Effect of interferon on exogenous, endogenous, and chronic murine leukemia virus infection

VIROLOGY 70, 324-338

(1976)

Effect of Interferon

PAULA

on Exogenous, Endogenous, Leukemia Virus Infection

M. PITHA,’

WALLACE

P. ROWE,2

AND

and Chronic

MICHAEL

Murine

N. OXMAN

’ Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, 2 Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, and 3 Virus Research Unit, Childrens Hospital Medical Center and the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts Accepted November

12, 1975

The effect of purified mouse interferon on the replication of AKR mouse leukemia virus (MLV) in a clonal line of AKR cells was studied, with emphasis on comparing the effect of interferon on the replication of exogenous virus, activation of endogenous virus by IdLJrd, and production of virus by chronically infected cells. It was found that interferon inhibited replication of virus in all three systems. Interferon did not abort exogenous infection or virus induction by IdUrd, but only delayed appearance of infectious virus. Virus production by chronically infected cells also was suppressed in the presence of interferon; however, after removal of interferon, rapid recovery of virus production occurred. Under conditions where interferon treatment inhibited virus yield as measured both by infectious virus and virion-associated reverse transcriptase activity, no significant inhibition of synthesis of virus specific (gs) antigen was observed (as measured by immunofluorescence and radioimmunoassay for p30 protein). These results indicate that unlike its effect on the majority of viruses, interferon does not inhibit MLV by a general inhibition of viral protein synthesis; rather, it appears to inhibit one or more of the later steps in MLV replication which occur after the expression of viral gs antigen. The interferon block of acute exogenous or IdUrd-induced endogenous infection appeared to occur prior to virus assembly, resulting in a marked decrease in the number of free and cell-associated particles. In the chronic infection, however, the interferon treatment only partially prevented assembly and release of virus particles, but these had markedly reduced infectivity.

were inhibited when the cells were treated with interferon prior to infection. However, once the cells were transformed [and the SV40 genome integrated into cellular DNA (Sambrook et al., 196811, the synthesis of SV40 T antigen was completely resistant to interferon (Oxman et al., 1967a). It was shown that the inhibition of SV40 infection in permissive systems by interferon is probably due to a block in virus transcription (Oxman and Levin, 1971). In the present work, we examined whether the ability of the interferon systern to recognize the exogenous viral genome, but not the integrated one, also applies to murine leukemia virus (MLV). It, has been shown previously that both replication and cellular transformation caused

INTRODUCTION

It is generally believed that interferon inhibits the multiplication of oncogenic viruses and blocks virus-induced cell transformation by interfering with the expression of nonintegrated viral genes (Oxman, 1973). This hypothesis has been based predominantly on work with Simian virus 40 (SV40). In permissive cells, pretreatment with interferon was shown to inhibit the production of SV40 T antigen (an early viral gene function), V antigen (a late viral

gene function),

and infectious

virions

(Oxman et al., 1967b). In nonpermissive cells, synthesis of T antigen (Oxman and Black, 1966) and SV40-induced cell transformation (Todaro and Baron, 1965) also 324 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

EFFECT

OF INTERFERON

by MLV and murine sarcoma virus (MSV) are inhibited in cells pretreated with interferon (Fitzgerald, 1969; Sarma et al., 1969; Peries et al., 1968; Gresser et al., 1967). However, in those studies, virus replication was not measured under single-cycle conditions and the involvement of helper viruses (Hartley and Rowe, 1966) makes the systems examined too complex to permit this question to be answered unambiguously. Furthermore, these studies were performed with unpurified interferon, and thus, the observed effects might have been due to inhibition of cellular metabolism [by interferon itself (Gresser et al., 1970)or by impurities in the interferon preparations used] rather than to the direct antiviral action of interferon. This is a particularly crucial point in studies with C-type RNA viruses, where the efficiency of infection is dependent upon the competence of the cell to synthesize DNA and to undergo mitosis (Temin, 1969). The development of new procedures for a high degree of purification of mouse interferon (>lO’ units/mg protein) by Paucker and his co-workers (Ogburn et al., 1973) and DeMaeyer and his co-workers (Sippe et al., 1973) and the existence of virus-free AKR mouse embryo cell lines in which the MLV genome is part of the cell genome and can be induced by IdUrd treatment (Rowe et al., 1971) provided an opportunity to study, in a single virus-cell system, the effect of interferon on acute exogenous virus infection, on the induction of the endogenous (integrated) viral genome, and on virus production by chronically infected cells. The results reported here indicate that interferon produces a temporary inhibition of the replication of both exogenous and endogenous MLV. However, in contrast to the effect of interferon observed in other virus-cell systems, the inhibition of MLV replication is not due to a general inhibition of viral protein synthesis, but rather to interference with some later step (or steps) in the virus growth cycle. During the course of the investigations described here, Billiau et al. (1973, 1974) and Friedman and Ramseur (1974), reported the effect of interferon on cells persistently infected with mouse leukemia virus. There is a basic correlation between

ON MLV

325

INFECTION

their findings here.

and the results reported

MATERIALS

AND

METHODS

Cells and viruses. The virus-negative AKR mouse embryo clonal cell line AKR2B (Rowe et al., 1971) was grown in McCoy’s 5a medium supplemented with 10% heated fetal bovine serum (FBS). The SC-l mouse cell line (Hartley and Rowe, 1976), primary NIH Swiss mouse embryo cells, mouse L cells, and XC cells (Svoboda et ccl., 1963) were grown in Eagle’s minimal essential medium with 10% heated FBS. Chronically infected AKR cells were obtained by infecting virus-negative AKR2B cells with 0.1 PFUlcell of AKR-Ll virus. The cells were dispersed with trypsin (0.75%) 24 hr after infection and seeded in 250-ml Falcon plastic flasks in Eagle’s minimal essential medium with 10% FBS (approximately lo” cells/ff ask). The cells were passaged at weekly intervals and the medium was changed at 3-day intervals. The chronic infection was well established by the third passage, at which time the virus titer of the culture fluid was 1.5 x 10” PFU/ml. Virus production remained stable at this level for an additional 20 passages (over a period of 5 months) and then decreased to 1.5 x lo5 PFU/ml. Fluorescent antibody staining revealed that when chronic infection was well established (passage 5 to 20), 100% of the cells contained gs antigen. The AKR-Ll virus was isolated from a spontaneously leukemic AKR mouse (Hartley et al., 1970) and adapted to growth in tissue culture by serial passage in NIH Swiss mouse embryo cells. The virus employed in the present investigations consisted of clarified (10,000 g; 10 min; 4”) tissue-culture supernatant. The titer was 10’ PFU/ml in SC-1 cells. Vesicular stomatitis virus (VSV; New Jersey serotype) was propagated in DEAE-dextran (10 pg/ml) treated mouse L cells which were infected at low multiplicity (0.01 PFU/cell) and harvested 24 hr after infection. The titer of the clarified cell supernatant was 10%PFUI ml in mouse L cells. Assays of infectious MLV. Titrations of cell-free virus were done with the SC-I.

326

PITHA,

ROWE

mouse cell line using the standard UV-XC plaque procedure (Rowe et al., 1970). For quantitation of cells which had become virus-producers at various times following exogenous infection or IdUrd induction, the UV-ME test was used. This procedure is an in situ infectious center assay which takes advantage of the fact that virus-producing cells remain infectious for some time after lethal uv-irradiation. The culture, in a 60-mm plastic petri dish, is uvirradiated and 2 x lo5 SC-1 cells are added; 4 days later, the dish is again uv-irradiated and XC cells are added (the UV-XC procedure). The culture is fixed and stained with hematoxylin 3 days later. Cells which were producing virus at the time of the first uv-irradiation infect the SC-1 cells, producing a local focus of infection which subsequently registers as a plaque in the UV-XC test. A single virusproducing cell or a focal area of infected cells will register as a single plaque, while an infected cell which has not yet begun virus production will not induce a plaque. For infectious center assays, infected cultures were dispersed with 0.75% trypsin at 16 hr after infection; the cells were counted and appropriate numbers of cells were plated on SC-1 cell cultures which had been seeded with 3.5 x lo5 cells/dish 24 hr earlier. These cultures were then incubated for 4 days and lethally uv-irradiated, and the foci of MLV infection were detected by the UV-XC test as described above. Induction of endogenous virus from AKR-2B cells. AKR-2B cells, seeded at 3.5

x lo5 cellsl60-mm petri dish 24 hr earlier, were treated with IdUrd (20 pg/ml) for 24 hr at 37” as previously described (Teich et al., 1973; Pitha et al., 1975). They were then washed and refed with medium. In all tables, times of assay refer to hours from the initiation of IdUrd treatment. Assays for group-specific

(gs) antigens.

AND

OXMAN

Virion-associated

reverse transcriptase.

The virion-associated reverse transcriptase activity of tissue-culture supernatant fluids was measured using synthetic template poly(rA)*oligo(dT) (Collaborative Research) as previously described (Pitha et al., 1975). After the fluids were clarified (3000g; 10 min; 4”; followed by 10,000g; 30 min; 4”), the virus was pelleted by centrifugation at 100,000g for 60 min at 4”. In some experiments, the activity was high enough that assays could be performed on the clarified fluids themselves. Interferon. Interferon, a generous gift of Dr. K. Paucker, was induced in mouse L cells by uv-irradiated Newcastle disease virus (Youngner et al., 1966), concentrated by Amicon filtration, and purified by affinity chromatography on anti-L cell interferon globulin coupled to sepharose 4B (Ogburn et al., 1973). The two preparations employed in these studies contained 1 x lo7 and 1 x lo* international reference units per milligram of protein. At the highest concentration of interferon employed (150 units/ml), neither preparation of interferon affected cell DNA synthesis (as measured by incorporation of i3Hlthymidine) or protein synthesis (as measured by incorporation of 13H]leucine) in AKR-2B cells. Furthermore, at a concentration of 150 units/ml, these interferon preparations did not alter the growth rate of the AKR-2B cells, as measured by the increase in the number of viable cells in nonconfluent cultures over a period of 120 hr (Fig. 1). Assay of interferon-induced antiviral effeet. The interferon-induced antiviral state

was measured by single-cycle VSV yield assays. Interferon-treated and control cultures were washed, exposed to VSV (10 PFU/cell) for 1 hr at 37”, washed again, and refed with medium. The yield of VSV at 15 hr after infection was determined by plaque assay on mouse L cells.

Cells were stained for gs antigens utilizing a direct immunofluorescence test, as previRESULTS ously described (Hartley and Rowe, 1975; Hartley, personal communication). Ra- The Antiviral Effect Induced by Interferon under Various Experimental Condidioimmunoassays for MLV p30 antigens tions were performed by Dr. W. D. Parks as Because cell division is essential for the previously described (Parks and Scolnick, establishment of MLV infection, all exper1972).

EFFECT

OF INTERFERON

ON MLV

327

INFECTION TABLE

PERSISTENCE

Interferon dose (units/ ml)

VSV yield (PFUkulture

iments were carried out in dividing cells. However, it has been shown that newly seeded, dividing cells are less sensitive to the antiviral effect of interferon than are the confluent nondividing cells usually employed in interferon research (Rossman and Vilcek, 1969). Furthermore, other experimental manipulations, such as treatment with IdUrd, superinfection with MLV, and trypsinization after interferon treatment, also might alter the response to interferon. Consequently, the antiviral effect of interferon under these experimental conditions was measured by the single-cycle VSV yield assay. The results in Table 1 show that while the dividing cells were relatively sensitive to interferon (3 units/ ml reduced the yield of VSV to 47% of that in untreated cultures), the antiviral effect decreased markedly within 24 hr after the removal of interferon. The residual antiviral activity, which appears to be comparable at 24 and 48 hr after removal of the interferon, may represent the effect of interferon bound to the cells and not removed by washing, which subsequently elutes to produce a low level of interferon in the medium. The antiviral effect induced by interferon was not altered in cells treated with

x 10-Y

Interval between removal of interferon and VSV challenge

0 3 30

FIG. 1. Effect of interferon on the growth of AKR-2B cells. Cells were seeded at a density of 3.5 x lo” tells/60-mm petri dish in growth medium (10% FBS) with or without interferon (150 units/ml) and the viable cells were counted at the times indicated. The ability of the cells to exclude trypan blue was used as a criterion of viability (Phillips and Terry berry, 1957); each value represents the average of triplicate cultures. O-O, cells grown in interferon; O-----O, cells grown in medium.

1

OF ANTIVIRAL EFFECT OF INTERFERON IN DIVIDING AKR-2B CELL@

0 hr

24 hr

48 hr

21 9.8 0.1

20 15 2.4

22 15 2.0

a Nonconfluent cultures (seeded 6 hr earlier with 3.5 x 10” tells/60-mm petri dish) were exposed to interferon for 24 hr at 37”; cells were then washed and either immediately infected with VSV (10 PFU/ cell) or incubated in interferon-free medium for 24 or 48 hr prior to challenge. Fluids were collected 15 hr after challenge and VSV was titrated by plaque assay in L cells.

IdUrd or in cells acutely or chronically infected with AKR-Ll virus (Table 2). However, trypsinization after interferon treatment led to a rapid decay in the interferon-induced antiviral state (Table 2). Trypsinization completely abolished the antiviral effect induced by low doses of interferon (3 units/ml) and markedly decreased that induced by high doses. Effect of Interferon Infection

on Exogenous

MLV

As measured by standard single-cycle yield assays in cells pretreated with interferon for 24 hr, the replication of AKR-Ll virus in AKR-2B cells was relatively sensitive to interferon (Fig. 2). Pretreatment with 8 units/ml of interferon produced a 50% reduction in the yield of extracellular MLV. It required only 2.2 units/ml to produce a 50% reduction in the yield of VSV in the same cells. Thus, the replication of AKR-Ll virus was only three- to fourfold less sensitive to interferon than the replication of VSV. However, further analysis with other methods of assay and at different times after MLV infection showed that the response to interferon was complex. Table 3 presents the results of two experiments in which the yield of infectious virus and the proportion of the infected cells that had begun producing virus (i.e., had become uv-resistant in situ infectious centers)

328

PITHA, ROWE AND OXMAN TABLE 2

EFFECT OF IdUrd, TBYPBINIZATION, AND ACUTE AND CHRONIC AKR-~1 VIRUEI INFECTION ON THE ANTIVIRAL EFFECT OF INTERFERON IN AKR-2B CELL@

Treatment

Interferon dose

VSV Yield (PFU/culture x lo-*) Treated

Untreated control

0 3 30

23 9.0 0.25

21 9.8 0.1

Acute AKR-Ll” virus infection

0 30 150

16 7.0 0.1

19 8.0 0.08

Chronic AKR-Lid virus infection

0 30 150

13 6.0 0.14

10 5.2 0.21

Trypsinization’

0 3 30 150

15 15 2.9 4.0

16 8.9 0.33 0.07

YlP IdUrd*

a AKR-2B cells were plated in medium with or without interferon; 24 hr later, cells were washed and treated as described in the following footnotes. In all cases, VSV in the fluid was collected 15 hr postinfection and plaque titrated in L cells. * Incubated with IdUrd (20 pg/ml) with or without interferon for 24 hr; washed and infected with VSV (10 PFU/cell). o Treated with DEAE-dextran (20 pg/ml) for 1 hr at 37”; washed and infected with AKR-Ll virus (1 PFU/cell) for 2 hr at 37”; and overlayed with medium with or without interferon and incubated for 48 hr. Cells were washed and infected with VSV. d Infected with VSV. p Trypsinized with 0.75% trypsin, plated without dilution in interferon free medium, and infected with VSV 4 hr later.

were examined at 24, 48, and 72 hr after infection. The proportion of cells actually infected (infectious centers) was also measured. In these experiments, interferon treatment was from 24 hr before until 16 hr after infection. It can be seen that the degree of inhibition induced by interferon was dependent upon the time at which the assays were performed; that is, it appears that many of the cells in which

INTERFERON,unitr,m,,

FIG. 2. Interferon sensitivity

of VSV and AKRLl virus in AKR-2B cells. Cells were plated (3.5 x lo5 cells/dish) either in medium (10% FBS) or medium with interferon as indicated. Twenty-four hours later, interferon was removed, cells were washed with medium and treated with DEAE-dextran (20 pg/ml) for 1 hr at 37”. After treatment, cells were washed and infected either with VSV (10 PFU/ cell) or AKR-Ll virus (1 PFU/cell). After virus absorption (2 hr at 37”), cells were washed and overlayed with 3 ml of medium without interferon. VSV was collected 15 hr later and titrated by plaque assay in L cells. Culture fluid with MLV was collected 36 hr later and virus was titrated by the XC test in SC-l cells. O-O, VSV; O-0, MLV.

virus replication was inhibited at 24 hr had escaped from interferon inhibition and had become virus producers by 48 or 72 hr. This phenomenon was investigated further using a different experimental system, namely, cultures containing only 200 AKR-2B cells per 60-mm petri dish at the time of high multiplicity (3 PFU/cell) infection with AKR-Ll virus. This technique obviates the need for trypsin dispersal of the infected cultures (which produces a rapid decrease in the interferon-induced antiviral effect as shown in Table 2) while also minimizing secondary infections. In this experiment, interferon treatment was from 24 hr before until 48 hr after infection. The number of cells producing virus at 48 and 96 hr was measured by the UVME test (Table 4). Again, the number of virus-producing cells in the interferontreated cultures was significantly reduced when measured at 48 hr. However, at 96 hr (48 hr after removal of the interferon), the number was the same in the interferontreated and control cultures. These results indicate that interferon has a marked inhibitory effect on MLV

EFFECT

OF INTERFERON

ON MLV

TABLE ERECT

OF INTERFERON

merit

YYml) 1alz

Cells positive (%)

1

j

48 hr

12 hr

Inhibition (5%) 24 br

48

hr

CENTERS,

Cells positive (96)

72 hr

‘i.3,!$I~ii~;~3i~

‘:i

Inhibition (46)

;

Inhibition (%)

log PFUlml 24 hr

1

AND VIRUS

Virus yield

Infectious centers

proportion of cells producing virus

24hP

:

3

ON THE PROWRTION OF CELLS PRODUCING VIRUS, INFECTIOUS YIELD FOLLOWING Exogenous INFECTIO~P

Interferon

Experi-

329

INFECTION

48 br

12 hr

24

48

12

;i!Jjjj.

(1AKR-2B cells were plated in medium with or without interferon 24 hr before infection, washed, DEAEtreated (25 wg/ml, 1 hr at 37”), and infected with AKR-Ll virus (1 PFU/cell, in medium) for 2 hr at 37”. Then they were overlayed again with medium or interferon. To remove the exogenous virus, cells were trypsinized 16 hr later (0.75% trypsin) and plated in medium without interferon. To determine the number of cells producing virus at given times after infection, 100 cells were plated per 60-mm dish, and at the times indicated, cultures were uv irradiated and overlayed with SC-1 cells; the UV-XC test was performed 4 days later. For the infectious center assay, 100 cells were plated over SC-I cells and the UV-XC test was performed 4 days later. To determine virus yield, trypsinized cells were plated in fresh medium at a density of 5 x lo5 tells/60-mm dish and the culture fluid was harvested at the times indicated. Infectious center and virus yield determinations were performed as described in Methods. * All times represent hours after initiation of infection. TABLE EFFECT OF INTEFERON

Interferon treatment (hr)*

-24 -+ 48

-24 + 48 -

-24 + 48

ON THE ESTABLISHMENT

Assay

4

OF INFECTION

BY EXOGENOUS

Virus-producing cells (in situ plaques)

ME at 48 hr ME at 48 hr

AKR-Ll

VIRUS”

Inhibition

(%)

Exnt 1

Exnt 2

Exnt 1

Exnt 2

186 175

150 150

6

0

UV-ME UV-ME

at 48 hr at 48 hr

71 40

64 28

44

56

UV-ME UV-ME

at 96 hr at 96 hr

204 212

185 180

0

3

n AKR-2B cells were seeded (200 cells/dish) either in interferon (150 units/ml) or medium. Twenty-four hours later, interferon was removed, and cells were washed and infected with AKR-Ll virus (5 x 10” PFUi dish). After virus adsorption (1 hr at 37”), the cells were washed and overlayed with 3 ml of interferon or medium. At given times, cells were uv irradiated and overlayed with SC-l cells (UVME assay) or overlayed with SC-l cells without uv irradiation (ME assay). The UV-XC test was done 3 days later. Under these conditions, no adsorption of virus to the plates was observed in the absence of cells. Values represent the average of results from triplicate cultures. * Time measured from the initiation of infection.

production following exogenous infection. However, the MLV infection is not aborted in the interferon-treated cells; when the antiviral effect decays, MLV produc-

tion commences. Thus, while the yield of extracellular virus and the proportion of virus-producing cells are both reduced when measured 48 hr after infection, the

330

PITHA,

ROWE

AND

OXMAN

number of cells in which MLV infection is function of the timing of the interferon established (as measured by infectious treatment. While pretreatment with intercenters) is not affected by interferon treat- feron had little effect upon the subsequent induction of endogenous MLV by IdUrd, ment. To determine the effect of interferon on marked inhibition was observed when inother parameters of MLV infection, we terferon was present during the induction compared its effect on the synthesis of and the postinduction periods. This effect MLV gs antigen (measured by immuno- of interferon was not due to inhibition of fluorescent staining of infected cells and incorporation of IdUrd into DNA, since by radioimmunoassay of p30 protein in cell there was no detectable inhibition of the extracts done by Dr. Wade D. Parks) and incorporation of [3H]IdUrd (20 pg/ml; 0 to on virus production and release (deter- 24 hr) into cellular DNA in interferon mined by thin section electron microscopy treated (150 units/ml; 0 to 24 hr) cultures of infected cells by Dr. Nelson A. Wivel, (data not shown). To determine whether interferon treatand by assay of infectious virus and reverse transcriptase activity in culture ment prevented the induction of infectious fluids). The results (Table 5) showed that virus, or only delayed its replication, while interferon produced a generally par- IdUrd-treated cells were either incubated in the continuous presence of interferon or allel reduction in the yield of infectious virus and reverse transcriptase and in the had the interferon removed 48 hr after innumber of cell-associated virions seen by duction. The number of virus-producing electron microscopy, it appeared to have cells was assayed by in situ plaque formalittle effect upon the synthesis of gs antition (UV-ME test) at four different time gen. Under conditions where virion pro- intervals. The results of a representative duction was inhibited 20- to 600-fold, gs experiment are shown in Table 7. Interantigen synthesis was reduced by less feron significantly inhibited MLV production, as measured by the number of cells than twofold. These observations indicate that the inhibition of MLV production by producing virus at given time intervals, when it was continuously present in the interferon is not due to an overall inhibimedium. However, after interferon was tion of synthesis of virus-specific proteins, or to a block in virus release. Rather, it removed, there was a rapid recovery of appears to involve selective inhibition of virus production. The degree of recovery some unknown late step (or steps) in MLV depended upon the amount of interferon that was initially present. With a small replication. Furthermore, since interferon treatment (150 units/ml) did not affect cel- dose (15 units/ml), complete recovery of MLV production occurred within 48 hr lular DNA synthesis or cell multiplication after removal of interferon. With a high (Fig. l), this anti-MLV effect of interferon would not appear to be secondary to any dose (150 units/ml), a residual antiviral effect was observed for as long as 72 hr. toxic effect on cellular metabolism. This rapid decay in the antiviral effect Effect of Interferon on Activation of (similar to that observed in experiments Endogenous MLV by IdUrd with exogenous MLV and VSV infection) is probably due to the fact that the experiTreatment of the AKR-2B cells with IdUrd results in the production of detecta- ments described were all performed in ble endogenous MLV within 48 to 72 hr dividing cells, a condition required for (Lowy et al., 1971; Pitha et al., 1975). All both the establishment of exogenous infecavailable information indicates that this tion and for the activation of endogenous activation results from the transcription of virus production by IdUrd. These results indicate that interferon chromosomally located MLV DNA sequences (Teich et al., 19731. The effect of does not block the initial activation of the treatment with interferon during the early endogenous MLV genome by IdUrd, but stages of induction is shown in Table 6. rather interferes with a step in the virus The degree of inhibition observed was a replicative cycle which occurs after IdUrd

+ + -

-

from

duplicate

5

150 units activity

98 80

0

852 356

656 476 4

p30 protein (ng/mg cell protein)

antigens

58

27

Inhibition (46)

of interferon for 24 hr before infection were measured at 48 hr after infection

19

30

70 50

Viral Inhibition (96)

FA positive (96)

Cells

b Hours measured from initiation of infection. ” Electron microscopic examination of thin sectioned cells was done by Dr. Nelson ’ Picomoles of TTP incorporated per milliliter of culture fluids.

results

treated with transcriptase cultures.

-24 + 48 -

-

-

-24 4 48

+

(hrP

Interferon treatment

+

virus

TABLE

95

Inhibition (96) titer

6.7 4.0

3.9

6.7

(logmqFU

Virus

99

99

Inhibition (5%)

Fluids

0.1

70 2.6

0.07

1.0

50

Reverse transcriptase activity’l

A. Wivel.

96

98

Inhibition (%I

OF INTRACELLULAR GROUP-SPECIFIC VIRAL ANTIGENS, ACTIVITY IN CULTURE FLUIDS~

with AKR-Ll virus (m.o.i. 1). The gs antigens, p30 protein, as described in Methods. Values represent the average of the

0

1

20

Cell-associated’ virions (no. 50 cells)

DIFFERENTIAL EFFECT OF INTERFERON ON THE INDUCTION PRODUCTION OF INFECTIOUS VIRUS, AND REVERSE TRANSCRIPTASE

VIRUS INFECTION:

* AKR-2B cells were MLV yield, and reverse

2

1

EXpWimerit

EXOGENOW

33.2

PITHA,

ROWE

AND

OXMAN

activation. To further explore this phe- interferon treatment suppressed the pronomenon, the effect of interferon on the duction of both cell-associated and extrainduction of MLV gene expression (as cellular infectious virus. However, no efmeasured by the synthesis of gs antigen fect was observed on the induction of gs and by the production of infectious virus antigen. and virion-associated reverse transcriptase) was examined (Table 8). Continuous The Effect of Interferon on MLV ProducTABLE 6 EFFECT OF INTERFERON ON THE ACTIVATION OF ENDOGENOUS VIRUS BY IDURLI”

iE~“z? 0 15

150

Timing of interferon treatment (hr)b

MLV-producing cells (in situ UVME plaques)

Inhibition

tion by Chronically

(%)

40 hr

72 hr

48 hr

72 hr

18 18 2 6 1 8 0 1 0

2,000 2,000 1,500 1,500 600 1,000 187 360 127

0 89 67 94 56 100 94 100

0 25 25 70 50 91 82 94

-24+ 0 -24 + 24 0 + 24 O-+48 -24+ 0 -24 + 24 0 + 24 0 + 48

D AKR-2B cells were seeded (3.5 x 105/60-mm petri dish) in medium or interferon. Twenty-four hours later, cells were washed and treated with IdUrd (20 pg/ml) either in medium or interferon as indicated. The cells were then washed and incubated in medium or interferon. Twenty-four or 48 hr later, cells were washed, uv-irradiated, and overlayed with SC1 cells. The UV-XC test was done 4 days later. The 0 time indicates the time of start of IdUrd treatment. The values represent the average of duplicate cultures; values higher than 400 were estimated by comparison with standards (cultures infected with known quantities of AKR-Ll virus). b All times refer to hours from the initiation of IdUrd treatment. TABLE

(units/ 0 15 15 150 150

treatment

Virus-producing

cells (in situ plaques)

OF

THE INDUCTION

Inhibition

(hr)

48 hr

72 hr

96 hr

120 hr

48 hr

72 hr

-

63 11 8 3 2

2,000 1,000 200 120 50

2,000 2,000 200 400 65

2,000 2,000 400 1,000 lop

83 87 95 97

50 90 94 97

-24 -24 -24 -24

+ 48 + 120 + 48 + 120

Cells

7

REQUIREMENT FOR THE CONTINUOUS PRESENCE OF INTERFERON FOR INHIBITION ENDOGENOIJS MLV BY IDURD@ Interferon

Infected

Interferon treatment also inhibited the production of infectious MLV by chronically infected AKR-ZB cells. Again, the degree of inhibition depended upon the duration of interferon treatment, as well as upon the concentration of interferon. Treatment with interferon (150 units/ml) for 24 hr resulted in a 2.5 log reduction in infectious MLV production. When the cells were exposed to the same concentration of interferon for 72 hr, a 4 log inhibition of virus production was observed (data not shown). In both cases, the antiviral effect disappeared rapidly when interferon was removed from the cultures (Fig. 3). Within 48 hr after the interferon was removed, the production of MLV had returned to normal. As in the acutely infected AKR-2B cells, this rapid recovery of MLV production presumably reflected the rapid decay in the interferon-induced antiviral effect. Next we examined whether interferon inhibited virus production by the persistently infected AKR-2B cells without inhibiting the synthesis of p30 protein. However, since the chronically infected cells are virus positive, there are no negative controls for these experiments. The effect OF

(8) 96 hr

120 hr

-

-

0 90 80 97

0 80 50 95

a AKR-2B cells were seeded either in medium or interferon, and endogenous virus was induced by IdUrd a8 described in the footnotes to Table 6. Maintenance medium and interferon were replaced every 48 hr; at the times indicated, the cells were lethally irradiated and virus-producing cells were detected by the UV-ME test. The values represent the average of duplicate cultures.

EFFECT

OF INTERFERON

ON MLV

TABLE

8

INDUCTION OF ENWGENOUS MLV BY IDURD: DIFFERENTIAL GROUP-SPECIFIC VIRAL ANTIGENS, INFECTIOUS VIRUS, Treatment

Time” (hr) IdUrdb

48 72

+ + + + -

Cell-associated viral antigens

Interferon (150 units/ ml) (hr)”

FA posi-

-

1 1

-24 -+ 48 -24 + 72 -

tive (96)

EFFECT OF INTERFERON ON THE INDUCTION AND REVERSE TRANSCRIPTASE ACTIVITY Virus prcducing cells (irt situ plaques)

p30 protein

(ngimg cell protein)

3 3 0

194 157 190

237 4

of interferon was examined in cultures seeded at both high and low cell densities, the latter being used to attempt to reduce the background level of preformed ~30. Cells were trypsinized to remove cell-associated virus and plated at either density in interferon or medium. After 24 hr, the cultures were washed and refed with medium without interferon. Three or eight hours

Extracellular

Infectious virus (log PFUlml)

400 4 2,000 310 0

1.4 0 5.0 2.9 -

u Time of virus assay after initiation of IdUrd treatment. * AKR-2B cells were treated with IdUrd (20 fig/ml) for 24 hr. ” Time measured from initiation of IdUrd treatment. d Picomoles of [3HlTTP incorporated per milliliter culture fluid; values represent cultures.

FIG. 3. The effect of interferon treatment on virus production in chronically infected cells. Chronically infected AKR-2B cells were trypsinized (0.75% trypsin) and plated (3.5 x lo5 cells/dish) in medium (10% FBS) with or without interferon (150 units/ml). Twenty-four hours later, interferon (or medium) was removed, and cells were washed and incubated with 3 ml of interferon-free medium. At the times indicated (hours post-removal of interferon) the medium was collected and virus was assayed by XC test in SC-1 cells.

333

INFECTION

OF

virus

Reverse transcriptase activiw 0.43 0.05 3.2 0.4 0.02

the average of duplicate

after removal of interferon, the culture supernatants were assayed for infectious virus and virion-associated reverse transcriptase, and the cells were harvested for p30 assay (Table 9). It can be seen that the interferon-induced inhibition of MLV production was comparable at both time periods and was relatively independent of cell density. It also can be seen (Table 9) that in these chronically infected cells, interferon inhibited the production of infection virus to a greater degree than the production of virus particles (as measured by virion-associated reverse transcriptase activity). For example, when interferon reduced infectious MLV production by 25- to lOO-fold, reverse transcriptase activity was reduced by only three- to fourfold. In contrast, no inhibition of p30 protein was observed in the interferon treated cultures. Similar results were obtained in two different chronically infected AKR-2B cell lines. This dissociation between the effect of interferon on the production of infectious MLV and on the synthesis of p30 protein is similar to that observed with exogenous AKR-Ll infection and during IdUrd activation of the endogenous AKR MLV genome. DISCUSSION

The results presented here indicate that at least in the case of AKR leukemia virus, exogenous, IdUrd-induced endogenous,

334

PITHA,

ROWE

AND

TABLE CHRONIC AKR-Ll Chroni-

tally in-

Number of cella

fected

planted

AKR-2B cell line

A

5x105 1.5 x 108

B

1 x 105 1 x 108

INFECTION: TRANSCRIPTASE

9

EFFECT OF INTERFERON ON INFECTIOUS VIRUS PRODUCTION, REVERSE ACTIVITY

AND INTRACELLULAR

p30 VIRAL

PROTEI@

Culture Fluid

Interferon

treatment (150 units/ml) (-24 + 0 hrs)

OXMAN

Infectious virus (log PFU/ml) 3 hr

+ +

3.6 1.7 (1%) 3.7 2.3 (4%)

+ +

3.5 1.0 (0.4%) 4.2 1.0 (0.06%)

8 hr 4.2 1.7 (0.3%) 4.5 2.4 (0.8%) ND ND

Cells

Reverse transcriptaseb OhI

ND ND 1.5 0.3 (20%) 3.2 0.25 (8%)

3 hr 1.6 0.6 (38%) 3.9 0.45 (12%)

8 hr 3.1 1.7 (55%) 4.8 2.1 (44%)

p30 Protein (ng/mg cell protein) 3 hr 1,273 1,454 (114%) 933 875 (94%)

8 hr 818 625 (71%) 653 720 (110%)

1.6 0.3 (19%) it

(6%)

a Chronically infected AKRPB cells were trypsinised (0.75% trypsinl and plated either in medium or interferon (150 units/ml) at the indicated concentration of cells per dish, 24 hr later, interferon was removed and the cells were washed and incubated with 3 ml of media for an additional 3 or 8 hr. At this time, infectious virus and reverse transcriptase activity were assayed in the fluids and intracellular p30 protein in the cells. Two experiments were performed on independently derived chronically infected AKR-2B cell lines. The percentage of the control values are given in parentheses. * Picomoles of 13Hl’TTP incorporated per milliliter of culture fluid.

and chronic MLV infections are all com- tion was manifested as long as interferon was present in the culture medium, but parably sensitive to interferon. However, when the interferon was removed, there in contrast to its action on most other was a rapid recovery of virus production. viruses, interferon does not block MLVTable 10 summarizes the patterns of respecific protein synthesis or prevent the establishment of infection either by exoge- sults observed with the three types of infection. With all three, the amount of infecnous MLV genome. Rather, in these MLV tious virus in culture fluids was markedly systems, interferon appears to reversibly reduced by interferon, and in all cases, the inhibit some later step (or steps) in the effect was temporary. In general, there virus replicative cycle required for the was no clear-cut difference in the effect of assembly of infectious virions. interferon on the acute exogenous and enPrevious studies with murine leukemia dogenous infections; there was a proporvirus (MLV) and murine sarcoma virus (MSV) have suggested that there may be a tionate reduction in virus infectivity and reverse transcriptase activity in the culdifference between the effect of interferon ture fluids. The fact that both the number on acute exogenous and on established inof virus-producing cells and the quantity fections (Fitzgerald, 1969; Sarma et al., 1969; Peries, et al., 1968; Gresser et al., of released virus were reduced after exogenous infection and IdUrd induction, 1967). Treatment with interferon inhibited focus formation induced by exogenous implies that interferon does not merely prevent the release of virus from infected MLV and MSV infection, but prolonged passage in interferon did not decrease vicells, but blocks virus replication at a point prior to the assembly of infectious rus production, as detected by electron mivirions. If the inhibition of extracellular croscopy, in Balb/c mouse embryo fibroblasts productively infected with MLV and virus production was due only to a block in the release of infectious virions from the MSV (Chany and Vignal, 1970). However, cells, the number of in situ plaques in it has been reported recently that interferon does cause a temporary inhibition of interferon-treated cultures would not be reduced, because cells which contain virus virus production or release in cells chronibut release it poorly should still register as cally infected with MLV (Billiau et al., 1973, 1974; Friedman and Ramseur, 1974; in situ plaques in the UV-ME test. These findings, in conjunction with the electron Van Griensven et al., 1971). This inhibi-

EFFECT

OF INTERFERON

ON MLV

TABLE

335

INFECTION

10

THE EFFECTOFINTERFERONON MLV INFECTION Degree of inhibition

Culture fluids Infectious virus Virus particles* Cells Cells producing infectious infectious centers) Virus particles’ gs antigen (~30)

virus (uv-resistant

Acute exogenous infection

Induction of endogenous virus

Chronic infection

++ ++

++ ++

++ t

++

++

++ +

-

0 ++ > IOO-fold inhibition; + = two- to tenfold inhibition; - = less than twofold b By reverse transcriptase activity. ” Friedman and Ramseur (1974). d By PHluridine incorporation; Billiau et al. (1974). ’ By PHluridine incorporation; van Griensven et al. (1974). ’ By electron microscopy.

microscopic observations (Table 51, indicate that virus maturation is blocked. There may have been some difference in inhibition of gs antigen synthesis, the exogenous infection showing a slight (1.3- to 2-fold) reduction, while the IdUrd-induced infection showed none; this could reflect the possibility that some of the gs antigen synthesis following IdUrd induction is from chromosomal viral genomes other than the mouse-tropic AKR virus. The nature of the interferon effect on late stages of virus synthesis in the chronically infected cells may not be identical in all respects to that in the acute infections. In the former, we observed a disproportionate decrease in infectivity (RIO-fold), as compared to reverse transcriptase activity (2- to &fold) in culture fluids, suggesting that some virion maturation was occurring, but resulting in noninfectious particles; preliminary electron microscopic studies (in collaboration with Dr. Nelson A. Wivel) support this interpretation. The failure of interferon to inhibit the establishment of MLV infection by exogenous virus indicates that there is no interferon-sensitive step required for the synthesis of the DNA (provirus) copy of the MLV genome or for the integration of the provirus into the cell genome. Furthermore, the failure of interferon to block gs

by interferon5

-

Chronic infection (literature) ++ + +“’ d. 6

-d -I

inhibition.

(~30) antigen synthesis in chronic infection and following IdUrd-induction of the endogenous AKR-MLV genome, indicates that the transcription of at least a portion of the integrated provirus into mRNA and the translation of that mRNA into protein is not affected by interferon treatment. It is possible, however, that interferon does block the transcription or translation of other portions of the provirus genome and that the inhibition of virus production observed in interferon-treated cells results from the absence of one or more viral proteins other than ~30. Further studies of MLV RNA and protein synthesis in interferon-treated cells should clarify this point. The observations on chronic infection are somewhat at variance with other reported studies (Table 10). Friedman and Ramseur (19741 found that in interferontreated AKR-2B cells chronically infected with AKR virus, there is a marked inhibition of virus production and a simultaneous accumulation of viral p30 protein in the cells; however, there was no disproportion between infectivity and reverse transcriptase activity in their experiments. On the other hand, Billiau et al. (1974) found that in both JLSVS cells (cells derived from the spleen and thymus of Balb/c mice and chronically infected with Rauscher

336

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ROWE

MLV) and in C3H mouse embryo cells chronically infected with Kirsten MSV, interferon inhibited virus production without altering the number of cell-associated virus particles; they interpreted these findings as indicating that the effect of interferon was not on maturation, but on release of virus from the cell surface. Our data are most compatible with the report of van Griensven et al. (1971), who found that interferon treatment of JLSV5 cells led to the production of incomplete virions lacking part of the MLV genome. Some of the discrepancies in the results obtained in these MLV- and MSV-cell systems may be related to differences in the interferon preparations used, in the virus-cell systems studied, or in experimental design. Our experiments were done with the same cell line and the same interferon preparation used by Friedman and Ramseur (1974), but the AKR virus strains used had markedly different tissue-culture passage histories; whether this difference in virus strain can explain the different results obtained remains to be determined. Although the exact mechanism of interferon’s antiviral action is still unclear, studies with viruses other than oncornaviruses have consistently shown that when interferon inhibits virus replication, it inhibits the synthesis of all detectable virusspecific proteins (Joklik and Merigan, et al., 1972; Metz and Es1966; Jungwirth teban, 1972; Taylor, 1965). Thus, the transcription of at least a portion of the integrated provirus into mRNA and its translation to gs antigens (p30) in interferontreated cells is in sharp contrast to results obtained in other virus systems. The possibility that some other portion of the provirus genome is not transcribed or translated in interferon-treated cells cannot be completely eliminated; however, this would require postulating polycistronic transcription or translation of the viral genome, with one cistron sensitive to interferon, and another resistant. Thus, our findings are difficult to interpret in terms of current models of interferon action (e.g., general inhibition of translation or transcription of viral genomes), and suggest a unique mode of interferon action in this

AND

OXMAN

system. It is conceivable that the interferon effect does not involve inhibition of MLV expression through action of the interferon-induced antiviral protein; rather, interferon could be altering cell physiology, such as cell membrane properties (for example, see Stewart et al., 1971, 1972; Lindahl et al., 1973) in a manner that interferes with the complex process of viral assembly. ACKNOWLEDGMENTS We wish to thank Dr. K. Paucker for the gift of purified interferon, Dr. W. P. Parks for assaying the p30 protein, Dr. N. A. Wivel for performing the electron microscopic examination, and Miss M. Lander for technical assistance. This work was supported by grants from National Science Foundation (BMS-74-21025), National Institutes of Health (AI 10944-031, and partially by Virus Cancer Program of National Cancer Institute. P.M.P. is a Scholar of Leukemia Society of America, Inc. and M.N.O. is a recipient of American Cancer Society Faculty Research Award (PRA-89). REFERENCES A., EDY, V. G., SOBIS, H., and DESOMMER, P. (1974). Influence of interferon on virus particle synthesis in oncornavirus carrier line. Evidence for a direct effect on particle release. Znt. J. Cancer 14, 335-340. BILLIAU, A., SOBIS, H., and DESOMMER, P. (1973). Influence of interferon on virus particle formation in different oncornavirus carrier cell lines. Znt. J. Cancer 12, 646-653. CHANY, C., and VIGNAL, M. (19701. Effect of prolonged interferon treatment on mouse embryonic tibroblasts transformed by murine sarcoma virus. J. Gen. Viral. 7, 203-210. FITZGERALD, G. R. (1969). The effect of interferon on focus formation and yield of murine sarcoma virus in uitro. Proc. Sot. Exp. Biol. Med. 130, 960-965. FRIEDMAN, R. M., and RAMSEUR, J. M. (1974). Inhibition of murine leukemia virus production in chronically infected AKR cells; a novel effect of interferon. Proc. Nat. Acad. Sci. USA 71, 35423544. GRESSER, I., COPPEY, J., FONTAINE-BROUTY-BOYE, D., and FALCOFF, R. (1967). Interferon and murine leukemia. Efficacy of interferon preparations administered after inoculation of Friend virus. Nuture (London) 215, 174-175. GRESSER, I., BROUTY-BOYE, D., THOMAS, M. T., and MACIEIRA-COELHO, A. (1970). Interferon and cell division; inhibition of multiplication of mouse leukemia L1210 cells in vitro by interferon preparations. Proc. Nat. Acad. Sci. USA 66, 1052-1053. BILLIAU,

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