ase, endonuclease L. Activated endonuclease L ... through the endonuclease L pathway, in this action of ..... Cell extracts ment incubation (cpm) RNase L activity.
JOURNAL OF VIROLOGY, Mar. 1983, p. 1017-1027 0022-538X/83/031017-11$02.00/0 Copyright © 1983, American Society for Microbiology
Vol. 45, No. 3
Differential Antiviral Effects of Interferon in Three Murine Cell Lines GANES C. SEN* AND RUTH E. HERZ Sloan-Kettering Cancer Center, and Sloan-Kettering Division of Graduate School of Medical Sciences, Cornell University, New York, New York 10021 Received 26 August 1982/Accepted 1 December 1982
Infectious leukemia virus production by two chronically infected NIH/MOL lines was strongly inhibited by interferon treatment of the cells. The corresponding degree of inhibition in JLSV-11 cells was much lower. Multiplication of encephalomyocarditis virus in all three cell lines was barely affected by interferon treatment. Replication of vesicular stomatitis virus, on the other hand, was highly sensitive to interferon in the JLSV-11 line and in one NIH/MOL line but was practically insensitive in the other NIH/MOL line. Anticellular actions of interferon were more pronounced in the JLSV-11 line than in the others. In response to interferon treatment, 2',5'-oligoadenylate synthetase activity was induced to a high level in JLSV-11 cells and to lower levels in the NIH/MOL lines. We failed to detect any 2',5'-oligoadenylate-dependent endonuclease activity in extracts of these cells. Double-stranded RNA-dependent protein kinase activity was present in extracts of interferon-treated NIH/MOL cells, but it was barely detectable in extracts of interferon-treated JLSV-11 cells. The above studies demonstrated that interferon could differentially affect the replication of three different viruses in three different cell lines, including two seemingly identical NIH/MOL lines, and that certain tentative conclusions can be drawn regarding the roles of different interferon-inducible enzyme markers in the different antiviral actions of interferons.
Treatment of cells in culture with interferons (IFNs) makes them incapable of supporting replication of a broad range of viruses (37). Encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) are two of the major cytopathic viruses whose susceptibility to IFN action has been studied in detail. The biochemical basis for inhibition of replication of both of these viruses in IFN-treated cells appears to be an inhibition of viral RNA or protein synthesis or both. Several pathways have been suggested which are operative in IFN-treated, virus-infected cells and which lead to the inhibition of viral protein synthesis (for review, see 4, 21, 28, 32, 41). The most notable among them are the double-stranded (ds) RNA-dependent protein kinase pathway and the dsRNA-dependent endonuclease pathway. IFN treatment of many cell lines increases the level of a dsRNA-dependent protein kinase which, when activated by dsRNA, can phosphorylate the peptide chain initiation factor eIF-2 and thereby inhibit protein synthesis. Another enzyme whose level is boosted by IFN treatment of cells is 2',5'-oligoadenylate [2,5(A)] synthetase. In the presence of dsRNA this enzyme polymerizes ATP into 2,5(A), which can activate the latent endonucle-
ase, endonuclease L. Activated endonuclease L hydrolyzes single-stranded RNAs, including mRNAs, and inhibits protein synthesis (11). In addition to their antiviral effects, IFNs also have anticellular effects. The rates of multiplication of many cell lines are decreased with IFN treatment (38). It has also been shown that human IFN--y has cytolytic activity against certain transformed cell lines (31). The mechanism of anticellular actions of IFN is yet to be elucidated. However, there is some experimental evidence to suggest a role of 2,5(A), presumably through the endonuclease L pathway, in this action of IFN as well (17, 19, 24, 40). Retroviruses can infect cells and establish a symbiotic relationship with them (6). These chronically infected cells grow normally, but they also release infectious virions into the culture media. IFN treatment of such cells causes a reduction in the amount of extracellular virus production (12, 32). In some cells IFN treatment also causes production of noninfectious virion particles (26, 42). It is well established that the antiretroviral effect of IFN is not exerted at the level of viral protein and RNA syntheses. Retrovirus production is blocked at the plasma membrane level, where the virions assemble and
1017
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SEN AND HERZ
from where they bud (13, 22, 25, 34). Therefore, the antiretroviral effect of IFN can be considered to be an index of the perturbance in the plasma membrane of an IFN-treated cell. As more and more cell lines are being tested for IFN action, it has become apparent that all cell lines do not respond to IFN similarly. In extreme cases, such as the mouse L1210R line (1) or the human HEC-1 line (39), IFN-a or IFN,B does not elicit any response that has been tested for. In some other lines a partial response to IFN is observed. For example, in undifferentiated embryonal carcinoma cells, IFN binds to the receptors (2) and induces 2,5(A) synthetase activity (43), but the antiviral state is not achieved with respect to at least some viruses (2, 7, 23, 43). Similarly, retrovirus production by human RD-114 cells is inhibited by IFN-cx or INF--y, but replication of VSV or EMCV in the same cells is insensitive to either species of IFN (R. E. Herz, B. Y. Rubin, and G. C. Sen, Virology, in press). Czarniecki et al. have described a Swiss/3T3 line and an NIH/3T3 line, both chronically infected with Moloney murine leukemia virus (MuLV), in which MuLV production is sensitive to IFN but EMCV replication is insensitive (8). These cells are also resistant to the growthinhibitory effects of IFN. In further studies with the NIH/3T3 line Epstein et al. have found that VSV replication is also insensitive to IFN in this line (10). Moreover, these cells lack a functional endonuclease L activity and they do not respond to exogenously added 2,5(A) (24). We report here the properties of another Moloney MuLVinfected NIH/3T3 line and compare it with the line described by Czarniecki et al. (8). The major difference between the two lines is that, in the one described here, VSV replication is sensitive to IFN, although these cells also lack endonuclease L activity. Allen et al. (3) observed that Rauscher MuLV production by JLSV-9R cells is not inhibited by IFN but VSV replication in the same cells is quite sensitive to IFN. These authors, however, did not measure infectivity of the MuLV particles produced by IFN-treated JLSV-9R cells, and the possibility remains open that noninfectious virus particles are produced by these cells in response to IFN. We tested for IFN sensitivity of a Moloney MuLV-infected JLSV-9 line (JLSV-11) and report here that, compared with NIH/MOL lines, infectious MuLV production by the JLSV-11 line was much less sensitive to IFN, VSV replication was extremely sensitive to IFN, and EMCV replication was insensitive. We could not detect any endonuclease L activity in extracts of JLSV-11 cells, although a high level of 2,5(A) synthetase activity was induced by IFN treatment of these cells.
J. VIROL.
(Part of this work is included in the doctoral thesis of R.E.H., Cornell University, New York, N.Y., 1983.) MATERIALS AND METHODS IFN. Partially purified mouse IFN (specific activity, 107 U/mg of protein) was a gift of Peter Lengyel of Yale University. Cells. All cell cultures were grown in Dulbecco minimum essential medium containing 10% fetal bovine serum. The JLSV-11 line and the NIH/MOL C line were obtained from Peter Besmer of this institute (5). The NIH/MOL B line was obtained from Robert Friedman of Uniformed Services University. All of these lines were chronically infected with Moloney MuLV. The NIH/MOL C line was established in Cambridge, Mass. (C for Cambridge), and the NIH/ MOL B line was established in Bethesda, Md. (B for Bethesda), by infecting NIH/3T3 cells with Moloney MuLV (P. Besmer and R. Friedman, respectively, personal communications). The JLSV-11 line was established by infecting JLSV-9 cells with Moloney MuLV. A sister line infected with Rauscher MuLV is called JLSV-9R or JLSV-10 (45). Reverse transcriptase assay. Cell culture media containing the virions were first clarified by centrifugation at 1,600 x g for 15 min. The supernatant was directly used for the enzyme assay, or the virus was pelleted first by centrifugation at 160,000 x g for 60 min. The assay mixture contained, in addition to the virus, 5 IXCi of [3H]TTP (60 to 80 Ci/mmol; New England Nuclear), 0.01 absorbancy (at 260 nm) unit of poly A-(dT)12 18 (PL Biochemicals) as the template-primer, 0.5 mM MnCl2, 50 mM Tris-chloride (pH 7.8), 1 mM dithiothreitol, 50 mM KCI, 0.2% Nonidet P-40, and 0.01% bovine serum albumin in a total volume of 0.1 ml. Incubations were at 37°C for 20 min. At the end of the reaction, radioactivity incorporated into trichloroacetic acid-insoluble material was measured. MuLV infectivity assay. The number of infectious MuLV present in the culture medium was determined by the plaque assay method as described by Rowe et al. (30). NIH/3T3 cells were infected with the test MuLV preparation, allowed to grow to confluence, inactivated with UV light, and overlaid with XC cells. Each infectious center gave rise to a syncytium. VSV and EMCV yield reduction assay. IFN-treated or untreated cells were infected with VSV or EMCV at a multiplicity of infection of 10. After 1 h of virus adsorption, cells were incubated overnight in culture media. Virions were released from the cells by alternate freezing and thawing three times. Virus yield was measured by standard plaque assays on L929 cells. [3H]thymidine incorporation. [3Hlthymidine incorporation was measured by labeling IFN-treated or untreated cells in medium containing 5 ,uCi of [3H]thymidine per ml for 2 h. At the end of labeling, cells were washed with cold phosphate-buffered saline and then lysed in the same buffer containing 0.1% sodium dodecyl sulfate. Trichloroacetic acid-soluble and -insoluble counts in the lysates were determined. Ceil multiplication. About 104 cells were seeded into each 60-mm dish. IFN at 200 U/ml was added, if needed, only after the cells had plated out. For each time point, duplicate plates were used. The number of
VOL. 45, 1983
DIFFERENTIAL ANTIVIRAL EFFECTS OF IFN
cells in a dish was determined by trypsinization and counting under a microscope. 2,5(A) synthetase assay. Enzyme activity was measured in detergent lysates of cells following the procedure of Revel et al. (29). The enzyme was adsorbed on poly(I) * poly(C) agarose beads and incubated overnight with [c-32P]ATP and other components of the reaction mixture. Bacterial alkaline phosphatase was then added directly to the reaction mixture containing the beads and incubated for 60 min at 37°C. A 400-,ul amount of 1 M glycine-HCl (pH 2) was added, and the products were analyzed on alumina columns. dsRNA-dependent protein kinase assay. Detergent extracts of cells were prepared according to Jarvis et al. (18). Endogenous phosphorylation of the extracts was measured in a mixture of a 30-,ul total volume containing 25 mM Tris-chloride (pH 7.6), 120 mM KCI, 5 mM Mg acetate, 7 mM 2-mercaptoethanol, 15 to 20 ,Ci of [y-32P]ATP, and 0.155 absorbancy (at 260 nm) unit of cell extracts. A 100-ng portion of poly(I) * poly(C) was added when indicated. Incubations were at 30°C for 30 min. Phosphorylated proteins were analyzed by gel electrophoresis (20). Endonuclease L assay. The 30,000 x g supernatants of cell extracts made by Dounce homogenization were used for endonuclease L assays (33). 32P-labeled 12.S VSV mRNA was used as the substrate (27). dsRNAdependent nuclease assays were done in reaction mixtures of 30 ,ul containing 17 mM Tris (pH 7.5), 80 mM KCI, 3.3 mM 2-mercaptoethanol, 3.3 mM Mg acetate, 1 mM ATP, 600 ng of poly(I) * poly(C), 32p_ labeled VSV 12S mRNA, and 0.15 to 0.5 absorbancy (at 260 nm) unit of cell extract. The reaction mixtures were incubated for 15 min at 30°C before adding the mRNA and for 30 min after adding the mRNA. Reactions were stopped by adding 200 RI of 10 mM Trischloride (pH 7.5)-0.1 M NaCl-5 mM EDTA-0.5% sodium dodecyl sulfate. The mixtures were deproteinized by phenol-chloroform extraction, and 150 RI1 of the aqueous phase was used for size analysis of the RNA by centrifugation through a continuous gradient of 15 to 30% sucrose in 50 mM NaCl-10 mM Tris (pH 7.5)-10 mM EDTA-0.2% sodium dodecyl sulfate. Centrifugation was for 17 h in an SW41 rotor at 38,000 rpm and 24°C. Fractions were collected from the bottom of the tubes and counted for radioactivity. Reaction mixtures for the nuclease assay by the trichloroacetic acid precipitation method (10) contained 30 mM HEPES (N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid)-KOH (pH 7.5), 135 mM KCI, 3.5 mM Mg acetate, 1 mM ATP, 10 mM P3mercaptoethanol, 0.11 absorbancy (at 260 nm) unit of cell extract, 32P-labeled VSV mRNA, and 0.45 nmol of 2,5(A), where needed, in a total volume 30 ,ul. The reaction mixtures were incubated at 30°C for 3 h, and trichloroacetic acid-precipitable counts were determined.
RESULTS Inhibition of MuLV production by IFN treatment. Moloney MuLV-infected NIH/3T3 C line and Moloney MuLV-infected JLSV-9 line (JLSV-11) cells were tested for sensitivity to IFN with respect to their capacities to produce extracellular MuLV. Cells were treated with
1019
different doses of IFN, and virus production was estimated by two different assays: by measuring virion-associated reverse transcriptase activity and by measuring virus infectivity (Fig. 1). Increasing doses of IFN caused increasing inhibition of MuLV production by the NIH/MOL C cells. About 100 U of IFN per ml caused 50% inhibition of MuLV production. About 85% inhibition was obtained at 200 U of IFN per ml as measured by reverse transcriptase assays. Higher doses of IFN did not increase the degree of inhibition further (Fig. IA). When infectivity of the MuLV particles produced by IFN-treated NIH/MOL C cells was measured, the degrees of inhibition were very similar, although the maximum inhibition of infectious virus production was about 95% (Fig. 1B), suggesting production of some noninfectious particles, with associated reverse transcriptase activity, by IFN-treated cells. The degree of inhibition of MuLV production by JLSV-11 cells in response to IFN treatment was much lower. Even at the maximum dose tested (500 U/ml) there was about 35% inhibition of MuLV production by JLSV-11 cells, as measured by virion-associated reverse transcriptase activity. The extent of inhibition was also similar (maximum, 50%) when viral infectivity was measured, indicating that IFNtreated JLSV-11 cells did not produce a large number of noninfectious virions with reverse transcriptase activity. Moloney MuLV production by JLSV-11 cells was therefore less sensitive to IFN treatment than was that by NIH/ MOL C cells. The same pattern was also true for Rauscher MuLV production by Rauscher MuLV-infected NIH/3T3 C and JLSV-9 cell lines (data not shown). Another Moloney MuLV-infected NIH/3T3 cell line, NIH/MOL B, behaved similarly to the NIH/MOL C line (data not shown; 8) with respect to the extent of inhibition of MuLV production in response to IFN. Effect of IFN on replication of VSV and EMCV. We measured the effects of IFN treatment on VSV and EMCV replication in JLSV-11, NIH/ MOL C, and NIH/MOL B cells (Fig. 2). EMCV replication was relatively insensitive to IFN in all three cell lines. The maximum inhibition was about 0.7 log at 500 U of IFN per ml. In most commonly used mouse cell lines (e.g., L929 or Ehrlich ascites tumor), this amount of IFN would cause 3- to 4-log inhibition of EMCV yield. VSV replication was affected differently by IFN treatment of the three cell lines we were testing. At the highest dose tested (500 U/ml), VSV yield was reduced by 3.3 logs in JLSV-11 cells and by about 3 logs in NIH/MOL C cells, but by only about 0.4 log in NIH/MOL B cells. Uninfected JLSV-9 and NIH/3T3 C cells behaved similarly to the corresponding MuLV-
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remarkable difference in the sensitivity of VSV replication to IFN in the two NIH/MOL cell lines, lines which did not have any known phenotypic difference. Effects of IFN treatment on [3H]thymidine * NIH/MOL,C z incorporation into DNA and on the rate of cell x JLSV-11 O 50 multiplication. Since the antiviral effects of IFN were different in the three different cell lines, we ai. were interested to learn whether IFN treatment would also affect the rate of DNA synthesis and the rate of multiplication of these cells differento o ly. The results of the relevant experiments are shown in Fig. 3 and 4. [3H]thymidine incorpooz10 (B) t m 0 ration into DNA of NIH/MOL B cells was z I0 barely inhibited by IFN treatment even at a high dose. Treatment with 500 and 1,000 U of IFN z per ml appreciably inhibited [3H]thymidine H50 incorporation into DNA of NIH/MOL C cells. JLSV-11 was the most sensitive cell line in this respect: even 100 U of IFN per ml inhibited thymidine incorporation by about 20%, whereas 1,000 U/ml caused 60% inhibition (Fig. 3). A 200-U/ml amount of IFN was used to exam0 100 1Q00 ine the effects on the rate of multiplication of the IFN DOSE (U/ml) three cell lines (Fig. 4). Untreated cells multiFIG. 1. Effect of increasing doses of IFN on MuLV plied logarithmically for 6 days. Inclusion of production by NIH/MOL C and JLSV-11 cell lines. IFN in the culture media slowed down the NIH/MOL C (3 x 105) and JLSV-11 (106) cells were multiplication rate of both of the NIH/MOL plated. On the next day the indicated doses of IFN lines slightly. There were about 40% fewer cells were added. After 12 h, old culture medium was in the IFN-treated NIH/MOL cultures than in replaced by 3 ml of fresh medium containing the the untreated cultures on day 5. In the case of original doses of IFN, and the cells were incubated for line the corresponding degree of inhibianother 12 h. Culture media were collected, and the JLSV-11 tion was about 80%. amounts of virus shed into the media were measured We inferred from the above results that IFN either by (A) reverse transcriptase assay or (B) infectivity assay as described in the text. Results are had pronounced anticellular effects on the presented as percent inhibition of virus production as JLSV-11 line as demonstrated by its effects on compared with virus production by cells not treated both thymidine incorporation and rate of cell with IFN. Amounts of virus production by untreated multiplication. On the other hand, anticellular cells were as follows: (A) JLSV-11, 480 pmol of TTP effects of IFN in both NIH/MOL lines were incorporated per ml of culture medium, and NIH/ comparable and not as pronounced as in the MOL C, 405 pmol of TTP incorporated per ml of JLSV-11 line. culture medium; (B) JLSV-11, 5.8 x 106 PFU/ml, and Status of the dsRNA-dependent endonuclease NIH/MOL C, 4.6 x 106 PFU/ml. system in the three cell lines. A dsRNA-dependent endonuclease system is present in many infected lines with respect to sensitivity of VSV IFN-treated cell lines (21, 32, 41). This system is and EMCV replication to IFN (data not shown). probably responsible for IFN-mediated inhibiThe following conclusions could be drawn tion of virus replication in some cell lines. We from the above set of experiments. (i) In the inquired whether this system is operative in the JLSV-11 and NIH/MOL lines. Extracts were same cells VSV replication could be sensitive to IFN treatment while EMCV replication was made from these cells after they had been treatinsensitive. (ii) Whereas MuLV production by ed with IFN, and we measured the rate of JLSV-11 cells was less inhibited than was that degradation of 32P-labeled VSV mRNA in these by NIH/MOL C cells, VSV replication was extracts in the presence and absence of dsRNA slightly more sensitive to IFN in the JLSV-11 (Fig. 5). VSV mRNA was degraded much more line. (iii) The level of the classical antiviral state in the presence of dsRNA than in its absence in extracts of IFN-treated L929 cells, which are was very low in IFN-treated NIH/MOL B cells known to have the dsRNA-dependent endonuas manifested by the low sensitivity of VSV and EMCV replication to IFN; however, the antiret- clease system (Fig. 5A). But no such degradaroviral state was as pronounced in these cells as tion was observed in extracts of IFN-treated in any other cells (8, 10). (iv) There was a NIH/MOL C cells (Fig. SB) or in extracts of z
0
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0
z w
0-
DIFFERENTIAL ANTIVIRAL EFFECTS OF IFN
VOL. 45, 1983
1021
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cn5 0'
KO 0
50-
K)0 500 10 IFN DOSE (U/ml)
K) i'O
500
FIG. 2. Effect of increasing doses of IFN on multiplication of VSV and EMCV in (A) JLSV-11, (B) NIH/MOL C, and (C) NIHtMOL B cell lines. Confluent monolayers of cells were treated with various doses of IFN for 12 h and then infected with either VSV (x) or EMCV (m) at a multiplicity of 10. Cells were further incubated for 12 h, and all virions were released into the medium by alternate freezing and thawing of the cells. Virus yields were measured by standard plaque assays on L929 cells.
IFN-treated JLSV-11 cells (Fig. 5C). Also, no activity was detected in extracts of IFN-treated NIH/MOL B cells (data not shown). The dsRNA-dependent endonuclease system consists of two key enzymes: 2,5(A) synthetase and 2,5(A)-dependent endonuclease L (11). The absence of dsRNA-dependent endonuclease activity could be due to the absence of one or both of these enzymes in IFN-treated JLSV-11 and NIH/MOL cells. We therefore measured the activity of the two enzymes individually. Cells were treated with different doses of IFN, extracts were made, and 2,5(A) synthetase activities in these extracts were measured (Fig. 6).
100'
.
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100
1000
IFN DOSE (U/mi)
FIG. 3. Effect of increasing doses of IFN on [3H]thymidine incorporation into DNA of NIH/MOL B, NIH/MOL C and JLSV-11 cells. Nonconfluent monolayers were treated with different doses of IFN for 18 h and then labeled with [3H]thymidine for 2 h as described in the text. Amounts of label incorporated into DNA were measured. Results are presented as percent incorporation as compared with cells not treated with IFN.
There were very low levels of the enzyme activity in extracts of untreated cells. In JLSV-11 cells this activity was elevated by about 15-fold upon treatment with 50 U of IFN per ml and by about 50-fold upon treatment with 500 U of IFN per ml. On the other hand, treatment of either NIH/MOL B or NIH/MOL C line cells with up to 100 U of IFN per ml did not increase the level of 2,5(A) synthetase in these cells. Higher doses of IFN raised the levels noticeably, but the levels were much lower than the corresponding levels of the enzymes in extracts of IFN-treated JLSV-11 or L929 cells. The observed lower levels of 2,5(A) synthetase activity in extracts in NIHIMOL cells was not due to the presence of an inhibitor of this enzyme or to the presence of a potent 2,5(A)-degrading activity in these cells. The above conclusions were reached based on the observation that when equal amounts of extracts of IFN-treated NIH/MOL and L929 cells were mixed and tested for 2,5(A) synthetase activity, the total activity observed was a sum of the individual activities (data not shown). To examine the possibility that the observed absence of dsRNA-dependent endonuclease activity in extracts of IFN-treated NIH/MOL cells was due to the very low level of 2,5(A) synthesis, we tested directly the levels of 2,5(A)dependent endonuclease L activity in extracts of L929, NIH/MOL C, and JLSV-11 cells. 32P_ labeled VSV mRNA was incubated with various cell extracts in the absence and presence of added 2,5(A), and the amounts of residual trichloroacetic acid-precipitable counts after 3 h of incubation were determined (Table 1). In the case of extracts of IFN-treated L929 cells, there was about twofold more acid-insoluble RNA left after the incubation without 2,5(A) than in the one with 2,5(A), demonstrating the presence of a 2,5(A)-dependent endonuclease in these ex-
1022
SEN AND HERZ
J. VIROL.
w
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0
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2
4
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2 4 DAYS IN CULTURE
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4
6
FIG. 4. Effect of IFN on rate of cell multiplication. About 104 cells were plated in 60-mm dishes. After the cells had plated out, media were changed and some cultures received 200 U of IFN per ml. Cell numbers in duplicate dishes were counted on the days indicated, the day of plating being day 0. Numbers of cells per dish were measured by trypsinization and counting. All dishes received a medium change on day 3. Symbols: *, cultures with no IFN; x, cultures with 200 U of IFN per ml.
tracts. No such activity was present, however, in extracts of IFN-treated or untreated NIH/ MOL C and JLSV-11 cells. (The low levels of RNase L activity recorded in these extracts [Table 1] were within the range of experimental errors and they cannot be regarded as true signals.) We concluded from the above set of experiments that (i) a high level of 2,5(A) synthetase activity was induced by IFN-treatment of JLSV11 cells, whereas much lower levels of this enzyme were induced in NIH/MOL B or NIH/
MOL C cells; (ii) none of the cell lines had a level of 2,5(A)-dependent endonuclease L activity detectable under the present assay conditions. Status of the dsRNA-dependent protein kinase system in extracts of JLSV-11 and NIH/MOL cells. Another pathway of inhibition of translation of viral mRNAs in IFN-treated cells is mediated through a dsRNA-dependent protein kinase system (11). When extracts of IFN-treated Ehrlich ascites tumor cells are incubated with [_y-32P]ATP, phosphorylation of at least two pro-
FRACTION NUMBER
FIG. 5. dsRNA-dependent endonuclease activity in extracts of IFN-treated cells. Dounce extracts were made from IFN-treated (200 U/ml) L929, NIH/MOL C, and JLSV-11 cells as described in the text. Purified 32p_ labeled VSV mRNA (12.5S) was incubated with these extracts in the absence (0) or presence (x) of dsRNA for 30 min as described before. Incubation mixtures were deproteinized by phenol extraction, and size of the labeled RNA was analyzed by centrifugation through sucrose gradients. Fractions were collected from the bottom of the tube, and the amount of radioactivity in each fraction was determined. Amounts of total counts in reactions were (A) 3,200, (B) 7,500, and (C) 2,300 cpm.
VOL. 45, 1983
DIFFERENTIAL ANTIVIRAL EFFECTS OF IFN
1023
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10 100 1000 IFN DOSE (U/ml) FIG. 6. 2,5(A) synthetase activities in cells treated with different doses of IFN. Cells were treated with IFN for 18 h, and detergent extracts were made from them. 2,5(A) synthetase activity was measured as described in the text. Results are presented as nanomoles of AMP polymerized per 106 cells, with the assumption that only trimers were synthesized.
teins, P1 and P2, is enhanced by inclusion of dsRNA in the incubation mixture (20). This is due to a dsRNA-dependent protein kinase whose level is elevated by IFN treatment of cells (36). We tested for the presence of this enzyme in extracts of L929, NIH/MOL C, and JLSV-11 cells (Fig. 7). There was strong dsRNA-dependent phosphorylation of P, in two sets of IFNtreated L929 cell extracts (lanes 1 to 4). A lower level of activity was detectable in extracts of IFN-treated NIH/MOL C cells (lanes 9 and 10). No such activity was present in extracts of untreated NIH/MOL C cells (data not shown) or TABLE 1. 2,5(A)-dependent endonuclease L activity in different cell extractsa IFN
Cell extracts
L929 NIH/MOL C
U/ml)
(X)
(Y)
+ -
629 917 822 651 472
295 895 838 647 460
+
JLSV-11
TCA-precipitable
treat- .RNA after ment incubation (cpm) RNase L activity (20- 2,5(A) + 2,5(A)([0(X-Y/}
-
+
53 2.5 0.5 2.5
a Incubation conditions are described in the text. Input was 1,200 cpm in each reaction. TCA, Trichloroacetic acid.
1
2 34 5 6 7 8
9 10
FIG. 7. dsRNA-dependent protein kinase activity in different cell extracts. dsRNA-dependent protein kinase activities in extracts of IFN-treated or untreated cells were measured as described in the text. Lanes 1 to 8 are from one experiment and lanes 9 and 10 are from another experiment. The arrow indicates the position of PI (molecular weight, 67,000), whose phosphorylation was enhanced by dsRNA. (Lanes 1-4) Extracts of IFN-treated (200 U/ml) L929 cells; (lanes 5 and 6) extracts of untreated JLSV-11 cells; (lanes 7 and 8) extracts of IFN-treated (200 U/ml) JLSV-11 cells; (lanes 9 and 10) extracts of IFN-treated (200 U/ml) NIH/MOL C cells. Reactions in odd-numbered lanes did not include any dsRNA, whereas those in even-numbered lanes included dsRNA.
JLSV-11 cells (lanes 5 and 6). In extracts of IFN-treated JLSV-11 cells (lanes 7 and 8), a very low level of dsRNA-dependent phosphorylation of P1 was observed. In several independent experiments with different concentrations of different sets of IFN-treated JLSV-11 extracts we have noticed this low level of P1 phosphorylation. The results from the experiments suggest that IFN treatment of NIH/MOL C cells induces a low level of dsRNA-dependent protein kinase activity in these cells. Epstein et al. (10) observed such an activity in extracts of IFNtreated NIH/MOL B cells, whereas in another NIH/3T3 line Hovanessian et al. (16) failed to detect it. Our results further suggest that IFN treatment (200 U/ml) of JLSV-11 cells causes the induction of a barely detectable level of dsRNAdependent protein kinase activity in extracts of these cells. DISCUSSION In the studies reported here we examined three biological effects of IFN: the classical
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SEN AND HERZ
antiviral effects against VSV and EMCV, its anticellular effects, and its antiretroviral effects. We also measured the levels of three enzyme activities, 2,5(A) synthetase, endonuclease L, and dsRNA-dependent protein kinase, in extracts of IFN-treated cells. All of these parameters were tested in three mouse cell lines: the JLSV-11 line and two NIH/MOL lines. Allen et al. (3) reported that IFN treatment of the JLSV-9R line does not inhibit MuLV production. They estimated MuLV production by two methods: by measuring virion-associated reverse transcriptase activity and by metabolically labeling the virions with [3H]uridine. Although they concluded that there was no inhibition of MuLV production in response to IFN, careful examination of the results of their uridine-labeling experiments reveals that at higher doses of IFN there was some inhibition of MuLV production. In our studies we found that IFN treatment of JLSV-11 cells resulted in only about 35% inhibition of MuLV production, as measured by reverse transcriptase activity. This result was therefore in general agreement with the results of Allen et al. (3), although we were studying JLSV-9 cells infected with a different MuLV. Allen et al. (3) did not measure infectivity of the virions produced by IFN-treated JLSV-9R cells. Since IFN treatment of cell lines such as SC-1 (26) or TB (42) results in the production of large quantities of noninfectious MuLV particles that have virion-associated reverse transcriptase activity, the possibility remained open that treatment of JLSV-9R cells with IFN actually caused a high degree of inhibition of infectious MuLV production. Our experiments with JLSV-11 cells ruled out that possibility. The low degree of IFN-mediated inhibition of MuLV production by JLSV-11 cells was exceptional. In all other cell lines tested, which include various murine lines infected with different leukemia viruses and mammary tumor viruses (reviewed in 32), a human line infected with RD-114 retrovirus (G. C. Sen, R. E. Herz, and B. Y. Rubin, J. Cell. Biochem. Suppl. 6:96, 1982), and a feline line infected with feline leukemia virus (our unpublished data), IFN treatment causes a more pronounced inhibition of retrovirus production. We confirmed that the lower sensitivity of MuLV production to IFN is an inherent property of the JLSV-9 line and not of the strain of leukemia virus, since both Rauscher and Moloney MuLVs behaved similarly in these cells and behaved differently in NIH/3T3 cells and other cells. MuLV production by the NIH/MOL C line was more sensitive to IFN treatment than was that by JLSV-11 cells. The degree of sensitivity was comparable to that of the NIH/MOL B line as reported by Czarniecki et al. (8). Neither of
J. VIROL.
the NIH/MOL lines produced a large number of noninfectious MuLV particles in response to IFN treatment. Rauscher MuLV production by Rauscher MuLV-infected NIH/3T3 C cells was also inhibited by IFN (data not shown). We measured the conventional antiviral responses of the three cell lines to IFN, using EMCV or VSV as the challenge virus. It should be pointed out that there are major differences in the protocols used for measuring the antiretroviral state and the classical antiviral state. For the latter, cells are challenged with viruses after IFN treatment, whereas for the former IFN treatment is done after the viral infection has been established. Moreover, VSV and EMCV are both cytopathic viruses and they shut off host protein synthesis shortly after infection, whereas MuLV infection does not cause any apparent harm to the cells. In contrast to the lower degree of sensitivity of MuLV production to IFN, VSV replication was more sensitive to IFN in JLSV-11 cells than in NIH/MOL C cells. This was also true for the corresponding pair of uninfected lines, NIH/3T3 C and JLSV-9. The above observations support the idea that antiMuLV effects and anti-VSV effects of IFN are mediated through different molecular mechanisms. High sensitivity of VSV to IFN in NIH/MOL C cells was surprising in view of the report by Epstein et al. (10) that VSV was insensitive to IFN in their NIH/MOL B line. We therefore compared the two NIH/MOL lines directly and observed that VSV replication was indeed sensitive to IFN to different degrees in the two lines. We are not aware of any other difference between the two lines. We also do not know whether the original parental lines had the same sensitivity to IFN with respect to VSV replication, and the two present variant cell populations have been selected out from them. As far as we can tell, the NIH/MOL C line was established in Cambridge, Mass., by infecting a NIH/3T3 line also established there; the NIH/ MOL B line was established in Bethesda, Md. from a different parent NIH/3T3 line. No matter what their origins are, the two lines represent a convenient couple for understanding how IFN inhibits VSV replication. These two lines differed from the NIH/3T3 line described by Hovanessian et al. (16) in which replication of both VSV and mengovirus, a close relative of EMCV, was inhibited by IFN. In contrast to differential sensitivity of VSV in the three cell lines, EMCV replication was uniformly resistant to IFN action in all three cell lines. In NIH/MOL B cells, therefore, only MuLV production and not VSV or EMCV replication was sensitive to IFN as documented previously by Friedman and co-workers (8, 10).
DIFFERENTIAL ANTIVIRAL EFFECTS OF IFN
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TABLE 2. Antiviral, anticellular, and enzyme-inducing actions of IFN in various cell lines' Cell line
NIH/MOL C NIH/MOL B JLSV-11 RD-114 RD-114
Source
Mouse Mouse Mouse Human Human
IFN
Anti-T
Antiretrovirus
Anti-
'vs'
AntiEMCV
incorapoa tion
Anti-cell plication
2,5(A) synthetase
Endonuclease L
dsRNA protein kinase
a +
++++
+++
-
+
+
+
-
+
a+ a +1
++++ ++
-
-
-
+ ++ + +
+ ++++ + +
-
+
++++ ++ ± + + a ++++ + + ++++ -y a Results are presented in a semiquantitative form. Data for NIH/MOL C and JLSV-11 lines are from this paper; those for the NIH/MOL B line are from this paper and references 8 and 10; and those for the RD-114 line are from Herz et al. (in press).
This is similar to the situation in human RD-114 cells in which both human IFN-a and IFN-y inhibit retrovirus production but do not inhibit VSV or EMCV replication (Herz et al., in press). In JLSV-11 and NIH/MOL C cells, on the other hand, IFN induced a different spectrum of antiviral effects. To our knowledge, no other cell line has been described before in which VSV replication is sensitive to IFN but EMCV replication is not. The JLSV-11 line was the most sensitive among the three to the anticellular actions of IFN. In our hands, the NIH/MOL B line was somewhat more sensitive than reported by Czarniecki et al. (8). However, the experimental conditions they used differed from ours. They serum starved the cells and treated them with IFN for only 48 h. We also observed that the extent of inhibition of cell multiplication would vary from experiment to experiment unless culture conditions were strictly controlled. Even in subconfluent cultures, the number of cells plated per dish and the doubling time of the cells were two important parameters in this respect. Our attempts to correlate the presence or absence of different antiviral actions and anticellular actions of IFN in a given cell line with the presence or absence of IFN-inducible enzyme markers were partially successful. The results from the studies presented here and elsewhere that are pertinent to differential antiviral (MuLV, VSV, and EMCV) actions, anticellular actions, and enzyme-inducing actions of IFN have been compiled in Table 2. The results have been tabulated in a semiquantitative fashion to provide an overview of the state of knowledge in this area. It appears that the antiretroviral action of IFN, which may be considered an index of IFN action on the plasma membrane, is one of the most universal actions of IFN. There was no correlation between the degree of antiretroviral effects and the levels of the enzymes of the dsRNA-dependent protein kinase system or the endonuclease system or both. It appears, there-
fore, that these biochemical pathways do not play a direct role in the antiretroviral action of IFN. The notion that the absence of endonuclease L would make a cell insensitive to the anticellular actions of IFN (24) holds true for the NIH/ MOL lines but is invalid for the JLSV-11 line. It is curious to note that the JLSV-11 line was highly sensitive to the anticellular actions of IFN, and it also had the highest level of IFNinduced 2,5(A) synthetase activity, suggesting the possibility that the two phenotypes may be related. 2,5(A) may cause inhibition of cell proliferation through some mechanisms other than activating endonuclease L. Panet et al. (24), however, have noted that deliberate introduction of 2,5(A) into NIH/MOL B cells does not cause inhibition of protein or DNA synthesis. Similar experiments with IFN-treated and untreated JLSV-11 cells will provide useful information along these lines. The high level of 2,5(A) synthetase in IFN-treated JLSV-11 cells in the absence of detectable endonuclease L activity is hard to justify from a physiological and evolutionary standpoint unless 2,5(A) has other functions in the cell. It is worthwhile to keep in mind, however, that our assay for endonuclease L in cell extracts is not a very sensitive one and we have not yet tested the enzyme activity in whole cells. The absence of sensitivity of EMCV replication in all of the cell lines described here was surprising, especially in view of the fact that mengovirus replication in another NIH/3T3 line is sensitive to IFN (16). It has been shown in other systems that EMCV replication can be sensitive in the absence of functional dsRNAdependent protein kinase activity (14-16), and the endonuclease pathway has been implicated to be responsible for inhibition of EMCV replication (44). The results presented here are consistent with these ideas. EMCV replication was insensitive in all three lines lacking endonuclease L activity. Resistance of VSV replication to IFN was
1026
SEN AND HERZ
correlated with the absence of endonuclease L activity in NIH/MOL B cells (10). This correlation obviously does not exist in NIH/MOL C and JLSV-11 cells. Our results and results of other workers (10, 16) indicate that the presence or absence of dsRNA-dependent protein kinase activity does not have any bearing on anti-VSV action of IFN. Both dsRNA-dependent pathways of inhibition of virus replication seem to be, therefore, inoperative against VSV replication. It is possible that some other pathway of inhibition of viral protein synthesis, such as inhibition of viral mRNA cap methylation (35), is the relevant route through which VSV replication is inhibited in IFN-treated cells. The observations by DeFerra and Baglioni (9) in HeLa cells are very much in line with this possibility. ACKNOWLEDGMENTS We are grateful to Peter Besmer and Robert Friedman for the cell lines, to Peter Lengyel for IFN and 2,5(A), to Amiya Banerjee for VSV mRNA, and to Sohan Gupta for helpful discussion. We are happy to acknowledge the technical assistance of Deborah Richardson and Wanda Zablocki. This work was supported by Public Health Service institutional core grant CA-08748 from the National Cancer Institute and by American Cancer Society grant MV-146.
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