Vol. 64, No. 5
JOURNAL OF VIROLOGY, May 1990, p. 2430-2432
0022-538X/90/052430-03$02.00/0 Copyright C 1990, American Society for Microbiology
Alpha/Beta Interferons Fail To Induce Antiviral Activity from within the Nucleus ISABELLE RIVIERE AND JAQUELINE DE MAEYER-GUIGNARD*
Centre National de la Recherche Scientifique, URA 1343, Institut Curie, Biologie, Bactiment 110, Universite de Paris-Sud, 91405 Orsay, France Received 13 October 1989/Accepted 29 January 1990
Electrophoretically pure murine alpha/beta interferons (IFN-a/$) were microinjected directly into the nuclei of mouse L cells, each nucleus receiving 10 fi containing about 20,000 (IFN) molecules, an amount sufficient to induce the antiviral state when added to the culture medium of control cells. Three, six or 24 h after intranuclear delivery, the cells were challenged with vesicular stomatitis virus or Semliki Forest virus and the appearance of cytopathic effects was scored for each individual cell. The scoring of more than 1,000 intranuclearly injected cells in nine different experiments showed unambiguously that the intranuclear delivery of IFN-a/e did not induce the antiviral state. The results argue strongly against the physiological importance of high-affinity nuclear binding sites for native IFN that have been recently described (V. M. Kushnaryov, H. S. MacDonald, G. P. Lemense, J. Debruin, J. J. Sedmak, and S. E. Grossberg, Cytobios 53:185-197, 1988). Together with earlier results of other groups describing the lack of IFN activity after intracytoplasmic injection (Y. Higashi and Y. Sokawa, J. Biochem. 91:2021-2028, 1982; G. Huez, M. Silhol, and B. Lebleu, Biochem. Biophys. Res. Commun. 110:155-160, 1983), these results lend weight to the hypothesis that the binding of IFN-a$/, to the plasma membrane receptor is sufficient to set into motion the complex mechanism of transmembrane signalling without requiring internalization of the bound IFN molecules. dependent efflux of RNA from these nuclei in a dosedependent manner, suggesting a direct physiological effect of IFN on the cell nucleus (15). On the basis of these results, the hypothesis has been advanced that IFN molecules are delivered into the nucleus to directly affect nuclear function, including gene expression (12). If the binding of IFN to the plasma membrane only serves as a mechanism for delivering IFN molecules into the nuclei and if there are specific nuclear receptors for IFN molecules that are responsible for IFN activity, it should be possible to obtain an IFN-induced antiviral state in cells by inoculating IFN molecules directly into the nucleus. We have tested this possibility by examining the antiviral state of cells after the intranuclear injection of pure Mu IFN-ca/p. The electrophoretically pure IFN preparation used in our experiments was obtained as described previously by two-step affinity chromatography of IFN derived from mouse C-243 cells induced with Newcastle disease virus (5). The titer of the preparation used for microinjection was 3 x 107 units per 0.2 ml after dialysis in phosphate-buffered saline in a Centiprep concentrator (Amicon, W. R. Grace & Co., Danvers, Mass.). L cells cultivated in Eagle minimal essential medium with 8% newborn calf serum were transferred onto glass slides (1 by 1 cm) that had been engraved with numbered squares (1.5 by 1.5 mm) to facilitate the identification of the cells. We found that a cell density of about 30 cells per square was convenient for performing microinjection for monitoring each cell individually. Only two to eight neighboring squares (among 40 per slide) were seeded on each individual slide. For each experiment, one slide was used for the cells that were microinjected in the nucleus, a second slide was used for intracytoplasmic injections, and a third slide consisted of uninjected control cells. Immediately after the cells were seeded, the slides were placed in a 3-cm-diameter petri dish, covered with 2 ml of medium, and left for 24 h in a CO2 incubator. To neutralize any IFN molecules that might have
To display their antiviral properties together with a broad of other biological activities, alpha/beta interferons (IFN-aod) bind to a common, specific plasma membrane receptor of high affinity (1), and the transcription of several IFN-induced genes can be observed as soon as 5 min after receptor binding (6, 8). The mechanisms of signal transduction following the interaction of IFN with its receptor (for a review see reference 4) have not yet been elucidated, and whether the internalization of IFN or of the IFN-receptor complex is necessary for IFN action is still a highly debated issue for which there is presently no definite answer. Electron microscopic observation of the fate of murine (Mu) IFN-W/p, as visualized by ferritin-labeled immunoglobulin G, has shown the presence of IFN in coated pits, coated vesicles, and receptosomes a few minutes after binding to mouse L-cell receptors (14), and human IFN-otA molecules labeled with colloidal gold for monitoring the endocytic pathway have been visualized successively on plasma membrane receptors, in clathrin-coated pits, in receptosomes inside the cell, and finally in lysosomes (17). Of particular interest are the observations that suggest the existence of IFN receptors within the cell nucleus. Indeed, electron microscopic studies have shown that, after binding to the plasma membrane receptor, some of the IFN molecules are translocated by coated vesicles and by receptosomes to nuclear receptors and to nuclear pores. Using postembedding immunolabeling, Kushnaryov et al. (11) showed that 3 min after binding to the plasma membrane receptor of mouse L cells, a majority of Mu IFN-P molecules were present in the cell nucleus. The same group published evidence that isolated L929 cell nuclei bound radiolabeled Mu IFN-P with a sevenfold higher affinity (Kd, 1.4 x 10-10 M) and higher receptor density (about 104 per nucleus) than did the plasma membrane (13). Moreover, treatment of isolated L929 cell nuclei with Mu IFN-P was shown to reduce the energy-
range
*
Corresponding author. 2430
NOTES
VOL. 64, 1990
leaked out of the micropipettes or of the cells during and after the injection, we supplemented the medium with 0.33% goat anti-IFN-a/p serum neutralizing 4 IFN units at a dilution of 10' (3). Microinjections were performed as described by Graessmann and Graessmann (7) under a phase-contrast microscope (Zeiss ICM 405) with 0.5- to 1-p.m-diameter glass capillaries (Femtotips; Eppendorf, Hamburg, Federal Republic of Germany). The microcapillary prefilled with the IFN preparation was directed into the nucleus or the cytoplasm with the aid of a micromanipulator (MR mot; Zeiss, Oberkochen, Federal Republic of Germany). The efficacy of the intranuclear injection procedure was verified by inoculating 10 fl of fluorescein isothiocyanate-labeled dextran (16); this procedure revealed that, on average, the fluorescein was limited to the nucleus in about 66% of the inoculated cells. The average volume of the IFN preparation injected either into the nucleus or into the cytoplasm was approximately 10 fl per cell, corresponding to about 20,000 IFN-o/IB molecules per cell. This amount of IFN resulted in a complete antiviral state against vesicular stomatitis virus (VSV) when added to the culture medium of uninjected controls at 10 fl per cell, despite the significant dilution into the culture medium. Following microinjection, the cultures were returned to the CO2 incubator for 3 or 6 h prior to viral infection. At these times, the total number of living cells on each slide was determined to be used as the 100% value against which the percentage of cells surviving viral infection was determined. About 20% of the cells died within a few hours of the microinjection procedure; we determined in one experiment that the surviving cell population subsequently had a normal multiplication rate, indicating that the microinjection procedure per se did not disrupt nuclear function. For infection of cells, the medium was removed and replaced by Eagle minimal essential medium containing 2% calf serum and, as the challenge virus, VSV at an input multiplicity ranging from 0.2 to 10 PFU/mm2, depending on the experiment. The development of the viral cytopathic effect was monitored for 48 h. The percentages of cells surviving viral infection are given in Table 1 (experiments 1 to 6). On the whole, the progression of the cytopathic effect was comparable in the six different experiments; although the cytopathic effect was less pronounced on one slide of intranuclearly injected cells (experiment 6) and on one slide of intracytoplasmically injected cells (experiment 5), we do not consider these results to be significant, because they were not regularly observed. To ascertain that we had allowed enough time for the antiviral state to develop, we carried out two additional experiments in which the cells were infected 24 h instead of 3 or 6 h after microinjection (experiments 7 and 8). Again, there was no difference in the development of the cytopathic effect in IFN-injected versus untreated cells. Finally, one experiment was done with a different challenge virus, Semliki Forest virus, at 2.3 PFU/mm2 (experiment 9). The absence of protection by intranuclearly inoculated IFN was also observed with Semliki Forest virus. To rule out the possibility that the absence of the antiviral response was the result of an aspecific harmful effect of the microinjection procedure, we verified that after intranuclear injection of IFN, cells retained their capacity to respond to IFN present in the culture medium. For this, L cells were microinjected in the nucleus with 10 fl of IFN. Six hours later, IFN was added to the culture medium at a total dose of 3 x io0 IFN units per 100 cells in 2 ml of culture medium. After overnight incubation, the challenge virus (VSV) was
2431
TABLE 1. Number of surviving cells after viral challengea Time (h) prior to infection
IFN injection
1
3
2
6
3
6
4
6
5
6
6
6
7
24
8
24
9
24
CON NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON NUC
Expt
siteb
No. (%) of surviving cells at indicated time after viral challengec: 12 h 24 h 48 h
100/243 (41) 24/77 (31) 2979 (37) 409/551 (74) 131/166 (79) 144/166 (87) 675/835 (81) 215/260 (83) 175/200 (87) 150/388 (39) 76/113 (67) 40/91 (44) 73/97 (75) 111/161 (69) 55/63 (87)
420/545 (77) 62/545 (11) 140/153 (91) 27/153 (18) 45/144 (31) 10/109 (9) 15/77 (19) 10/77 (13) 27/79 (34) 10/79 (13) 92/551 (17) 14/551 (3) 44/166 (27) 7/166 (4) 27/166 (16) 10/166 (6) 100/835 (12) 0/835 (0) 47/260 (18) 0/260 (0) 35/200 (17) 0/200 (0) 80/388 (21) 38/388 (10) 50/113 (44) 24/113 (21) 26/91 (29) 12/91 (13) 36/97 (37) 26/97 (27) 66/161 (41) 42/161 (26) 46/63 (73) 25/63 (40) 46/74 (62) 21/74 (28) 30/38 (79) 15/38 (39) 9/28 (32) 19/28 (68) 87/135 (64) 34/135 (25) 36/62 (58) 18/62 (29) 26/53 (49) 16/53 (30) 300/399 (75) 94/399 (24) 166/211 (79) 58/211 (27)
a The challenge viruses were VSV for experiments 1 to 8 and Semliki Forest virus for experiment 9. b CON, Uninjected control cells; CYT, intracytoplasmically injected cells; NUC, intranuclearly injected cells. I For uninjected infected control cells, surviving cells were enumerated on four slides, whereas only one slide was used for intranuclear or intracytoplasmic microinjection in each different experiment.
added and the cytopathic effect was monitored for 48 h. Controls consisted of cells receiving IFN either in the nucleus only or from the culture medium only. Cells microinjected with IFN in the nucleus were fully competent to replicate and to develop an antiviral state (Table 2). In conclusion, our results show unambiguously that Mu IFN-a/p molecules directly delivered into the nuclei of mouse L cells fail to induce an antiviral state. We think it therefore rather unlikely that nuclear binding sites of high affinity for native IFN-,B, as described by Kushnaryov et al. (12), are involved in the establishment of the antiviral state. However, and although we believe this to be unlikely, it cannot be ruled out that, after receptor binding, the internalized IFN molecules are processed and partially degraded TABLE 2. Lack of effect of intranuclear injection of IFN on cell replication and on the response to external IFN No. (%) of surviving cells at:
IFN treatment
Intranu- External IFN 6 h after added to clear intranuclear injection the culture injection mediumb of IFN'
+
+ +
+
-
133 112 133 97
Time of VSV in-
24 h after VSV in-
48 h after VSV in-
fection
fection
fection
199 176 211 167
(100) (100) (100) (100)
340 313 243 183
(171) 624 (314) (178) 540 (307) 0 (0) (115) 0 (0) (110)
Intranuclear injection of IFN was performed 30 h before VSV infection. External IFN was added to the culture medium 6 h after intranuclear injection and 24 h before VSV infection. a
b
2432
J. VIROL.
NOTES
and that some of these processed molecules then interact with other structures in the cell, including the nucleus. Arnheiter and Zoon (2) previously showed that microinjection of anti-human IFN-ax antibodies either into the cytoplasm or into the nucleus of MDBK cells did not inhibit the subsequent establishment of the antiviral state when these cells were exposed to human IFN-a These observations provide additional evidence against the possibility that native IFN molecules themselves act in the cytoplasmic compartment or in the nucleus to induce an antiviral state. Taken together, the earlier work of Higashi and Sokawa (9) and Huez et al. (10), who showed that Mu IFN-a/o or human IFN-P did not confer an antiviral state after intracytoplasmic injection, and the direct evidence for the lack of intranuclear activity provided by the present work strongly argue against the possibility that native IFN molecules are directly delivered into the nucleus to affect gene expression, resulting in the antiviral state. LITERATURE CITED 1. Aguet, M., and B. Blanchard. 1981. High affinity binding of 125I-labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties. Virology 115:249-261. 2. Arnheiter, H., and K. C. Zoon. 1984. Microinjection of antiinterferon antibodies into cells does not inhibit the induction of an antiviral state by interferon. Virology 52:284-287. 3. De Maeyer, E., and J. De Maeyer-Guignard. 1983. Delayed hypersensitivity to Newcastle disease virus in high and low interferon-producing mice. J. Immunol. 130:2392-2396. 4. De Maeyer, E., and J. De Maeyer-Guignard. 1988. Interferons and other regulatory cytokines, p. 67-90. John Wiley & Sons, Inc., New York. 5. De Maeyer-Guignard, J., M. G. Tovey, I. Gresser, and E. De Maeyer. 1978. Purification of mouse interferon by sequential affinity chromatography on poly(U)- and antibody-agarose columns. Nature (London) 271:622-625. 6. Friedman, R. L., S. P. Manly, M. McMahon, I. M. Kerr, and G. R. Stark. 1984. Transcriptional and posttranscriptional reg-
7. 8. 9. 10. 11.
12.
13.
14.
15. 16.
17.
ulation of interferon-induced gene expression in human cells. Cell 38:745-755. Graessmann, M., and A. Graessmann. 1983. Microinjection of tissue culture cells. Methods Enzymol. 101:482-492. Hannigan, G., and B. R. G. Williams. 1986. Transcriptional regulation of interferon-responsive genes is closely linked to interferon receptor occupancy. EMBO J 5:1607-1613. Higashi, Y., and Y. Sokawa. 1982. Microinjection of interferon and 2',5'-oligoadenylate into mouse L cells and their effects on virus growth. J. Biochem. 91:2021-2028. Huez, G., M. Silhol, and B. Lebleu. 1983. Microinjected interferon does not promote an antiviral response in HeLa cells. Biochem. Biophys. Res. Commun. 110:155-160. Kushnaryov, V. M., H. S. MacDonald, J. Debruin, G. P. Lemense, J. J. Sedmak, and S. E. Grossberg. 1986. Internalization and transport of mouse beta-interferon into the cell nucleus. J. Interferon Res. 6:241-246. Kushnaryov, V. M., H. S. MacDonald, G. P. Lemense, J. Debruin, J. J. Sedmak, and S. E. Grossberg. 1988. Quantitative analysis of mouse interferon-beta receptor-mediated endocytosis and nuclear entry. Cytobios 53:185-197. Kushnaryov, V. M., H. S. MacDonald, J. J. Sedmak, and S. Grossberg. 1985. Murine interferon-beta receptor-mediated endocytosis and nuclear membrane binding. Proc. Natl. Acad. Sci. USA 82:3281-3285. Kushnaryov, V. M., H. S. MacDonald, J. J. Sedmak, and S. E. Grossberg. 1983. Ultrastructural distribution of interferon receptor sites on mouse L fibroblasts grown in suspension: ganglioside blockade of ligand binding. Infect. Immun. 40: 320-329. MacDonald, H. S., V. M. Kushnaryov, R. Hevner, J. J. Sedmak, and S. E. Grossberg. 1986. Mouse beta-interferon reduces RNA efflux from isolated nuclei. J. Interferon Res. 6:247-250. Pepperkok, R., M. Zanetti, R. King, D. Delia, W. Ansorge, L. Philipson, and C. Schneider. 1988. Automatic microinjection system facilitates detection of growth inhibitory mRNA. Proc. Natl. Acad. Sci. USA 85:6748-6752. Zoon, K. C., H. Arnheiter, D. Zur Nedden, D. J. P. Fitzgerald, and M. C. Willingham. 1983. Human interferon alpha enters cells by receptor-mediated endocytosis. Virology 130:195-203.
JOURNAL OF VIROLOGY, May 1990, p. 2430-2432
0022-538X/90/052430-03$02.00/0 Copyright C 1990, American Society for Microbiology
Alpha/Beta Interferons Fail To Induce Antiviral Activity from within the Nucleus ISABELLE RIVIERE AND JAQUELINE DE MAEYER-GUIGNARD*
Centre National de la Recherche Scientifique, URA 1343, Institut Curie, Biologie, Bactiment 110, Universite de Paris-Sud, 91405 Orsay, France Received 13 October 1989/Accepted 29 January 1990
Electrophoretically pure murine alpha/beta interferons (IFN-a/$) were microinjected directly into the nuclei of mouse L cells, each nucleus receiving 10 fi containing about 20,000 (IFN) molecules, an amount sufficient to induce the antiviral state when added to the culture medium of control cells. Three, six or 24 h after intranuclear delivery, the cells were challenged with vesicular stomatitis virus or Semliki Forest virus and the appearance of cytopathic effects was scored for each individual cell. The scoring of more than 1,000 intranuclearly injected cells in nine different experiments showed unambiguously that the intranuclear delivery of IFN-a/e did not induce the antiviral state. The results argue strongly against the physiological importance of high-affinity nuclear binding sites for native IFN that have been recently described (V. M. Kushnaryov, H. S. MacDonald, G. P. Lemense, J. Debruin, J. J. Sedmak, and S. E. Grossberg, Cytobios 53:185-197, 1988). Together with earlier results of other groups describing the lack of IFN activity after intracytoplasmic injection (Y. Higashi and Y. Sokawa, J. Biochem. 91:2021-2028, 1982; G. Huez, M. Silhol, and B. Lebleu, Biochem. Biophys. Res. Commun. 110:155-160, 1983), these results lend weight to the hypothesis that the binding of IFN-a$/, to the plasma membrane receptor is sufficient to set into motion the complex mechanism of transmembrane signalling without requiring internalization of the bound IFN molecules. dependent efflux of RNA from these nuclei in a dosedependent manner, suggesting a direct physiological effect of IFN on the cell nucleus (15). On the basis of these results, the hypothesis has been advanced that IFN molecules are delivered into the nucleus to directly affect nuclear function, including gene expression (12). If the binding of IFN to the plasma membrane only serves as a mechanism for delivering IFN molecules into the nuclei and if there are specific nuclear receptors for IFN molecules that are responsible for IFN activity, it should be possible to obtain an IFN-induced antiviral state in cells by inoculating IFN molecules directly into the nucleus. We have tested this possibility by examining the antiviral state of cells after the intranuclear injection of pure Mu IFN-ca/p. The electrophoretically pure IFN preparation used in our experiments was obtained as described previously by two-step affinity chromatography of IFN derived from mouse C-243 cells induced with Newcastle disease virus (5). The titer of the preparation used for microinjection was 3 x 107 units per 0.2 ml after dialysis in phosphate-buffered saline in a Centiprep concentrator (Amicon, W. R. Grace & Co., Danvers, Mass.). L cells cultivated in Eagle minimal essential medium with 8% newborn calf serum were transferred onto glass slides (1 by 1 cm) that had been engraved with numbered squares (1.5 by 1.5 mm) to facilitate the identification of the cells. We found that a cell density of about 30 cells per square was convenient for performing microinjection for monitoring each cell individually. Only two to eight neighboring squares (among 40 per slide) were seeded on each individual slide. For each experiment, one slide was used for the cells that were microinjected in the nucleus, a second slide was used for intracytoplasmic injections, and a third slide consisted of uninjected control cells. Immediately after the cells were seeded, the slides were placed in a 3-cm-diameter petri dish, covered with 2 ml of medium, and left for 24 h in a CO2 incubator. To neutralize any IFN molecules that might have
To display their antiviral properties together with a broad of other biological activities, alpha/beta interferons (IFN-aod) bind to a common, specific plasma membrane receptor of high affinity (1), and the transcription of several IFN-induced genes can be observed as soon as 5 min after receptor binding (6, 8). The mechanisms of signal transduction following the interaction of IFN with its receptor (for a review see reference 4) have not yet been elucidated, and whether the internalization of IFN or of the IFN-receptor complex is necessary for IFN action is still a highly debated issue for which there is presently no definite answer. Electron microscopic observation of the fate of murine (Mu) IFN-W/p, as visualized by ferritin-labeled immunoglobulin G, has shown the presence of IFN in coated pits, coated vesicles, and receptosomes a few minutes after binding to mouse L-cell receptors (14), and human IFN-otA molecules labeled with colloidal gold for monitoring the endocytic pathway have been visualized successively on plasma membrane receptors, in clathrin-coated pits, in receptosomes inside the cell, and finally in lysosomes (17). Of particular interest are the observations that suggest the existence of IFN receptors within the cell nucleus. Indeed, electron microscopic studies have shown that, after binding to the plasma membrane receptor, some of the IFN molecules are translocated by coated vesicles and by receptosomes to nuclear receptors and to nuclear pores. Using postembedding immunolabeling, Kushnaryov et al. (11) showed that 3 min after binding to the plasma membrane receptor of mouse L cells, a majority of Mu IFN-P molecules were present in the cell nucleus. The same group published evidence that isolated L929 cell nuclei bound radiolabeled Mu IFN-P with a sevenfold higher affinity (Kd, 1.4 x 10-10 M) and higher receptor density (about 104 per nucleus) than did the plasma membrane (13). Moreover, treatment of isolated L929 cell nuclei with Mu IFN-P was shown to reduce the energy-
range
*
Corresponding author. 2430
NOTES
VOL. 64, 1990
leaked out of the micropipettes or of the cells during and after the injection, we supplemented the medium with 0.33% goat anti-IFN-a/p serum neutralizing 4 IFN units at a dilution of 10' (3). Microinjections were performed as described by Graessmann and Graessmann (7) under a phase-contrast microscope (Zeiss ICM 405) with 0.5- to 1-p.m-diameter glass capillaries (Femtotips; Eppendorf, Hamburg, Federal Republic of Germany). The microcapillary prefilled with the IFN preparation was directed into the nucleus or the cytoplasm with the aid of a micromanipulator (MR mot; Zeiss, Oberkochen, Federal Republic of Germany). The efficacy of the intranuclear injection procedure was verified by inoculating 10 fl of fluorescein isothiocyanate-labeled dextran (16); this procedure revealed that, on average, the fluorescein was limited to the nucleus in about 66% of the inoculated cells. The average volume of the IFN preparation injected either into the nucleus or into the cytoplasm was approximately 10 fl per cell, corresponding to about 20,000 IFN-o/IB molecules per cell. This amount of IFN resulted in a complete antiviral state against vesicular stomatitis virus (VSV) when added to the culture medium of uninjected controls at 10 fl per cell, despite the significant dilution into the culture medium. Following microinjection, the cultures were returned to the CO2 incubator for 3 or 6 h prior to viral infection. At these times, the total number of living cells on each slide was determined to be used as the 100% value against which the percentage of cells surviving viral infection was determined. About 20% of the cells died within a few hours of the microinjection procedure; we determined in one experiment that the surviving cell population subsequently had a normal multiplication rate, indicating that the microinjection procedure per se did not disrupt nuclear function. For infection of cells, the medium was removed and replaced by Eagle minimal essential medium containing 2% calf serum and, as the challenge virus, VSV at an input multiplicity ranging from 0.2 to 10 PFU/mm2, depending on the experiment. The development of the viral cytopathic effect was monitored for 48 h. The percentages of cells surviving viral infection are given in Table 1 (experiments 1 to 6). On the whole, the progression of the cytopathic effect was comparable in the six different experiments; although the cytopathic effect was less pronounced on one slide of intranuclearly injected cells (experiment 6) and on one slide of intracytoplasmically injected cells (experiment 5), we do not consider these results to be significant, because they were not regularly observed. To ascertain that we had allowed enough time for the antiviral state to develop, we carried out two additional experiments in which the cells were infected 24 h instead of 3 or 6 h after microinjection (experiments 7 and 8). Again, there was no difference in the development of the cytopathic effect in IFN-injected versus untreated cells. Finally, one experiment was done with a different challenge virus, Semliki Forest virus, at 2.3 PFU/mm2 (experiment 9). The absence of protection by intranuclearly inoculated IFN was also observed with Semliki Forest virus. To rule out the possibility that the absence of the antiviral response was the result of an aspecific harmful effect of the microinjection procedure, we verified that after intranuclear injection of IFN, cells retained their capacity to respond to IFN present in the culture medium. For this, L cells were microinjected in the nucleus with 10 fl of IFN. Six hours later, IFN was added to the culture medium at a total dose of 3 x io0 IFN units per 100 cells in 2 ml of culture medium. After overnight incubation, the challenge virus (VSV) was
2431
TABLE 1. Number of surviving cells after viral challengea Time (h) prior to infection
IFN injection
1
3
2
6
3
6
4
6
5
6
6
6
7
24
8
24
9
24
CON NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON CYT NUC CON NUC
Expt
siteb
No. (%) of surviving cells at indicated time after viral challengec: 12 h 24 h 48 h
100/243 (41) 24/77 (31) 2979 (37) 409/551 (74) 131/166 (79) 144/166 (87) 675/835 (81) 215/260 (83) 175/200 (87) 150/388 (39) 76/113 (67) 40/91 (44) 73/97 (75) 111/161 (69) 55/63 (87)
420/545 (77) 62/545 (11) 140/153 (91) 27/153 (18) 45/144 (31) 10/109 (9) 15/77 (19) 10/77 (13) 27/79 (34) 10/79 (13) 92/551 (17) 14/551 (3) 44/166 (27) 7/166 (4) 27/166 (16) 10/166 (6) 100/835 (12) 0/835 (0) 47/260 (18) 0/260 (0) 35/200 (17) 0/200 (0) 80/388 (21) 38/388 (10) 50/113 (44) 24/113 (21) 26/91 (29) 12/91 (13) 36/97 (37) 26/97 (27) 66/161 (41) 42/161 (26) 46/63 (73) 25/63 (40) 46/74 (62) 21/74 (28) 30/38 (79) 15/38 (39) 9/28 (32) 19/28 (68) 87/135 (64) 34/135 (25) 36/62 (58) 18/62 (29) 26/53 (49) 16/53 (30) 300/399 (75) 94/399 (24) 166/211 (79) 58/211 (27)
a The challenge viruses were VSV for experiments 1 to 8 and Semliki Forest virus for experiment 9. b CON, Uninjected control cells; CYT, intracytoplasmically injected cells; NUC, intranuclearly injected cells. I For uninjected infected control cells, surviving cells were enumerated on four slides, whereas only one slide was used for intranuclear or intracytoplasmic microinjection in each different experiment.
added and the cytopathic effect was monitored for 48 h. Controls consisted of cells receiving IFN either in the nucleus only or from the culture medium only. Cells microinjected with IFN in the nucleus were fully competent to replicate and to develop an antiviral state (Table 2). In conclusion, our results show unambiguously that Mu IFN-a/p molecules directly delivered into the nuclei of mouse L cells fail to induce an antiviral state. We think it therefore rather unlikely that nuclear binding sites of high affinity for native IFN-,B, as described by Kushnaryov et al. (12), are involved in the establishment of the antiviral state. However, and although we believe this to be unlikely, it cannot be ruled out that, after receptor binding, the internalized IFN molecules are processed and partially degraded TABLE 2. Lack of effect of intranuclear injection of IFN on cell replication and on the response to external IFN No. (%) of surviving cells at:
IFN treatment
Intranu- External IFN 6 h after added to clear intranuclear injection the culture injection mediumb of IFN'
+
+ +
+
-
133 112 133 97
Time of VSV in-
24 h after VSV in-
48 h after VSV in-
fection
fection
fection
199 176 211 167
(100) (100) (100) (100)
340 313 243 183
(171) 624 (314) (178) 540 (307) 0 (0) (115) 0 (0) (110)
Intranuclear injection of IFN was performed 30 h before VSV infection. External IFN was added to the culture medium 6 h after intranuclear injection and 24 h before VSV infection. a
b
2432
J. VIROL.
NOTES
and that some of these processed molecules then interact with other structures in the cell, including the nucleus. Arnheiter and Zoon (2) previously showed that microinjection of anti-human IFN-ax antibodies either into the cytoplasm or into the nucleus of MDBK cells did not inhibit the subsequent establishment of the antiviral state when these cells were exposed to human IFN-a These observations provide additional evidence against the possibility that native IFN molecules themselves act in the cytoplasmic compartment or in the nucleus to induce an antiviral state. Taken together, the earlier work of Higashi and Sokawa (9) and Huez et al. (10), who showed that Mu IFN-a/o or human IFN-P did not confer an antiviral state after intracytoplasmic injection, and the direct evidence for the lack of intranuclear activity provided by the present work strongly argue against the possibility that native IFN molecules are directly delivered into the nucleus to affect gene expression, resulting in the antiviral state. LITERATURE CITED 1. Aguet, M., and B. Blanchard. 1981. High affinity binding of 125I-labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties. Virology 115:249-261. 2. Arnheiter, H., and K. C. Zoon. 1984. Microinjection of antiinterferon antibodies into cells does not inhibit the induction of an antiviral state by interferon. Virology 52:284-287. 3. De Maeyer, E., and J. De Maeyer-Guignard. 1983. Delayed hypersensitivity to Newcastle disease virus in high and low interferon-producing mice. J. Immunol. 130:2392-2396. 4. De Maeyer, E., and J. De Maeyer-Guignard. 1988. Interferons and other regulatory cytokines, p. 67-90. John Wiley & Sons, Inc., New York. 5. De Maeyer-Guignard, J., M. G. Tovey, I. Gresser, and E. De Maeyer. 1978. Purification of mouse interferon by sequential affinity chromatography on poly(U)- and antibody-agarose columns. Nature (London) 271:622-625. 6. Friedman, R. L., S. P. Manly, M. McMahon, I. M. Kerr, and G. R. Stark. 1984. Transcriptional and posttranscriptional reg-
7. 8. 9. 10. 11.
12.
13.
14.
15. 16.
17.
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