VIROLOGY
65,10~111(1975)
Distinguishing Characteristics of Interferon Induction with Poly(l)-Poly(C) and Newcastle Disease Virus in Human Cells LEE Department
of Microbiology,
New
W. MOZES York
AND
University
Accepted
JAN
School
December
VILCEK
of Medicine,
New
York,
New
York
10016
24, 1974
Exposure of a human diploid foreskin cell strain (FS-4) to polyinosinate-polycytidylate [poly(I) poly(C) ] resulted in an early rise of interferon production that peaked at about 4 hr after induction and decreased rapidly thereafter. Irradiation of cells with low to moderate doses of ultraviolet (uv) light immediately before induction with poly(1) .poly(C) increased the amount of interferon produced up to about tenfold. This enhancement was apparently due to interference with the shut-off process which in unirradiated cells leads to early termination of interferon production; in irradiated cells interferon production continued for much longer. Inoculation of FS-4 cells with Newcastle disease virus (NDV) resulted in interferon production that showed a slower rise and peaked only at about lo-15 hr after inoculation. Irradiation of cells at the time of induction with NDV resulted in a dose-dependent decrease of interferon production. However, a small fraction of the total amount of interferon produced in response to NDV, which appeared by about 5 hr after virus inoculation, was resistant to uv. This uv-resistant early peak of NDV-induced interferon was greatly enhanced in cells which 6 hr before virus inoculation had either been induced with poly(1) .poly(C) or incubated with interferon, while the appearance of the major, uv-sensitive peak of NDV-induced interferon was inhibited or delayed after the same treatments. In its characteristics the early peak of NDV-induced interferon resembled the poly(1) .poly(C)-induced interferon response. Poly(1) .poly(C)-induced, as well as the early and late NDV-induced interferons were all neutralized by an antiserum raised against poly(1) .poly(C)-induced interferon, suggesting that they represent products of the same structural gene(s). It is concluded that there may be more than one mechanism of interferon induction by a single virus.
stimulated with NDV. In marked contrast, the production of interferon in the same type of cells after stimulation with polyinosinate-polycytidylate [poly(I) .poly(C)] was enhanced in cultures irradiated with a wide range of doses of uv (Mozes and VilEek, 1974). We suggested that the enhanced interferon synthesis in irradiated cultures could be explained by the preferential inhibition by uv of the synthesis of a posttranscriptional repressor (VilEek, 1970a; Tan et al., 1970; VilEek and Ng, 1971) exerting negative control over interferon synthesis. It was concluded that, although both poly(1) .poly(C) and NDV might stimulate the same interferon gene(s), the control of its expression ap-
INTRODUCTION
Ultraviolet (uv) irradiation of cells prior to inoculation with Newcastle disease virus (NDV) (DeMaeyer-Guignard and DeMaeyer, 1965; Cogniaux-Leclerc et al., 1966; Coppey and Markovits, 1969) or influenza virus (Burke and Morrison, 1966) was shown to result in a dose-dependent inhibition of interferon production. It was proposed that this inhibition was mainly the result of inactivation of a discrete site in the cellular DNA coding for interferon (Coppey and Muel, 1970). Our earlier work employing cultures of the RK13 continuous line of rabbit kidney cells confirmed the dose-dependent inhibitory effect of uv on interferon production 100 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
INTERFERON
INDUCTION
peared to be different for each inducer (Mozes and VilEek, 1974). In its first part, the present work extends the validity of the results obtained with uv light in RK13 cells to cultures of human diploid cells. As in RK13 cells, irradiation of human FS-4 cells prior to induction exerted strikingly different effects on NDV- and poly(1) . poly(C)-stimulated interferon production. New evidence is presented that supports the suggestion that uv preferentially inhibits repressor synthesis over interferon synthesis in poly(1) . poly( Cstimulated cells. The second part of this paper attempts to analyze the events occurring during NDV-induced interferon synthesis. The results indicate that NDV is, in fact, capable of eliciting two distinct types of interferon responses that differ in their kinetics and other characteristics. The characteristics of one of the two interferon responses elicited by NDV were similar to those of poly(I) -poly(C)-induced interferon production. MATERIALS
AND
METHODS
Cell cultures. A strain of diploid human fibroblasts (FS-4), derived in this laboratory from a single neonate foreskin, was used for interferon production in all experiments. This cell strain was identified as a high interferon producer (VilEek and Haveil, 1973). The cells were grown in Eagle’s minimal essential medium (MEM), buffered with N-2-hydroxyethylpiperazineN’2’-ethanesulfonic acid (HEPES; 13.2 mA4) and N-tris(hydroxymethy1) methylglycine (tricine; 6.6 mM) to pH 7.6, containing 100 ccg of gentamicin and 2.5 pug of amphotericin B (Fungizone) per ml, and supplemented with 5% heated fetal calf serum. All experiments were performed in 12-day-old cultures grown at 36” in 60-mm plastic petri dishes (Falcon Plastics, Los Angeles, CA) in a humidified incubator provided with 5% CO,. Cultures were routinely seeded with 200,000 cells per plate and the medium was changed on the sixth day. Another human foreskin cell strain, designated FS-7, which was employed in all
IN HUMAN
CELLS
101
interferon titrations, was grown in 250-ml plastic flasks in the same medium. Chicken embryo cells, which were employed in the assay of NDV, were obtained by trypsinization of 11-day-old embryos. These cells were grown in 60-mm plastic petri plates in MEM containing 5% heated fetal calf serum. All tissue culture media and sera were purchased from Grand Island Biological Co., Grand Island, NY. Preparation
and purification
of virus.
NDV (Hickman strain, originally obtained from E. D. Kilbourne, Mt. Sinai School of Medicine, New York, NY) was propagated by intraallantoic inoculation of 11-day-old chicken embryos. The virus was purified according to the method of Laver (1969), by adsorption of the virus to chicken red cells at 0” followed by its elution into the medium at 37”. The virus suspension was then centrifuged at 4,000 g for 15 min to remove any particulate contaminants. Finally, the supernatant was centrifuged at 40,000 g for 30 min to pellet the virus that was then resuspended in a small volume of phosphate-buffered saline (PBS; 0.13 M NaCl, 7 mM phosphate, 0.9 mM CaCl,, and 0.5 mM MgCl,.GH,O; pH 7.4). The stock solution, which contained 2 x 10” PFU per ml, was stored at -70”. Cultures of chick embryo cells were employed for the plaque assay of NDV. Interferon induction. FS-4 cells were washed once with PBS and incubated at 36” with the appropriate concentration of the inducer. Poly(1) .poly(C) (supplied by the Infectious Disease Branch, National Institute of Allergy and Infectious Diseases, Bethesda, MD) was diluted in MEM to give a final concentration of 100 pg of poly(1) .poly(C) per ml. NDV was diluted in PBS and 4 ml of the diluted virus was exposed to uv light (350 erg/mma) in a loo-mm plastic petri dish. It was further diluted in MEM to give a final concentration of lOa PFU/ml (as calculated from the titer of unirradiated virus). The dilution factor of 1:200, which was used throughout, resulted in an input multiplicity of about 100 PFU per cell. It was established in preliminary experiments that this dose of
102
MOZES
AND
irradiation of NDV, which reduced the infectivity of virus by about 90%, resulted in maximum interferon production. When employed for interferon induction, the virus was always irradiated before use, although, for the sake of simplicity, this fact is not always stated in the description of the results. FS-4 cells were incubated with 2 ml of poly(1) .poly(C) or 1 ml of NDV for 2 hr. Thereafter, the plates were washed four times with Earle’s balanced salt solution and each culture was refed with 2 ml of production medium which consisted of MEM supplemented with 0.5% human plasma-protein fraction (Armour Pharmaceutical Co., Chicago, IL). All NDV-induced interferon preparations were dialyzed against 0.1 M KCl-HCl buffer, pH 2.0, for 4 days at 4” to inactivate any residual live virus. The samples were next dialyzed 1 day against PBS and then 1 day with MEM. The samples were then centrifuged for 1 hr at 100,000 g (50,000 rpm in a Spinco 65 angle-head rotor) to pellet the inactivated virus. The supernatant fluids were used for the titration of interferon. Conditions of irradiation. A. G.E. G15T8 germicidal fluorescent lamp was used for all irradiations. Its calibration by an Eppley thermopile indicated that it gave a dose rate of 34.9 erg/mm%ec at a target distance of 40 cm. Cultures of FS-4 cells in opened 60-mm plates were irradiated through the cover of 2 ml of clear PBS. Interferon titrations. A modification of the semimicro method described by Armstrong (1971) was employed. The basic procedure was described in detail previously (Have11 and VilEek, 1972). However, instead of using FS-3 cells in the assay system, we employed another strain of diploid human fibroblasts derived in this laboratory, designated FS-7 (30,000 cells/ well). Preparation of interferon used for treatment of cultures. The preparation of hightitered human interferon in roller-bottle cultures of FS-4 cells induced with poly(1) poly( C) and treated with cycloheximide and actinomycin D was described elsewhere (Have11 et al., 1974). The interferon
VILCEK
was concentrated about tenfold by ultrafiltration using a PM10 membrane filter (Amicon Corp., Lexington, MA). The preparation employed in the experiments titered 60,000 reference units/ml. Preparation of rabbit antiserum against interferon. The interferon used for immunization of New Zealand white female rabbits was prepared in serum-free MEM using roller-bottle cultures of FS-4 cells induced with poly(1) . poly( C) and treated with cycloheximide and actinomycin D. The details of the induction process and the schedule of immunizations were previously described (Have11 et al., 1974). Neutralization assay with interferon antiserum. A modification of the microassay for human interferon was used for the titration of the neutralizing activity of the anti-interferon serum. The details of the procedure are described elsewhere (Have11 titer is et al., 1974). The neutralizing expressed in terms of the highest dilution of the serum that inhibited the antiviral action of ten units of interferon (final concentration). RESULTS
The Kinetics of Appearance of Interferon in &irradiated FS-4 Cells Stimulated with Poly(I) .Poly(C) or NDV Interferon production in FS-4 cells stimulated with poly(1) .poly(C) rose early and then declined to undetectable levels by 8 hr (Fig. 1). The rise of NDV-stimulated interferon production was more gradual, reaching a peak only between 10 and 15 hr after inoculation. Similar differences in the kinetics of poly(1) .poly(C)-induced and NDV-induced interferon production were described earlier in cultures of rabbit (Vilcek, 1970b; Tan et al., 1970; Mozes and VilEek, 1974) or human (Bausek and Merigan, 1970) cells. Effects of UV Irradiation of FS-4 Cells on Interferon Production Stimulated with Poly(I) .Poly(C) or NDV In accord with the findings in other cell systems (DeMaeyer-Guignard and DeMaeyer, 1965; Burke and Morrison, 1966;
INTERFERON
INDUCTION
IN HUMAN
103
CELLS
hancement was maximal (about tenfold) in cultures irradiated with approximately 500-700 erg/mm2. Analysis of the Effects of UV Irradiation of FS-4 Cells on Interferon Production Stimulated with Poly(I) .Poly(C) In control unirradiated cultures stimulated with poly(1) .poly(C), interferon production peaked at about 4 hr and then rapidly declined. However, in cells which were irradiated with 700 erg/mm* immediately before induction with poly(1) .poly(C) the peak of production was not reached until 7 hr and interferon production was still detectable by 50 hr after stimulation (Fig. 3). The rapid decline of poly(1) .poly(C)induced interferon production in unirradiated cells is thought to be due to the
‘r, 1
5
IO
HOURS AFTER
I
I
15
20
25
STIMULATION
FIG. 1. Kinetics of interferon production in FS-4 cells stimulated with poly(I).poly(C) or NDV. Cultures were incubated for 2 hr commencing at time 0 with poly(1). poly(C) (0) or NDV (0) as described in Materials and Methods. Samples of culture fluid were collected at the times indicated, the cells were washed once with buffered saline, replenished with fresh production medium and reincubated until the next interval. Interferon yields are expressed in reference units/ml divided by the number of hr which had elapsed from the preceding interval.
\
\
\
\
I \ 0 1 \
0
\ ‘\
‘\
‘\ ’ \\
‘\ ?J,
Cogniaux-Leclerc et al., 1966; Coppey and Markovits, 1969; Mozes and VilEek, 1974), irradiation of FS-4 cells immediately before stimulation with NDV resulted in a dose-dependent decrease in interferon production (Fig. 2). A dose of 250 erg/mm’ produced a 50% inhibition in the yield of interferon obtained at 28 hr; irradiation with 750 erg/mm2 inhibited interferon production by 90%. In contrast to the inhibitory effect on NDV-induced interferon production, irradiation of FS-4 cells with low to moderate doses of uv immediately before induction with poly(1) .poly(C) had a stimulatory -. effect on interferon production. ‘l’his en-
\ ‘\\ ‘\ ‘\
Ii.
‘1
0
loo0
/
I
2000
0
!-
3ooo
ERG/mm2 2. Interferon production in FS-4 cells stimulated with poly(I).poly(C) (0) or NDV (0) after irradiation of cultures with various doses of uv. Cultures were irradiated with the indicated dose of uv as described in Materials and Methods and immediately thereafter induced as in Fig. 1. Interferon yields represent total amounts of interferon produced by 28 hr after stimulation. FIG.
104
MOZES
AND
VILEEK
less than 50% increase over the control total interferon yield (not shown). In FS-4 cells uv-irradiated at different intervals before stimulation with poly(1). poly( C) interferon production decreased with the length of the interval between irradiation and induction (Table 2). Although some of this decreased interferon production might be attributed to nonspecific cell damage produced by irradiation, the results of this and the preceding experiment show that in order to produce maximum enhancement of poly(1) .poly (C)stimulated interferon production, cells had to be irradiated within a specific time interval relative to induction.
1000 t
,ol! , !?,;; 215 ;5 415 0
5HOURS
10
AFTER
STIMULATION
FIG. 3. Effect of uv irradiation of FS-4 cells on the kinetics of interferon production induced with poly(1). poly(C). Control cultures (0) and cultures irradiated with 700 erg/mm* immediately before induction (0) were induced with poly(1) .poly(C) as in Fig. 1. Interferon yields are expressed in reference units/ml divided by the number of hr which had elapsed from preceding interval.
action of a posttranscriptional repressor protein whose synthesis would be expected to begin prior to the onset of the shut-off, i.e., no later than 4-5 hr after induction (VilEek, 1970a; Tan et al., 1970; VilEek and Ng, 1971). The results shown in Fig. 3 and the results of our earlier studies employing cultures of rabbit RK13 cells (Mazes and VilEek, 1974) suggested that uv irradiation of cells leads to preferential inhibition of repressor synthesis over interferon synthesis. If this conclusion is correct, then irradiation at various times after induction, when repressor synthesis is already underway, should result in a gradually decreasing enhancement of interferon production. The experiment summarized in Table 1 showed that this was indeed the case. Total interferon production was the highest in cells irradiated no later than 2 hr after induction and gradually decreased to control levels in cultures irradiated at subsequent intervals. In a separate experiment irradiation of cultures at 8.5 hr after stimulation with poly(1) .poly(C) resulted in a
Multiplication of NDV in &irradiated and W-irradiated FS-4 Cells
As in many other mammalian cells (Ruiz-Gomez and Isaacs, 1963; Youngner and Scott, 1968; Thacore and Youngner, 1969; Huppert et al., 1974), the replication of NDV in FS-4 cells was inefficient. The maximum yield of virus from cultures inoculated with a high multiplicity of live NDV was less than 1 PFUjcell. When the cells were uv-irradiated with up to 1,745 erg/mm* prior to inoculation with live NDV, the yield of infectious virus was neither significantly decreased nor increased (not shown here). These findings were similar to those reported for rabbit RK 13 cells, except that the increase in the yield of NDV from uv-irradiated cultures seen in RK 13 cells (Mozes and VilEek, 1974) was not observed in FS-4 cultures. Effect of W Irradiation of FS-4 Cells at Various Times after Induction with NDV
From the results shown in Tables 1 and 2 it is evident that the time of irradiation is an important factor in determining the yield of interferon with poly(1) . poly(C). In the experiment summarized in Table 3, plates were first inoculated with NDV and at various times after induction irradiated with 700 erg/mm2. Whereas irradiation immediately before induction (0 hr) was strongly inhibitory, interferon production became resistant to irradiation by 8 hr. A
INTERFERON
INDUCTION
IN HUMAN
TABLE INTERFERON
PRODUCTION
IN FS-4
CELLS W-IRRADIATED INDUCTION
Hr of irradiatior+
105
CELLS
1
AT THE TIME OF INDUCTION
WITH
Posy.
UW
Interferon At time irradiation
AND AT VARIOUS TIMES AFTER
POLY(C)’
of
At5hr
yield At 28 hr
Total
768 4,096
64 2.048
832 6,144
768 2,560
48 4,096
816 6,656
896 1.280
56 6.144
952 7.424
0
No Yes
1
No Yes
2
No Yes
3
No Yes
192 192
896 768
56 3.584
1,144 4,544
4
No Yes
512 512
256 320
64 2,560
832 3,392
5
No Yes
1,024 1,024
-
96 1,792
1,120 2,816
-
a Induction with poly(1) .poly(C) and other procedures were as described in Fig. 1. b Relative to the time of exposure of cells to poly(I) poly(C). c 700 erg/mm* at the indicated time. d In fluids collected from the cultures prior to irradiation. e Interferon produced from the time of irradiation until 5 hr after the exposure of cells to poly(1). ’ Not done. TABLE INTERFERON PRODUCTION AT VARIOUS TIMES POLY(1)
Hr of irradiatiorP
23.5 19 6 3.5 0 No irradiation
2
TABLE
IN FS-4 CELLS W-IRRADIATED BEFORE INDUCTION WITH
Interferon
Hr of irra-
yield
29
40 112 64 224 > 4,096 3,072
65,536 160
0 Induction with poly(1). poly(C) and other procedures were as described in Fig. 1. b Exposed to 700 erg/mm* at indicated hr before addition of poly(I) poly(C).
modest, approximately threefold increase in the interferon yield was observed in cultures irradiated at 10 hr after stimulation with NDV.
Interferon
TIMES
AFTER
yield
diation”
at hr
5
3
EFFECT OF uv IRRADIATION AT VARIOUS INDUCTION WITH NDV~
.POLY(C)O
poly(C).
-
None 0 6 8 9 10 11 12 13
At time of irradiation’
At 30 hr
Total
-* 21 42 96 200 342
684 43 384 768 768 2,048 512 684 256
684 43 384 768 789 2,090 608 884 598
Percent total of unirradiated control 100 6 56 112 116 306 89 129 87
a Induction with NDV as described in Fig. 1. b Relative to the time of exposure of cells to NDV. All plates were irradiated with 700 erg/mm’. ‘In fluids collected from the cultures prior to irradiation. d Not done.
106
MOZES
AND
In another experiment (not shown) irradiation of cultures at 10 hr after induction with 700 erg/mm2 produced a fourfold enhancement of the interferon yield, while higher or lower doses of uv light produced only a twofold increase or no increase at all. The finding that the enhancement of NDVinduced interferon production after uv irradiation of cells was, at best, of a small magnitude suggested that posttranscriptional control might be less important in NDV-stimulated than in poly(1) .poly(C)induced interferon production. Dual
Induction Poly(I) -Poly(C)
of Interferon and NDV
with
In another attempt to examine the role of the posttranscriptional control mechanism in NDV-induced interferon synthesis, cultures were induced sequentially with both poly(1) .poly(C) and NDV. Although the antiviral activities and other properties of the interferons made in FS-4 cells after exposure to either NDV or poly(1). poly(C) are indistinguishable, the two responses are discernible on the basis of their different kinetics (cf. Fig. 1). We anticipated that if the cultures were first stimulated with poly(1) .poly(C), followed 6 hr later by NDV, there might be a decrease in the NDV-induced interferon yield due to the presence of the repressor formed as a result of poly(1) .poly(C) induction. However, the addition of poly(1). poly(C) prior to NDV inoculation did not produce a detectable decrease in total NDV-induced interferon production. In fact, this dual induction resulted in the appearance of an early peak of interferon production at 5 hr after inoculation with NDV, unlike in control cultures stimulated with NDV alone which showed a characteristic peak of production at a later time (Fig. 4). The earlier appearance of interferon in the doubly induced cells was reminiscent of a similar shift of interferon production observed in L cells or primary mouse kidney cells “primed” by treatment with interferon prior to their stimulation with NDV (Stewart et al., 1971). Indeed, treatment of FS-4 cells with interferon for 6 hr
VILCEK
prior to induction with NDV produced a similar shift in the time of production as previous exposure to poly(1) .poly(C) (Fig. 4). In view of this finding it is very likely that the early interferon production in doubly induced cultures was also due to priming of the NDV response as a result of the interferon produced by the prior poly(1) . poly( C) stimulation. In addition to the early peak of interferon, there was also a late burst of production between 24 and 48 hr after inoculation with NDV, detectable only in the primed cells. Earlier studies suggested that the repressor of poly(1) .poly(C)-induced interferon production may be a labile protein (VilEek 1970a; Tan et al., 1970; VilEek and 5001
loo-
I-
5
IO
15
72
202r--G--
Hours After
Incwlot~on
wth
NDV
FIG. 4. Kinetics of NDV-induced interferon production in FS-4 cells after prior induction with poly(1) .poly(C) or incubation with interferon. At 6 hr before inoculation with NDV, one group of cultures was incubated with poly(1) .poly(C) (100 pg/ml) as described in Fig. 1 except that the contact with the polynucleotide was only for 1 hr (0). Another group of cultures was incubated for 6 hr with 2,000 units of interferon per ml (prepared as described in the Materials and Methods) in 2 ml of MEM supplemented with 2% fetal calf serum (A), while a third group was incubated with 2 ml of control medium for 6 hr prior to induction (0). At time 0 all three groups of cultures were washed and induced with NDV as described in Fig. 1. Interferon yields are expressed in reference units/ml divided by the number of hr which had elapsed since the preceding interval of medium collection. Another control group of cultures exposed to poly(I).poly(C) at -6 hr, but not induced with NDV at time 0, produced undetectable amounts of interferon during the periods of sampling recorded in the graph.
INTERFERON
INDUCTION
IN HUMAN
107
CELLS
Ng, 1971). It seemed possible that the failure to observe a decrease in NDVinduced interferon production in the doubly induced cultures was perhaps due to the lag between poly(1) .poly(C) induction and the later synthesis of the NDVinduced interferon.- However, the addition within the first hours of POlY(I) .POlY(C) after NDV inoculation also did not produce a detectable inhibition of NDV-induced interferon production. In fact, poly(1) . poly(C) added as late as 2 hr after the exposure of cells to NDV still produced an enhancement of the interferon production in the doubly induced cells (results not shown).
after inoculation. The delay could be due to the fact that a viral function, susceptible to the inhibitory action of interferon, is required for the induction of this interferon response. The nature of the early interferon peak seen in NDV-induced cells which had been preincubated with poly(1). poly(C) or interferon was further investigated by examining the effect of uv irradiation of cells prior to induction with NDV on interferon production in doubly induced or primed cells (Table 4). Interferon produced in response to inoculation with NDV in control cells was reduced by about 95% after irradiation of the cells. However, even in these cells, the 5- and B-hr yields were not completely eliminated by the uv light. In primed or Nature of Interferon Responses to NDV doubly induced cells irradiation failed to In its time of appearance, the early peak decrease the early production of interferon (which, on the contrary, seemed to be of NDV-induced interferon in the doubly induced or primed cells resembled poly(1) . protracted in the irradiated cells), whereas poly( C) -induced interferon production. later production was greatly reduced. The late production between 24 and 48 hr Thus, the early NDV-induced response in in primed cells could represent the delayed primed cells showed a similar characterisappearance of the interferon response tic resistance to uv as interferon production which in unprimed cells peaks at lo-15 hr in response to poly(1) .poly(C). TABLE EFFECT
Treatment at -6 hr
NOI@
OF cv
uvD
IRRADIATION
Induction (time 0)
NO Yes
NDV
Polyp poly(C 1:
NO Yes
Nonex
Poly(I) poly(C1’
NO Yes
KDV
Interfenm”
Y 2s
ND\’
ON NDV-INDUCED INDUCTION WITH
4
INTERFERON PROWCTIO~; IN FS-4 Posy. PoLY(C) OR INTERFERON”
Interferon
CELLS TREATED BEFORE Total yield
yield at hr after induction’ 73
Percent total yield from unirradiated controls
3.5
5
8
10
12.7
24
48
~24 < 24
36 36
144 36
96 C24
576 x24
384
65,10~111(1975)
Distinguishing Characteristics of Interferon Induction with Poly(l)-Poly(C) and Newcastle Disease Virus in Human Cells LEE Department
of Microbiology,
New
W. MOZES York
AND
University
Accepted
JAN
School
December
VILCEK
of Medicine,
New
York,
New
York
10016
24, 1974
Exposure of a human diploid foreskin cell strain (FS-4) to polyinosinate-polycytidylate [poly(I) poly(C) ] resulted in an early rise of interferon production that peaked at about 4 hr after induction and decreased rapidly thereafter. Irradiation of cells with low to moderate doses of ultraviolet (uv) light immediately before induction with poly(1) .poly(C) increased the amount of interferon produced up to about tenfold. This enhancement was apparently due to interference with the shut-off process which in unirradiated cells leads to early termination of interferon production; in irradiated cells interferon production continued for much longer. Inoculation of FS-4 cells with Newcastle disease virus (NDV) resulted in interferon production that showed a slower rise and peaked only at about lo-15 hr after inoculation. Irradiation of cells at the time of induction with NDV resulted in a dose-dependent decrease of interferon production. However, a small fraction of the total amount of interferon produced in response to NDV, which appeared by about 5 hr after virus inoculation, was resistant to uv. This uv-resistant early peak of NDV-induced interferon was greatly enhanced in cells which 6 hr before virus inoculation had either been induced with poly(1) .poly(C) or incubated with interferon, while the appearance of the major, uv-sensitive peak of NDV-induced interferon was inhibited or delayed after the same treatments. In its characteristics the early peak of NDV-induced interferon resembled the poly(1) .poly(C)-induced interferon response. Poly(1) .poly(C)-induced, as well as the early and late NDV-induced interferons were all neutralized by an antiserum raised against poly(1) .poly(C)-induced interferon, suggesting that they represent products of the same structural gene(s). It is concluded that there may be more than one mechanism of interferon induction by a single virus.
stimulated with NDV. In marked contrast, the production of interferon in the same type of cells after stimulation with polyinosinate-polycytidylate [poly(I) .poly(C)] was enhanced in cultures irradiated with a wide range of doses of uv (Mozes and VilEek, 1974). We suggested that the enhanced interferon synthesis in irradiated cultures could be explained by the preferential inhibition by uv of the synthesis of a posttranscriptional repressor (VilEek, 1970a; Tan et al., 1970; VilEek and Ng, 1971) exerting negative control over interferon synthesis. It was concluded that, although both poly(1) .poly(C) and NDV might stimulate the same interferon gene(s), the control of its expression ap-
INTRODUCTION
Ultraviolet (uv) irradiation of cells prior to inoculation with Newcastle disease virus (NDV) (DeMaeyer-Guignard and DeMaeyer, 1965; Cogniaux-Leclerc et al., 1966; Coppey and Markovits, 1969) or influenza virus (Burke and Morrison, 1966) was shown to result in a dose-dependent inhibition of interferon production. It was proposed that this inhibition was mainly the result of inactivation of a discrete site in the cellular DNA coding for interferon (Coppey and Muel, 1970). Our earlier work employing cultures of the RK13 continuous line of rabbit kidney cells confirmed the dose-dependent inhibitory effect of uv on interferon production 100 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
INTERFERON
INDUCTION
peared to be different for each inducer (Mozes and VilEek, 1974). In its first part, the present work extends the validity of the results obtained with uv light in RK13 cells to cultures of human diploid cells. As in RK13 cells, irradiation of human FS-4 cells prior to induction exerted strikingly different effects on NDV- and poly(1) . poly(C)-stimulated interferon production. New evidence is presented that supports the suggestion that uv preferentially inhibits repressor synthesis over interferon synthesis in poly(1) . poly( Cstimulated cells. The second part of this paper attempts to analyze the events occurring during NDV-induced interferon synthesis. The results indicate that NDV is, in fact, capable of eliciting two distinct types of interferon responses that differ in their kinetics and other characteristics. The characteristics of one of the two interferon responses elicited by NDV were similar to those of poly(I) -poly(C)-induced interferon production. MATERIALS
AND
METHODS
Cell cultures. A strain of diploid human fibroblasts (FS-4), derived in this laboratory from a single neonate foreskin, was used for interferon production in all experiments. This cell strain was identified as a high interferon producer (VilEek and Haveil, 1973). The cells were grown in Eagle’s minimal essential medium (MEM), buffered with N-2-hydroxyethylpiperazineN’2’-ethanesulfonic acid (HEPES; 13.2 mA4) and N-tris(hydroxymethy1) methylglycine (tricine; 6.6 mM) to pH 7.6, containing 100 ccg of gentamicin and 2.5 pug of amphotericin B (Fungizone) per ml, and supplemented with 5% heated fetal calf serum. All experiments were performed in 12-day-old cultures grown at 36” in 60-mm plastic petri dishes (Falcon Plastics, Los Angeles, CA) in a humidified incubator provided with 5% CO,. Cultures were routinely seeded with 200,000 cells per plate and the medium was changed on the sixth day. Another human foreskin cell strain, designated FS-7, which was employed in all
IN HUMAN
CELLS
101
interferon titrations, was grown in 250-ml plastic flasks in the same medium. Chicken embryo cells, which were employed in the assay of NDV, were obtained by trypsinization of 11-day-old embryos. These cells were grown in 60-mm plastic petri plates in MEM containing 5% heated fetal calf serum. All tissue culture media and sera were purchased from Grand Island Biological Co., Grand Island, NY. Preparation
and purification
of virus.
NDV (Hickman strain, originally obtained from E. D. Kilbourne, Mt. Sinai School of Medicine, New York, NY) was propagated by intraallantoic inoculation of 11-day-old chicken embryos. The virus was purified according to the method of Laver (1969), by adsorption of the virus to chicken red cells at 0” followed by its elution into the medium at 37”. The virus suspension was then centrifuged at 4,000 g for 15 min to remove any particulate contaminants. Finally, the supernatant was centrifuged at 40,000 g for 30 min to pellet the virus that was then resuspended in a small volume of phosphate-buffered saline (PBS; 0.13 M NaCl, 7 mM phosphate, 0.9 mM CaCl,, and 0.5 mM MgCl,.GH,O; pH 7.4). The stock solution, which contained 2 x 10” PFU per ml, was stored at -70”. Cultures of chick embryo cells were employed for the plaque assay of NDV. Interferon induction. FS-4 cells were washed once with PBS and incubated at 36” with the appropriate concentration of the inducer. Poly(1) .poly(C) (supplied by the Infectious Disease Branch, National Institute of Allergy and Infectious Diseases, Bethesda, MD) was diluted in MEM to give a final concentration of 100 pg of poly(1) .poly(C) per ml. NDV was diluted in PBS and 4 ml of the diluted virus was exposed to uv light (350 erg/mma) in a loo-mm plastic petri dish. It was further diluted in MEM to give a final concentration of lOa PFU/ml (as calculated from the titer of unirradiated virus). The dilution factor of 1:200, which was used throughout, resulted in an input multiplicity of about 100 PFU per cell. It was established in preliminary experiments that this dose of
102
MOZES
AND
irradiation of NDV, which reduced the infectivity of virus by about 90%, resulted in maximum interferon production. When employed for interferon induction, the virus was always irradiated before use, although, for the sake of simplicity, this fact is not always stated in the description of the results. FS-4 cells were incubated with 2 ml of poly(1) .poly(C) or 1 ml of NDV for 2 hr. Thereafter, the plates were washed four times with Earle’s balanced salt solution and each culture was refed with 2 ml of production medium which consisted of MEM supplemented with 0.5% human plasma-protein fraction (Armour Pharmaceutical Co., Chicago, IL). All NDV-induced interferon preparations were dialyzed against 0.1 M KCl-HCl buffer, pH 2.0, for 4 days at 4” to inactivate any residual live virus. The samples were next dialyzed 1 day against PBS and then 1 day with MEM. The samples were then centrifuged for 1 hr at 100,000 g (50,000 rpm in a Spinco 65 angle-head rotor) to pellet the inactivated virus. The supernatant fluids were used for the titration of interferon. Conditions of irradiation. A. G.E. G15T8 germicidal fluorescent lamp was used for all irradiations. Its calibration by an Eppley thermopile indicated that it gave a dose rate of 34.9 erg/mm%ec at a target distance of 40 cm. Cultures of FS-4 cells in opened 60-mm plates were irradiated through the cover of 2 ml of clear PBS. Interferon titrations. A modification of the semimicro method described by Armstrong (1971) was employed. The basic procedure was described in detail previously (Have11 and VilEek, 1972). However, instead of using FS-3 cells in the assay system, we employed another strain of diploid human fibroblasts derived in this laboratory, designated FS-7 (30,000 cells/ well). Preparation of interferon used for treatment of cultures. The preparation of hightitered human interferon in roller-bottle cultures of FS-4 cells induced with poly(1) poly( C) and treated with cycloheximide and actinomycin D was described elsewhere (Have11 et al., 1974). The interferon
VILCEK
was concentrated about tenfold by ultrafiltration using a PM10 membrane filter (Amicon Corp., Lexington, MA). The preparation employed in the experiments titered 60,000 reference units/ml. Preparation of rabbit antiserum against interferon. The interferon used for immunization of New Zealand white female rabbits was prepared in serum-free MEM using roller-bottle cultures of FS-4 cells induced with poly(1) . poly( C) and treated with cycloheximide and actinomycin D. The details of the induction process and the schedule of immunizations were previously described (Have11 et al., 1974). Neutralization assay with interferon antiserum. A modification of the microassay for human interferon was used for the titration of the neutralizing activity of the anti-interferon serum. The details of the procedure are described elsewhere (Have11 titer is et al., 1974). The neutralizing expressed in terms of the highest dilution of the serum that inhibited the antiviral action of ten units of interferon (final concentration). RESULTS
The Kinetics of Appearance of Interferon in &irradiated FS-4 Cells Stimulated with Poly(I) .Poly(C) or NDV Interferon production in FS-4 cells stimulated with poly(1) .poly(C) rose early and then declined to undetectable levels by 8 hr (Fig. 1). The rise of NDV-stimulated interferon production was more gradual, reaching a peak only between 10 and 15 hr after inoculation. Similar differences in the kinetics of poly(1) .poly(C)-induced and NDV-induced interferon production were described earlier in cultures of rabbit (Vilcek, 1970b; Tan et al., 1970; Mozes and VilEek, 1974) or human (Bausek and Merigan, 1970) cells. Effects of UV Irradiation of FS-4 Cells on Interferon Production Stimulated with Poly(I) .Poly(C) or NDV In accord with the findings in other cell systems (DeMaeyer-Guignard and DeMaeyer, 1965; Burke and Morrison, 1966;
INTERFERON
INDUCTION
IN HUMAN
103
CELLS
hancement was maximal (about tenfold) in cultures irradiated with approximately 500-700 erg/mm2. Analysis of the Effects of UV Irradiation of FS-4 Cells on Interferon Production Stimulated with Poly(I) .Poly(C) In control unirradiated cultures stimulated with poly(1) .poly(C), interferon production peaked at about 4 hr and then rapidly declined. However, in cells which were irradiated with 700 erg/mm* immediately before induction with poly(1) .poly(C) the peak of production was not reached until 7 hr and interferon production was still detectable by 50 hr after stimulation (Fig. 3). The rapid decline of poly(1) .poly(C)induced interferon production in unirradiated cells is thought to be due to the
‘r, 1
5
IO
HOURS AFTER
I
I
15
20
25
STIMULATION
FIG. 1. Kinetics of interferon production in FS-4 cells stimulated with poly(I).poly(C) or NDV. Cultures were incubated for 2 hr commencing at time 0 with poly(1). poly(C) (0) or NDV (0) as described in Materials and Methods. Samples of culture fluid were collected at the times indicated, the cells were washed once with buffered saline, replenished with fresh production medium and reincubated until the next interval. Interferon yields are expressed in reference units/ml divided by the number of hr which had elapsed from the preceding interval.
\
\
\
\
I \ 0 1 \
0
\ ‘\
‘\
‘\ ’ \\
‘\ ?J,
Cogniaux-Leclerc et al., 1966; Coppey and Markovits, 1969; Mozes and VilEek, 1974), irradiation of FS-4 cells immediately before stimulation with NDV resulted in a dose-dependent decrease in interferon production (Fig. 2). A dose of 250 erg/mm’ produced a 50% inhibition in the yield of interferon obtained at 28 hr; irradiation with 750 erg/mm2 inhibited interferon production by 90%. In contrast to the inhibitory effect on NDV-induced interferon production, irradiation of FS-4 cells with low to moderate doses of uv immediately before induction with poly(1) .poly(C) had a stimulatory -. effect on interferon production. ‘l’his en-
\ ‘\\ ‘\ ‘\
Ii.
‘1
0
loo0
/
I
2000
0
!-
3ooo
ERG/mm2 2. Interferon production in FS-4 cells stimulated with poly(I).poly(C) (0) or NDV (0) after irradiation of cultures with various doses of uv. Cultures were irradiated with the indicated dose of uv as described in Materials and Methods and immediately thereafter induced as in Fig. 1. Interferon yields represent total amounts of interferon produced by 28 hr after stimulation. FIG.
104
MOZES
AND
VILEEK
less than 50% increase over the control total interferon yield (not shown). In FS-4 cells uv-irradiated at different intervals before stimulation with poly(1). poly( C) interferon production decreased with the length of the interval between irradiation and induction (Table 2). Although some of this decreased interferon production might be attributed to nonspecific cell damage produced by irradiation, the results of this and the preceding experiment show that in order to produce maximum enhancement of poly(1) .poly (C)stimulated interferon production, cells had to be irradiated within a specific time interval relative to induction.
1000 t
,ol! , !?,;; 215 ;5 415 0
5HOURS
10
AFTER
STIMULATION
FIG. 3. Effect of uv irradiation of FS-4 cells on the kinetics of interferon production induced with poly(1). poly(C). Control cultures (0) and cultures irradiated with 700 erg/mm* immediately before induction (0) were induced with poly(1) .poly(C) as in Fig. 1. Interferon yields are expressed in reference units/ml divided by the number of hr which had elapsed from preceding interval.
action of a posttranscriptional repressor protein whose synthesis would be expected to begin prior to the onset of the shut-off, i.e., no later than 4-5 hr after induction (VilEek, 1970a; Tan et al., 1970; VilEek and Ng, 1971). The results shown in Fig. 3 and the results of our earlier studies employing cultures of rabbit RK13 cells (Mazes and VilEek, 1974) suggested that uv irradiation of cells leads to preferential inhibition of repressor synthesis over interferon synthesis. If this conclusion is correct, then irradiation at various times after induction, when repressor synthesis is already underway, should result in a gradually decreasing enhancement of interferon production. The experiment summarized in Table 1 showed that this was indeed the case. Total interferon production was the highest in cells irradiated no later than 2 hr after induction and gradually decreased to control levels in cultures irradiated at subsequent intervals. In a separate experiment irradiation of cultures at 8.5 hr after stimulation with poly(1) .poly(C) resulted in a
Multiplication of NDV in &irradiated and W-irradiated FS-4 Cells
As in many other mammalian cells (Ruiz-Gomez and Isaacs, 1963; Youngner and Scott, 1968; Thacore and Youngner, 1969; Huppert et al., 1974), the replication of NDV in FS-4 cells was inefficient. The maximum yield of virus from cultures inoculated with a high multiplicity of live NDV was less than 1 PFUjcell. When the cells were uv-irradiated with up to 1,745 erg/mm* prior to inoculation with live NDV, the yield of infectious virus was neither significantly decreased nor increased (not shown here). These findings were similar to those reported for rabbit RK 13 cells, except that the increase in the yield of NDV from uv-irradiated cultures seen in RK 13 cells (Mozes and VilEek, 1974) was not observed in FS-4 cultures. Effect of W Irradiation of FS-4 Cells at Various Times after Induction with NDV
From the results shown in Tables 1 and 2 it is evident that the time of irradiation is an important factor in determining the yield of interferon with poly(1) . poly(C). In the experiment summarized in Table 3, plates were first inoculated with NDV and at various times after induction irradiated with 700 erg/mm2. Whereas irradiation immediately before induction (0 hr) was strongly inhibitory, interferon production became resistant to irradiation by 8 hr. A
INTERFERON
INDUCTION
IN HUMAN
TABLE INTERFERON
PRODUCTION
IN FS-4
CELLS W-IRRADIATED INDUCTION
Hr of irradiatior+
105
CELLS
1
AT THE TIME OF INDUCTION
WITH
Posy.
UW
Interferon At time irradiation
AND AT VARIOUS TIMES AFTER
POLY(C)’
of
At5hr
yield At 28 hr
Total
768 4,096
64 2.048
832 6,144
768 2,560
48 4,096
816 6,656
896 1.280
56 6.144
952 7.424
0
No Yes
1
No Yes
2
No Yes
3
No Yes
192 192
896 768
56 3.584
1,144 4,544
4
No Yes
512 512
256 320
64 2,560
832 3,392
5
No Yes
1,024 1,024
-
96 1,792
1,120 2,816
-
a Induction with poly(1) .poly(C) and other procedures were as described in Fig. 1. b Relative to the time of exposure of cells to poly(I) poly(C). c 700 erg/mm* at the indicated time. d In fluids collected from the cultures prior to irradiation. e Interferon produced from the time of irradiation until 5 hr after the exposure of cells to poly(1). ’ Not done. TABLE INTERFERON PRODUCTION AT VARIOUS TIMES POLY(1)
Hr of irradiatiorP
23.5 19 6 3.5 0 No irradiation
2
TABLE
IN FS-4 CELLS W-IRRADIATED BEFORE INDUCTION WITH
Interferon
Hr of irra-
yield
29
40 112 64 224 > 4,096 3,072
65,536 160
0 Induction with poly(1). poly(C) and other procedures were as described in Fig. 1. b Exposed to 700 erg/mm* at indicated hr before addition of poly(I) poly(C).
modest, approximately threefold increase in the interferon yield was observed in cultures irradiated at 10 hr after stimulation with NDV.
Interferon
TIMES
AFTER
yield
diation”
at hr
5
3
EFFECT OF uv IRRADIATION AT VARIOUS INDUCTION WITH NDV~
.POLY(C)O
poly(C).
-
None 0 6 8 9 10 11 12 13
At time of irradiation’
At 30 hr
Total
-* 21 42 96 200 342
684 43 384 768 768 2,048 512 684 256
684 43 384 768 789 2,090 608 884 598
Percent total of unirradiated control 100 6 56 112 116 306 89 129 87
a Induction with NDV as described in Fig. 1. b Relative to the time of exposure of cells to NDV. All plates were irradiated with 700 erg/mm’. ‘In fluids collected from the cultures prior to irradiation. d Not done.
106
MOZES
AND
In another experiment (not shown) irradiation of cultures at 10 hr after induction with 700 erg/mm2 produced a fourfold enhancement of the interferon yield, while higher or lower doses of uv light produced only a twofold increase or no increase at all. The finding that the enhancement of NDVinduced interferon production after uv irradiation of cells was, at best, of a small magnitude suggested that posttranscriptional control might be less important in NDV-stimulated than in poly(1) .poly(C)induced interferon production. Dual
Induction Poly(I) -Poly(C)
of Interferon and NDV
with
In another attempt to examine the role of the posttranscriptional control mechanism in NDV-induced interferon synthesis, cultures were induced sequentially with both poly(1) .poly(C) and NDV. Although the antiviral activities and other properties of the interferons made in FS-4 cells after exposure to either NDV or poly(1). poly(C) are indistinguishable, the two responses are discernible on the basis of their different kinetics (cf. Fig. 1). We anticipated that if the cultures were first stimulated with poly(1) .poly(C), followed 6 hr later by NDV, there might be a decrease in the NDV-induced interferon yield due to the presence of the repressor formed as a result of poly(1) .poly(C) induction. However, the addition of poly(1). poly(C) prior to NDV inoculation did not produce a detectable decrease in total NDV-induced interferon production. In fact, this dual induction resulted in the appearance of an early peak of interferon production at 5 hr after inoculation with NDV, unlike in control cultures stimulated with NDV alone which showed a characteristic peak of production at a later time (Fig. 4). The earlier appearance of interferon in the doubly induced cells was reminiscent of a similar shift of interferon production observed in L cells or primary mouse kidney cells “primed” by treatment with interferon prior to their stimulation with NDV (Stewart et al., 1971). Indeed, treatment of FS-4 cells with interferon for 6 hr
VILCEK
prior to induction with NDV produced a similar shift in the time of production as previous exposure to poly(1) .poly(C) (Fig. 4). In view of this finding it is very likely that the early interferon production in doubly induced cultures was also due to priming of the NDV response as a result of the interferon produced by the prior poly(1) . poly( C) stimulation. In addition to the early peak of interferon, there was also a late burst of production between 24 and 48 hr after inoculation with NDV, detectable only in the primed cells. Earlier studies suggested that the repressor of poly(1) .poly(C)-induced interferon production may be a labile protein (VilEek 1970a; Tan et al., 1970; VilEek and 5001
loo-
I-
5
IO
15
72
202r--G--
Hours After
Incwlot~on
wth
NDV
FIG. 4. Kinetics of NDV-induced interferon production in FS-4 cells after prior induction with poly(1) .poly(C) or incubation with interferon. At 6 hr before inoculation with NDV, one group of cultures was incubated with poly(1) .poly(C) (100 pg/ml) as described in Fig. 1 except that the contact with the polynucleotide was only for 1 hr (0). Another group of cultures was incubated for 6 hr with 2,000 units of interferon per ml (prepared as described in the Materials and Methods) in 2 ml of MEM supplemented with 2% fetal calf serum (A), while a third group was incubated with 2 ml of control medium for 6 hr prior to induction (0). At time 0 all three groups of cultures were washed and induced with NDV as described in Fig. 1. Interferon yields are expressed in reference units/ml divided by the number of hr which had elapsed since the preceding interval of medium collection. Another control group of cultures exposed to poly(I).poly(C) at -6 hr, but not induced with NDV at time 0, produced undetectable amounts of interferon during the periods of sampling recorded in the graph.
INTERFERON
INDUCTION
IN HUMAN
107
CELLS
Ng, 1971). It seemed possible that the failure to observe a decrease in NDVinduced interferon production in the doubly induced cultures was perhaps due to the lag between poly(1) .poly(C) induction and the later synthesis of the NDVinduced interferon.- However, the addition within the first hours of POlY(I) .POlY(C) after NDV inoculation also did not produce a detectable inhibition of NDV-induced interferon production. In fact, poly(1) . poly(C) added as late as 2 hr after the exposure of cells to NDV still produced an enhancement of the interferon production in the doubly induced cells (results not shown).
after inoculation. The delay could be due to the fact that a viral function, susceptible to the inhibitory action of interferon, is required for the induction of this interferon response. The nature of the early interferon peak seen in NDV-induced cells which had been preincubated with poly(1). poly(C) or interferon was further investigated by examining the effect of uv irradiation of cells prior to induction with NDV on interferon production in doubly induced or primed cells (Table 4). Interferon produced in response to inoculation with NDV in control cells was reduced by about 95% after irradiation of the cells. However, even in these cells, the 5- and B-hr yields were not completely eliminated by the uv light. In primed or Nature of Interferon Responses to NDV doubly induced cells irradiation failed to In its time of appearance, the early peak decrease the early production of interferon (which, on the contrary, seemed to be of NDV-induced interferon in the doubly induced or primed cells resembled poly(1) . protracted in the irradiated cells), whereas poly( C) -induced interferon production. later production was greatly reduced. The late production between 24 and 48 hr Thus, the early NDV-induced response in in primed cells could represent the delayed primed cells showed a similar characterisappearance of the interferon response tic resistance to uv as interferon production which in unprimed cells peaks at lo-15 hr in response to poly(1) .poly(C). TABLE EFFECT
Treatment at -6 hr
NOI@
OF cv
uvD
IRRADIATION
Induction (time 0)
NO Yes
NDV
Polyp poly(C 1:
NO Yes
Nonex
Poly(I) poly(C1’
NO Yes
KDV
Interfenm”
Y 2s
ND\’
ON NDV-INDUCED INDUCTION WITH
4
INTERFERON PROWCTIO~; IN FS-4 Posy. PoLY(C) OR INTERFERON”
Interferon
CELLS TREATED BEFORE Total yield
yield at hr after induction’ 73
Percent total yield from unirradiated controls
3.5
5
8
10
12.7
24
48
~24 < 24
36 36
144 36
96 C24
576 x24
384
