 hr before infection, progeny release was still affected. INTRODUCTION

Interferon (IF) has been reported as capable of inhibiting the replication of RNA tumor viruses in exogenously (Peries et al., 1968; Pitha et al., 1976; Sarma et al., 1969), endogenously (Liberman et al., 1974; Pitha et al., 1976; Ramseur and Friedman, 1976; Wu et al., 19761, and chronically (Aboud et aZ., 1976; Allen et al., 1976; Billiau et al., 1973, 1974, 1975, 1976; Friedman et al., 1974, 1975, 1976, 1977; Liberman et al., 1974) infected cells. Several studies aimed at exploring the mechanism of IF effect on the chronic infection of these viruses have revealed that IF does not inhibit the synthesis of viral RNA (Salzberg et al., manuscript in preparation; Billiau et al., 1974) and virus-coded proteins (Friedman et aZ., 1974, 1975; Liberman et aZ., 1974; Pitha et al., 19761,nor does it interfere with the virion assembly (Billiau et al., 1974, 1976; Friedman et al., 1974, 19771,suggesting that IF blocks the final release of the virions from the cell surface. 1 Author to whom requests for reprints should be addressed. 134 0042-6822/78/0841-0134 $02.00/O Copyright 8 1978 by Academic F’rees, Inc. All rights of reproduction in any form reserved.

When cells are infected with exogenous RNA tumor viruses, the viral RNA is first transcribed into a DNA copy which then integrates into the cellular genome (Ali and Baluda, 1974; Khoury and Hanafusa, 1976; Temin and Baltimore, 1972; Varmus et al., 1973, 1973a, 1974, 1974a). The subsequent process of the virus development is supposedly identical to that of the chronic infection (Temin and Baltimore, 1972). For the sake of clarity we propose to define the events occurring up to the integration step as the. early stage and the subsequent events, which are common to both types of infection, as the late stage. In this study we present data which strongly suggest that IF exerts its effect at the early stage of the exogenous infection of murine leukemia virus. MATERIALS

AND METHODS

Cell lines. NIH/3T3 mouse fibroblasts were used for studies of exogenous infection, whereas NIH/3T3 cells chronically producing Moloney murine leukemia virus [NIH/STS(M-MLV)] were used for preparation of infecting virus. XC cells were

IF EFFECT

ON EXOGENOUS

applied for estimation of infectious centers. All cells were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated calf serum. Only when used for examination of IF effect was the serum content reduced to 2.5%. Preparation of infecting virus. M-MLV for exogenous infection was obtained from NIH/3T3(M-MLV) cell cultures. About 1 x lo6 cells were plated per g-cm tissue culture dish (Nunc, Denmark) in 10 ml of medium. After 48 hr the medium was replaced by 5 ml of fresh medium and the cells were further incubated for 16 hr. The medium was collected and centrifuged at 1000g for 20 min to remove cellular debris. The clear supernatant was brought to 8 pglml in Polybrene (Aldrich) and was warmed to 37” before use. Exogenous infection. NIH/3T3 cells were plated at a density 1.5 x lo6 cells per g-cm dish with 8 pg/ml of Polybrene present in the medium. After 18-24 hr the medium was removed and the cells were overlaid with 1 ml of virus suspension at a multiplicity of about 1 PFU per cell. After 1 hr at 37” the cells were washed three times with PBS and provided with fresh medium. Assay of infective centers. The efficiency of the exogenous infection was estimated by analyzing infectious center formation. NIH/3T3 cells were infected with M-MLV as described above. One hour after infection the cells were washed with PBS, dispersed by trypsinization, and plated at a density of about 400 cells per 5-cm dish in 5 ml of medium. After 4-5 days, clones of cells were microscopically visible. Some of the cultures were fixed with methanol and stained with Giemsa’s stain. The clones in these cultures were counted to determine cloning efficiency. The medium was removed from the rest of the cultures and the cells were covered with 1 x lo6 XC cells per dish (Rowe et al., 1970). After 2-3 days the cells were fixed and stained. The numbers of syncitia per dish were determined. Reverse transcriptase assay. Reverse transcriptase activity was measured in 50 ~1 of culture fluid as described elsewhere (Aboud et al., 1976).

MLV

Interferon

135

INFECTION

preparation

and

titration.

Mouse IF was prepared by infecting L,, cells with Newcastle disease virus (NDV) and was partially purified as previously described (Aboud et al., 1976). It was titreated by the reverse transcriptase reduction assay (Aboud et al., 1976). The IF preparation used throughout this study contained 640 RTRD,,, units/ml (RTRD,,, = reverse transcriptase-reduction dose 50%, an IF dose reducing reverse transcriptase activity by 50%). RNA extraction from infected cells. Total RNA was extracted from the infected cells at various intervals after infection as described by Salzberg et al. (1977a). Virus purification. NIH/3T3(M-MLV) cells were seeded in Roux bottles at a density of 4 x 10” cells/bottle in 100 ml of medium. The medium was changed every 12 hr until the cells detached from the bottles. The collected medium was centrifuged first at 1000g for 20 min to remove cell debris and then at 40,000 g for 2.5 hr to sediment virus particles. The sedimented virus was further purified as described by Salzberg et al. (1973). Preparation of cDNA. Purified virus was used to prepare the viral complementary DNA strand (cDNA) by the endogenous reverse transcriptase reaction in a final volume of 5 ml containing 50 mM Tris-HCl, pH 8.2, 5 mM dithiothreitol, 100 mM NaCl, 0.5 mM MnCl,, 0.1 mM dATP, dGTP, and dCTP, 0.01 mM [“H]TTP (50 Ci/mmol), 0.03% Nonidet P-40, 75 pg/ml of actinomycin D, and 350 pg/ml of purified virus protein. This mixture was incubated at 37”. Aliquots were taken at l-hr intervals to follow the reaction course. When DNA synthesis reached a plateau (usually after 3-4 hi-1 the reaction was stopped by adding 0.5% sodium dodecyl sulfate (SDS) and 0.01 M EDTA. After an additional 10 min at 37” nucleic acids were twice extracted by phenol (saturated with 100 mA4 NaC1, 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA) and by a mixture of chloroform-isoamyl alcohol (24:l). Escherichia coli tRNA (50 pg/ml; Sigma) was added as a carrier to the aqueous phase and the nucleic acids were precipitated overnight with 2.5 vol of ethanol at -20”.

136

ABOUD, SHOOR, AND SALZBERG

The precipitate was dissolved in 1 ml of 0.1~ SSC (IX SSC = 0.15 M NaCl and 0.015 M Na citrate, pH 5.5) and the RNA was hydrolyzed by treatment with 0.2 N NaOH for 60 min at 37”. After neutralization with HCl, 50 pg of E. coli DNA was added.as carrier and the DNA was precipitated with ethanol. The specific activity of the cDNA thus obtained was 2 x lo7 cpmlpg. It was 95% sensitive to Sl nuclease digestion and 88% resistant after hybridization to an excess of viral RNA. Quantitution of virus-specific RNA. Virus-specific RNA was determined by annealing about 600 cpm of cDNA to RNA samples for 40 hr at 68”, as previously described (Salzberg et al., 1977). The amount of viral RNA in each sample was computed from a standard curve. This standard curve was established by determining the percentage of the input cDNA hybridized as a function of the amount of purified 70 S viral RNA used in the annealing reaction (Salzberg et al., 1977a). Since, during RNA extraction, we used no internal standard for estimating the extraction efficiency, we expressed our resuits as nanograms of viral RNA per microgram of total RNA. RESULTS

TIME

AFTER

INFECTION

(hr)

FIG. 1. Effect of IF on virus productionin exogenously infectedNIH/3T3 cells. NIH/3T3 cells grown with or without 4 IF RTRD,, units/ml were infected as described in Materials and Methods. At the end of the adsorption period, the untreated control culture (0) and one of the IF-treated cultures (0) received IF-free medium. Two additional IF-treated cultures received medium with the same IF dose. The IF-containing medium was removed from one of the latter cultures 6 hr after infection. This culture was washed with PBS and received IF-free medium (A), whereas the second remained with IF throughout the experiment (01. At various intervals, aliquots of 50 ~1 were withdrawn from each culture for reverse transcriptase assay.

IF Effect on Infection

Efficiency

It was important to examine more diContinuous and Temporary Effects of IF rectly the possibility that IF might merely To demonstrate the effect of IF on exog- interfere with the establishment of the enous infection of M-MLV, NIH/3T3 cells infection rather than impair virus replicawere treated with IF for 24 hr before tion. For this, IF-treated and untreated infection. As can be seen from Fig. 1, in cells were infected with M-MLV, then disuntreated cells the first release of viral persed by trypsinization and seeded at a progeny to the medium was detected at 11 density of about 400 cells/dish without IF. hr after infection. In the IF-pretreated After 4-5 days some of the cultures were cultures no significant release of progeny stained and scored for clone formation to occurred even at 31 hr after infection, if determine the plating efficiency. The rest IF remained continuously in the medium. of the cultures were covered with XC It could be argued that the IF preparation cells. After an additional 2-3 days these only inhibits the adsorption or the pene- cultures were stained and scored for infectration of the virus to the cells rather tious centers. The results presented in than interfering with the virus replication Table 1 clearly show that our IF preparacycle. To examine this possibility IF was tion had no effect on the efficiency of eliminated either just prior to infection or infection. 6 hr later. In both cases only a temporary delay of virus release was observed, after IF Effect on Viral RNA Synthesis IF pretreated and untreated cultures which the normal rate was gradually rewere infected with M-MLV. Thereafter stored (Fig. 1).

IF EFFECT TABLE

ON EXOGENOUS

1

EFFICIENCY OF INFECTION IN IF-TREATED AND UNTREATED CELLS Cells Number of col- Number of in- Infection onies per dish” feetious centers efficiency per dish” (%) Untreated IF-treatedb

265 5 27 178 c 12

106 + 11 80 c 10

40 45

u Average of five plates -t standard deviation. b Cells were treated with 4 RTRD,,, units/ml 24 hr before infection.

for

the IF-pretreated cultures were supplied with IF-containing medium whereas the untreated cultures received IF-free medium. RNA was extracted from cells at different intervals after infection. Viral RNA was estimated by annealing appropriate amounts of the extracted RNA to viral cDNA. The results documented in Fig. 2 indicate that in the untreated cells viral RNA synthesis started at about 6 hr after infection. This finding is in accordance with other reports concerning similar systems (Salzberg et al., 1973; Schincariol and Joklik, 1973). In the IF-treated cells viral RNA accumulated at a much slower rate, suggesting either an inhibition of the viral RNA synthesis or its faster degradation. Effect of Cycloheximide on Virus and Viral RNA Synthesis

MLV

INFECTION

137

reduced accumulation of viral RNA, whereas an inhibition of a late step does not. It has been reported that cycloheximide (CH), a reversible inhibitor of protein synthesis, when added during the early stage of exogenous infection, blocks some early function required for the virus development (Gallis et al., 1976; Salzberg et al., 1977a). To examine its effect in our system, CH at a concentration of 10 pglml was added for 4-hr periods at various times before or after infection. This amount of the drug inhibited 95% of the cellular protein synthesis capacity as determined by the incorporation of [“Hlleucine into acid-insoluble material, whereas more than 90% of this capacity was restored within 1 hr after its removal (data not shown). As can be seen from Fig. 3, when this drug was present from 5 to 9 hr after infection, virus release was temporarily delayed. This is certainly a result of a temporary inhibition of viral protein synthesis normally occurring at this time. However, a delay in the virus release was also observed when CH was present from 1 to 5 hr after infection, namely, before viral RNA synthesis has started. This confirms the notion that CH inhibits some early step in the infectious

Release

The reduced accumulation of viral RNA in IF-treated cells could reflect a feedback effect of IF action on some late step participating in the utilization of viral RNA. However, this possibility is unlikely since we have observed that in chronically infected cells, where IF supposedly blocks the final virus release, IF treatment leads to accumulation of viral RNA above the normal level rather than to its degradation or to inhibition of its synthesis (Salzberg et al., manuscript in preparation). It is more likely that the reduced accumulation of viral RNA in the exogenous infection is a result of IF-induced inhibition of some early step. In order to substantiate these arguments it would be useful to provide another example in which inhibition of an early step in the infectious cycle leads to a

TIME

AFTER

INFECTION

FIG. 2. Effect of IF on viral RNA synthesis after exogenous infection. IF-treated (A) and untreated (0) NIH/3T3 cells were infected with M-MLV as in Fig. 1. At various times after infection RNA was extracted from cells and the amount of virus-specific RNA was determined by hybridization to cDNA as described in Materials and Methods. The dashed line represents the hybridization level obtained with RNA extracted from uninfected cells.

138

ABOUD,

TIME AFTER

SHOOR,

INFECTION (hr)

FIG. 3. Effect of CH on virus production in exogenous infection. NIH/3T3 cells were treated with CH for 4 hr before infection (A), at 1-5 hr (01, or at 5-9 hr (A) after infection. Untreated cells (0) served as a control. At various times after infection aliquots were taken from the culture medium for reverse transcriptase assay.

cycle. To exclude the possibility that these results reflected some nonspecific effects of CH, this drug was added for 4 hr before infection. No effect on virus release was detected in this case. In the experiment presented in Fig. 4 we examined the effect of this drug on viral RNA synthesis. As could be expected, inhibition of the early step by adding CH at 1 to 5 hr after infection led to a severe delay in viral RNA synthesis. However, when present for 5 to 9 hr after infection, namely when the early stage had supposedly been completed, or when present at 4 hr before infection, CH had no effect on viral RNA synthesis. IF Elimination Infection

at Various

Times

AND

SALZBERG

tion, namely, the final release of the virions. Since this event occurs 11 hr after infection, elimination of IF at any time prior to infection should result in no significant inhibition of viral progeny release. To determine whether this was indeed the case, cells were treated with IF for 24 hr and were infected with M-MLV at various times after IF removal. The results illustrated in Fig. 5 indicated a substantial effect of the IF pretreatment as long as IF was eliminated at any time shorter than 10 hr before infection. A slight effect was still found when IF was removed 10 hr before infection, but no effect could be detected when IF was removed 12 hr before infection. DISCUSSION

The exogenous infection of cells by oncornaviruses differs from chronic infection in that it includes additional steps, those involved in the viral DNA synthesis and in its integration into the cellular genome. These steps will be regarded as the early stage of the infectious cycle. After the integration has been completed, both types of infection are virtually identical with respect to the subsequent events consistT f

I

I

I

54

1

l-

before

Another approach was applied to determine whether IF exerted its effect at the early stage of the exogenous infection. This relied on our previous observation with chronically infected cells that the IF effect persisted for 10 hr after its removal (Salzberg et al ., manuscript in preparation). If IF exerted its effect on a late step of the exogenous infection, this would most likely be the same as in the chronic infec-

a >

0

TIME

I

I

I

5

IO

I5

AFTER

20

INFECTION

FIG. 4. Effect of CH on viral RNA synthesis in exogenously infected cells. NIH/3T3 cells were treated for 4 hr before infection (A), at 1-5 hr (O), or at 5-9 hr (m) after infection. Untreated cells served as a control (0). At various times after infection RNA was extracted from the cells and virus-specific RNA was determined as described in Materials and Methods.

IF EFFECT

ON EXOGENOUS

Ii ‘0 I

P . . A/ It

0

4 TIME

8 AFTER

12 INFECTION

16

20

24

lhr)

FIG. 5. Removal of IF at various times before exogenous infection of NIHi3T3 cells. Cells were plated with or without (0) IF as described in Fig. 1. At 12hr (O), 10 hr (A), 8 hr cm),4 hr CO),and 0 (A) hr before infection the IF-containing medium was removed and, after washing with PBS, the cells received IF-free medium. At zero time the cells were infected. One hour after infection the cells were washed with PBS and overlaid with IF-free medium. At various intervals thereafter aliquots were withdrawn for reverse transcriptase assay.

ing of the late stage of virus production. Thus, if IF affects the exogenous infection in its late stage, this is most likely at the same step as in the chronic infection, namely, the final virus release from the cell. On the other hand, if the affected event in the exogenous infection is different than that in the chronic one, this event most probably belongs to the early stage. When NIH/3T3 cells were treated with IF before infection with M-MLV no significant release of viral progeny occurred as long as IF was still present in the medium after infection. This was evidently not due to interference with adsorption or the penetration of the virus to the cells since, when IF was removed either before or later after infection, virus release gradually restored to normal after a temporary delay. This conclusion was further substantiated by the finding that IF treatment had no effect on the infection efficiency as determined by the formation of

MLV

INFECTION

139

infectious centers. Pitha et al. (19761, who also found no effect of IF on the formation of infectious centers, interpreted this finding as indicating that IF does not impair any of the early steps required for the synthesis of the viral DNA or for its integration into the cellular genome. It seems to us that alternative possibilities implying impairment of some early steps should not be excluded. For example, it is possible that the synthesis of the viral DNA is prevented due to inhibition of uncoating, thus preserving the viral RNA in a protected form until the cells recover from the IF effect. Similarly one can think of inhibition at the integration step while the synthesized viral DNA is maintained within the cells until recovery from the IF effect. Investigation of the effect of IF on viral RNA synthesis revealed that continuous treatment with IF before and after infection resulted in a remarkably reduced rate of viral RNA accumulation. This effect was already detected at 6-7 hr after infection before any release of virus particles could be expected. Therefore, the possibility that the effect of IF is, in fact, directed in this system at the virus release and that the reduced viral RNA accumulation is just a result of a feedback effect is excluded. Moreover, we have found that in chronically infected cells, where IF supposedly acts at the late stage, prolonged IF treatment leads to accumulation of viral RNA in the nucleus above the normal level rather than to its inhibition (Salzberg et al., manuscript in preparation). It is more likely that our findings with the exogenous infection indicate a reduced synthesis of viral RNA, resulting from IF-induced inhibition of some early step(s). The experiment with CH presented in Fig. 4 demonstrates another example where inhibition of an early function results in a delay in viral RNA synthesis while inhibition of a late step, such as viral protein synthesis, does not. The postulate that IF acts at the early stage of the exogenous infection was further supported by the experiment in which IF was removed at various times before infection. We have recently found that

ABOUD, SHOOR, AND SALZBERG

140

with chronically infected cells the antiviral state persisted for 10 hr after IF removal (Salzberg et al., manuscript in preparation). Accordingly, if in the exogenous infection IF acts at the final release of virus particles, no effect of IF pretreatment should be expected if IF is removed at any time before infection since this step occurs 11 hr after infection. It could indeed be argued that in this respect chronically infected cells do not necessarily resemble exogenously infected cells, but such an argument was eliminated by the results presented in Fig. 5. It was found that when IF was removed at any time shorter than 10 hr before infection, IF pretreatment was remarkably effective. When IF was eliminated 10 hr before infection a slight effect was still evident. However, when IF was eliminated 12 hr before infection IF pretreatment was no longer effective. This lo-hr limit precisely correlated with the lo-hr persistence of IF effect in chronically infected cells. It is unlikely that this correlation is just a coincidence. It is more reasonable to assume that the IF-induced antiviral state is maintained

t

LI

ACKNOWLEDGMENTS This investigation was supported by a grant from the National Council for Research and Development, Israel, and the KFA Jiilich, GSF Miinchen, GFK Karlsruhe, and GKSS Geesthacht-Tesperhude, Germany. REFERENCES

I

42 -IO 4

in NIH/3T3 cells for 10 hr after IF removal, regardless of whether or not they are infected with M-MLV. In Fig. 6 a model intended to explain the effect of IF when removed at various times before infection is presented. According to this model, the antiviral state persists for 10 hr after IF removal. When this period terminates before infection (i.e., when IF is removed 12 hr before infection) IF pretreatment is ineffective. However, when IF elimination is sufficiently close to infection the antiviral state is expanded into the early stage of infection. IF can thus act against several possible targets, such as uncoating, translation of parental viral RNA into early viral protein (Salzberg et al., 1977a), viral DNA synthesis, its transport to the cell nucleus, and its integration into the cellular genome. Direct studies on the molecular events sensitive to IF, which may provide more conclusive information, are now in progress.

I I II -6

-4

,

EARLY STAGE

0

2

LATE STAGE

I I I I I I I I I -2

TIME

OF

4

6

8

INFECTION

0

12 I4

I6

(hr)

FIG. 6. A schematic model for IF effect on exogenous infection. This model is based on the results presented in Fig. 5 and is intended to describe schematically the course of events occurring in NIH/ 3T3 cells pretreated with IF and infected with MMLV. The dotted bars represent the duration of persistence of the antiviral state after IF removal. The open bars represent the early stage of the exogenous infection. The black bars represent the late stage of Infection.

ABOUD, M., WEISS, O., and SALZBERG,S. (1976). Rapid quantitation of interferon with chronically oncornavirus-producing cells. Infect. Zmmun. 13, 1626-1632. ALI, M., and BALUDA, M. A. (1974). Synthesis of avian oncornavirus DNA in infected chicken cells. J. Viral. 13, 1005-1013. ALLEN, R. T., SCHELLEKENS,H., VAN-GRIENSVEN, L. J. L. D., and BILLIAU, A. (1976). Differential sensitivity of Rauscher murine leukemia virus (MuLV-Rl to interferon in two different interferon-responsive cell lines. J. Gen. Viral. 31, 429435. BILLIAU, A., SOBIS, H., and DE-SOMER, P. (1973). Influence of interferon on virus particles formation in different oncornavirus carrier lines. Znt. J. Cancer 12, 646-653. BILLIAU, A., EDY, V. G., SOBIS,H., and DE-SOMER, P. (1974). Influence of interferon on the synthesis of virus particles in oncornavirus carrier cell lines. II. Evidence for a direct effect on particle release. Znt. J. Cancer 14, 335-340. BILLIAU, A., EDY, V. G., DE-CLERCQ,E., HEREMANS, H., and DE-SOMER,P. (1975). Influence of inter-

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ON EXOGENOUS

feron on the synthesis of virus particles in oncornavirus carrier cell lines. III. Survey of effects on A-, B-, and C-type oncornaviruses. Int. J. Cancer 15, 947-953. BILLIAU, A., HEREMANS, H., ALLEN, P. T., DE-MAEYER-GUIGNARD, J., and DE-ISOMER, P. (1976). Trapping of oncornavirus particles at the surface of interferon treated cells. Virology 73, 537-542. FRIEDMAN, R. M., and RAMSEUR, J. M. (1974). Inhibition of murine leukemia virus production in chronicallly infected AKR cells. A novel effect of interferon. Proc. Nat. Acad. Sci. USA 71, 35423544. FRIEDMAN, R. M., CHANG, E. H., RAMSEUR, J. M., and MEYERS, M. W. (1975). Interferon directed inhibition of chronic murine leukemia virus production in cell cultures: Lack of effect on intracellular markers. J. Virol. 16, 569-574. FRIEDMAN, R. M., COSTA, J. C., RAMSEUR, J. M., MEYERS, M. W., JAY, F. T., and CHANG, E. H. (1976). Persistence of viral genome in interferontreated cells infected with oncogenic or nononcogenie viruses. J. Infect. Dis. 133, A43-A49. FRIEDMAN, R. M., JAY, F. T., CHANG, E. H., MEYERS, M. W., RAMSEUR, J. M., MIMS, S. J., TRICHE, T. J., and WONG, P. K. Y. (1977). Interferondirected inhibition of chronic murine leukemia virus production in cell cultures. In “Control of Neoplasis by Modulation of the Immune System” (M. A. Chirigos, ed.), pp. 347-359. Raven Press, New York. GALLIS, B. M., EISENMAN, R. N., and DIGGELMANN, H. (1976). Synthesis of the precursors to avian RNA tumor virus internal structural proteins early after infection. Virology 74, 302-313. KHOURY, A. T., and HANAFUSA, H. (1976). Synthesis and integration of viral DNA in chicken cells at different times after infection with various multiplicities of avian oncornavirus. J. Viral. 18, 383400. LIBERMAN, D., VOLOCH, Z., AVIV, H., NUDEL, U., and REVEL, M. (1974). Effects of interferon on hemoglobin synthesis and leukemia virus production in Friend cells. Mol. Biol. Rep. 1, 447-451. PERIES, J., CANIVET, M., GUILLEMA~N, B., and BOIRON, M. (1968). Inhibitory effect of interferon preparations on the development of foci of altered cells induced in vitro by mouse sarcoma virus. J. Gen. Virol. 3, 465-468. PITHA, P. M., Row, W. P., and OXMAN, M. N. (1976). Effect of interferon on exogenous, endogenous and chronic murine leukemia virus infection. Virology 70, 324-338. RAMSEUR, J. M., and FRIEDMAN, R. M. (1976). Interferon inhibits induction of endogenous murine leukemia virus production. Virology 73, 553556. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W. (1970). Plaque assay technique for murine leuke-

MLV

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141

mia viruses. Virology 42,1136-1139. SALZBERG, S., RABIN, M. S., and GREEN, M. (1973). Appearance of virus-specific RNA, virus particles and cell surface changes in cells rapidly transformed by murine sarcoma virus. Virology 53, 186-195. SALZBERG, S., LEVI, Z., ABOUD, M., and GOLDBERGER, A. (1977). Isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution. Biochemistry 16, 25-29. SALZBERG, S., ROBIN, M. S., and GREEN, M. (1977a). A possible requirement for protein synthesis early in infectious cycle of murine sarcoma-leukemia virus. Virology 76, 341-351. SARMA, P. S., SHIU, G., BARON, S., and HUEBNER, R. J. (1969). Inhibitory effect of interferon on murine sarcoma and leukemia virus in vitro. Nature (London) 223, 845-846. SCHINCARIOL, A. L., and JOKLIK, W. K. (1973). Early synthesis of virus specific RNA and DNA in cells rapidly transformed with Rous sarcoma virus. Virology 56, 532-548. TEMIN, H. M., and BALTIMORE, D. (1972). RNA directed DNA synthesis and RNA tumor viruses. Advan. Virus Res. 17, 129-186. VAN-GRIENSVEN, L. J., BAUDELAIRE, M. E., PERIES, J., EMANOIL-RAVICOVITCH, R., and BOIRON, M. (1971). Studies on the biosynthesis of murine leukemia virus RNA. Some properties of Rauscher leukemia virus isolated from interferon treated JLSV cells. In “Le Petit Coloquia,” pp. 145-153. North-Holland, Amsterdan. VARMUS, H. E., BISHOP, J. M., and VOGT, P. K. (1973). Synthesis and integration of Rous sarcoma virus specific DNA in permissive and non-permissive hosts. In “Virus Research” (C. F. Fox and L. Silvestri, eds.), pp. 373-379. Academic Press, New York. VARMUS, H. E., VOGT, P. K., and BISHOP, J. M. (1973a). Integration of deoxyribonucleic acid specific for Rous sarcoma virus after infection of permissive and non-permissive hosts. Proc. Nat. Acad. Sci. USA 70, 3067-3071. VARMUS, H. E., GUNTAKA, R. V., FAN, W. J., HEASLY, S., and BISHOP, J. M. (1974). Synthesis of viral DNA in the cytoplasm of duck embryo tibroblasts and in enucleated cells after infection of avian sarcoma virus. Proc. Nat. Acad. Sci. USA 71, 3874-3878. VARMUS, H. E., HEASLY, S., and BISHOP, J. M. (1974a). Use of DNA-DNA annealing to detect new virus specific DNA sequences in chicken embryo tibroblasts after infection with sarcoma virus. J. Virol. 14, 895-903. WV, A. M., SCHULTZ, A., and GALLO, R. C. (1976). Synthesis of type C virus particles from murine cultured cells induced by iododeoxyuridine. V. Effect of interferon and its interaction with dexamethasone. J. Viral. 19, 108-117.