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Unresponsiveness to the action of interferon developed in persistently infected L cells
Life Sciences No . 12, pp . 895-901, 1963 . Pergamon Press, Inc . Printed in the United States .
UNRESPC~TSTVENESS TO THE ACTIN OF INTERFERON DLVELOPED IN PEFSISTENTLY INFECTED L CELLS
Jan
Vil~ek and Daniel Stan~ek
Institute of Virology, Czechoslovak Academy of Sciences, Bratislava 9, Czechoslovakia (Received 25 October 1963) INTERFERON is supposed to play an important role in the establishment and maintenance of a virus carrier state in tissue culture
(1) . Recently Glasgow and Habel (2) demonstrated the effect
of interferon on the initial stage of persistent infection of mouse cells with vaccinia virus . The same authors were able to demonstrate the role of interferon in the establishment of a double carrier state in mouse cells persistently infected with polyoma virus and superinfected with herpes simplex virus (3) " L cells can be persistently infected with tick-borne encephalitis virus : cultures infected Kith e suitable dose of this virus continue to multiply and produce variable quantities of virus and interferon (4) . The possible role of interferon for the maintenance
of persistent infection of L cells vrith tick-
borne encephalitis virus was studied in the present work . some unexpected results were obtained which indicate that a state of unresponsiveness to interferon may develop in cells after a longlasting persistent viral infection. I~ aterie.ls and lriethods The origin of L cells persistently infected with tickborne encephalitis virus was described (4 ) . Three hundred ml .Roux bottles were seeded with approximately 1 .5x106 cells in 895
896
UNRESPONSIVENESS TO INTERFERON ACTION
No . 12
synthetic medium 199 with 10 ~ heated horse serum, 200 unite of penicillin, 200 ~. streptomycir. and 0 .001 ~ phenol red . The cells were trypsinized and passaged after 5 - 6 days cultivation . Cells which had undergone at least 15 passages after the establishment of persistent infection were used . Uninfected L cells were grown in the same type of medium . Both persistently infected and uninfected L cells were cultivated at a temperature of 36 - 37 ° C . The pH values of the medium, as roughly contro~ed by the inclusion of phenol red, varied during the cultivation between approximately 6 .8 -705, but no marked differences were observed in the rate of -acidification of the medium in persistently infected and uninfected L cells . Ten per cent suspensions of brPins from moribund mice intracerebrally inoculated with the Hypr strain of tick-borne encephalitis virus in which virus had been inactivated by acid ification were used as source of interferon . Hefore use the suspensions were centrifuged at 12,000 rpm for 30 minutes . Titrations of interferon were performed in tube-cultures of L cells . The interferon titre represents the reciprocal of the highest dilution of the tested materiel causing complete protection from the cytopathic effect of encephalomryocarditia (EI~C) virus (5) . Tick-borne encephalitis virus was titrated intracerebrally in mice . Results and Discussion In order to see whether interferon formed in L cells peraiatently infected with tick-borne encephalitis virus exerts any control over virus growth in the same cultures, large quantities of exogenous interferon were repeatedly added into the culture medium . It was expected that exogenous interferon would either cure the cultures from infection or at least efficiently suppress
üNRE8PON8IVENES$ TO INTERFERON ACTION
No . 12
897
virus multiplication, particularly in view of the fact that uninfected cultures of L cells were previously shown to be highly sensitive to the action of interferon (5) " In ane such experiment, 2 .5 ml . clarified mouse brain suspension containing 1,024 units of interferon per ml . was added to 22 .5 ml . medium at the tip of seeding of persistently infec ted cells and the same dose of interferon was applied on the third and fifth days of cultivation. Cells were trypsinised and peasaged after 6 days . Interferon eras applied in the same way for 4 passages, so that the cells were under the influence of appreciable quantities of interferon for a total of 24 days . Control cultures peasaged in parallel received no interferon . The titre of virus in the medium was estimated at the end of each passage in both interferon-treated and control cultures . Surprisingly, no marked differences were observed in the rate of virus multiplication in interferon-treated and control cultures (Table 1) . The failure of exogenous interferon given in high doses to suppress virus multiplication in persistently infected L cells could be explained in two ways : either by the failure of interfe ron to influence the multiplication of virus once the infection had been fully established, or by the fact that persistently infected L cells Per se become refractory to the action of interferon . The former possibility would agree with experimental data on the failure of interferon to suppress virus multiplication when applied after a critical time following the penetration of virus (6) and with the concept that interferon acts by preventing the transfer of information from the viral genor~e to the cell (7) . Qn the other hand, if the latter possibility were true, interferon should also fail to protect persistently infected L cells from
898
UNRESPONSIVENESS TO INTERFERON ACTION
No. 12
TALE 1 Failure of Exogenous Interferon to Influence Virus It:ultiplication in L Cells Persistently Infected with Tick-borne Encephalitis Virus
Culture
1
log LD5U/ml . in passage 210 . 2 3 4
Interferon-treated
3 .2
4 .U
3 .ir
2 .2
Untreated
3 .?
4 .ßs
2 .7
2 .8
superinfection with an other unrelated virus . Persistently infected and control uninfected L cells v:ere ~;roh~n in tubes . .lfter an ~lmost complete monolayer had been formed, the cultures vrere drained and refed ~:ith 1 uil . of twofold di lutions of mouse brain inter~fer~on in tissue culture medium, using tubes of each group for each dilution . The next day the cultures received tv~o different doses of r~i..C virus . Lit the same time, parallel titration: of Ei~:C vi rus titi~ere performed in persistently infeeted and previously uninfected tube-cultures of L cells . The interferon titres v,ere read v:hen control cultures receiving no interferon were completely degenerated (fable 2) . The same preparation of interferon which completely protected L cells from the cytopathic effect of E2r:C virus up to a dilution of 1 :2,048, was co~:pletely ineffective against the same doses of EP:iC virus in persistently infected L cells . It is to be noticed that there was no marked difference in the sensitivity of persistently infected and uninfected L cells to Eb :C virus, although the appearance of cytopathic effect in persistently infected cells was slightly delayed .
89 9
UNRESPONSIVENESS TO INTERFERON ACTION
No . 12
TABLE 2 Comparison of Interferon Titres in Persistently Infected and Uninfected L Cells
Interferon titre against
L cells
TCD50 of EB6C virus* 3 .2x102
Persistently infected Uninfected
3 .2x104
log TCD50/ml . of stock ENC virus
~ 16
< 16
7 .5
2,048
2,048
7 .8
*as estimated in uninfected L cells
It seems therefore that L cells persistently infected with tick-borne encephalitis virus are refractory to the action of interferon even against an unrelated virus ; consequently, exoge nous interferon could not cure them from persistent infection . It is not known at what stage of the persistent infection the unresponsiveness to interferon develops in L cells . Some results indicate, however,
that this change is gradual . L cells primari-
ly infected with tick-borne encephalitis vü~us are highly resistant to superinfection with high doses of ELiC virus ; this resistance gradually decreases in course of further passages, until practically no resistance remains . A similar tendency was also observed in interferon formation, but even at high passage levels when shown to be fully refractory to the action of interferon, the cells continue to produce barely detectable quantities of interferon (8 ) . It is not known whether the observed unresponsiveness of persistently infected L cells to interferon represents a more
900
IINRESPONSNENESS TO INTERFERON ACTION
No. 12
general phenomenon occurring also in other cells after a longlasting virus carrier state . It is therefore too early to make any definite generalisations . .Neverthelesa, the results obtained, in contradistinction to findings of other authors, suggest that come relationships might exist which could be of importance . After the excellent work by Isaacs et al (9) it seems very likely that interferon formation represents the control mechanism of the cell over invasion of foreign nucleic acid and,
con
sequently, over foreign genetic information. If a latent viral infection can suppress this mechanism then the same viral infection should also render the cell more sensitive to mutation and transformation . One possible implication of this state could be the greater sensitivity of cells to malignant transformation . It seem$ noteworthy in this respect to mention the recent results of De I~aeyer and De D~seyer-Guignard (10) showing that the carcinogenic substance methylcholanthrene can suppress interferon production . Similarly, an inhibitory effect of actinomycin D on the formation of interferon was observed (11) . Hermodsson (l2) observed an inhibition of the interferon mechanism by infection with parainfluerza-3 virus . The fact that some carcinogenic substances and some viruses cause inhibition of the interferon mechanism has to be taken into account . F~eferences 1.
~` . HEhLE, G. HENLE, F. DEIIJEiAFcDT and V. Y . HEFiGS, J. Exp . D~ed .
110, 525
(1959) "
2.
L. A . GLASGOw and K. HAGEL, J. Exp . ~ed . 11
3.
L. A . GIASGOW and K. HAGEL, Virolow l~, 328
4.
D . STANL~EK, Acta viral . (Prague)
3,
,
509 (1962) . (l960 .
225 (1963) .
No . 12
IINRE3PON3NENE33 TO INTERFERON ACTION
901
5.
J. VI?~EK and D. STANL~EK, Acta virol .
6.
& . R . WA($dER, Virolo~ ~,
?"
C . COCITO, E . DE MAEZER and P . DE SOAR, Life Sciences
(PraRUe)
323 (1961) .
3, 331 (1963) .
l, 753 (15x'2) . 8.
D . STdNL~EK and J. VIT.~EK, in preparation .
9.
A . ISAACS, R. A. COS and Z. ROTES, Lancet
10 .
ü, 113 (1963) "
E . DE MAEYh~t and J. DE MAE]~R-GUIQdARD, Yirolo~y 20,
(1963) . 11 .
E . HELhER, Biochem. J. ~, 18 P
12 .
S . HERI9~DSSON, ViroloAV 20,
(1963) "
333 (1963) "
536
UNRESPC~TSTVENESS TO THE ACTIN OF INTERFERON DLVELOPED IN PEFSISTENTLY INFECTED L CELLS
Jan
Vil~ek and Daniel Stan~ek
Institute of Virology, Czechoslovak Academy of Sciences, Bratislava 9, Czechoslovakia (Received 25 October 1963) INTERFERON is supposed to play an important role in the establishment and maintenance of a virus carrier state in tissue culture
(1) . Recently Glasgow and Habel (2) demonstrated the effect
of interferon on the initial stage of persistent infection of mouse cells with vaccinia virus . The same authors were able to demonstrate the role of interferon in the establishment of a double carrier state in mouse cells persistently infected with polyoma virus and superinfected with herpes simplex virus (3) " L cells can be persistently infected with tick-borne encephalitis virus : cultures infected Kith e suitable dose of this virus continue to multiply and produce variable quantities of virus and interferon (4) . The possible role of interferon for the maintenance
of persistent infection of L cells vrith tick-
borne encephalitis virus was studied in the present work . some unexpected results were obtained which indicate that a state of unresponsiveness to interferon may develop in cells after a longlasting persistent viral infection. I~ aterie.ls and lriethods The origin of L cells persistently infected with tickborne encephalitis virus was described (4 ) . Three hundred ml .Roux bottles were seeded with approximately 1 .5x106 cells in 895
896
UNRESPONSIVENESS TO INTERFERON ACTION
No . 12
synthetic medium 199 with 10 ~ heated horse serum, 200 unite of penicillin, 200 ~. streptomycir. and 0 .001 ~ phenol red . The cells were trypsinized and passaged after 5 - 6 days cultivation . Cells which had undergone at least 15 passages after the establishment of persistent infection were used . Uninfected L cells were grown in the same type of medium . Both persistently infected and uninfected L cells were cultivated at a temperature of 36 - 37 ° C . The pH values of the medium, as roughly contro~ed by the inclusion of phenol red, varied during the cultivation between approximately 6 .8 -705, but no marked differences were observed in the rate of -acidification of the medium in persistently infected and uninfected L cells . Ten per cent suspensions of brPins from moribund mice intracerebrally inoculated with the Hypr strain of tick-borne encephalitis virus in which virus had been inactivated by acid ification were used as source of interferon . Hefore use the suspensions were centrifuged at 12,000 rpm for 30 minutes . Titrations of interferon were performed in tube-cultures of L cells . The interferon titre represents the reciprocal of the highest dilution of the tested materiel causing complete protection from the cytopathic effect of encephalomryocarditia (EI~C) virus (5) . Tick-borne encephalitis virus was titrated intracerebrally in mice . Results and Discussion In order to see whether interferon formed in L cells peraiatently infected with tick-borne encephalitis virus exerts any control over virus growth in the same cultures, large quantities of exogenous interferon were repeatedly added into the culture medium . It was expected that exogenous interferon would either cure the cultures from infection or at least efficiently suppress
üNRE8PON8IVENES$ TO INTERFERON ACTION
No . 12
897
virus multiplication, particularly in view of the fact that uninfected cultures of L cells were previously shown to be highly sensitive to the action of interferon (5) " In ane such experiment, 2 .5 ml . clarified mouse brain suspension containing 1,024 units of interferon per ml . was added to 22 .5 ml . medium at the tip of seeding of persistently infec ted cells and the same dose of interferon was applied on the third and fifth days of cultivation. Cells were trypsinised and peasaged after 6 days . Interferon eras applied in the same way for 4 passages, so that the cells were under the influence of appreciable quantities of interferon for a total of 24 days . Control cultures peasaged in parallel received no interferon . The titre of virus in the medium was estimated at the end of each passage in both interferon-treated and control cultures . Surprisingly, no marked differences were observed in the rate of virus multiplication in interferon-treated and control cultures (Table 1) . The failure of exogenous interferon given in high doses to suppress virus multiplication in persistently infected L cells could be explained in two ways : either by the failure of interfe ron to influence the multiplication of virus once the infection had been fully established, or by the fact that persistently infected L cells Per se become refractory to the action of interferon . The former possibility would agree with experimental data on the failure of interferon to suppress virus multiplication when applied after a critical time following the penetration of virus (6) and with the concept that interferon acts by preventing the transfer of information from the viral genor~e to the cell (7) . Qn the other hand, if the latter possibility were true, interferon should also fail to protect persistently infected L cells from
898
UNRESPONSIVENESS TO INTERFERON ACTION
No. 12
TALE 1 Failure of Exogenous Interferon to Influence Virus It:ultiplication in L Cells Persistently Infected with Tick-borne Encephalitis Virus
Culture
1
log LD5U/ml . in passage 210 . 2 3 4
Interferon-treated
3 .2
4 .U
3 .ir
2 .2
Untreated
3 .?
4 .ßs
2 .7
2 .8
superinfection with an other unrelated virus . Persistently infected and control uninfected L cells v:ere ~;roh~n in tubes . .lfter an ~lmost complete monolayer had been formed, the cultures vrere drained and refed ~:ith 1 uil . of twofold di lutions of mouse brain inter~fer~on in tissue culture medium, using tubes of each group for each dilution . The next day the cultures received tv~o different doses of r~i..C virus . Lit the same time, parallel titration: of Ei~:C vi rus titi~ere performed in persistently infeeted and previously uninfected tube-cultures of L cells . The interferon titres v,ere read v:hen control cultures receiving no interferon were completely degenerated (fable 2) . The same preparation of interferon which completely protected L cells from the cytopathic effect of E2r:C virus up to a dilution of 1 :2,048, was co~:pletely ineffective against the same doses of EP:iC virus in persistently infected L cells . It is to be noticed that there was no marked difference in the sensitivity of persistently infected and uninfected L cells to Eb :C virus, although the appearance of cytopathic effect in persistently infected cells was slightly delayed .
89 9
UNRESPONSIVENESS TO INTERFERON ACTION
No . 12
TABLE 2 Comparison of Interferon Titres in Persistently Infected and Uninfected L Cells
Interferon titre against
L cells
TCD50 of EB6C virus* 3 .2x102
Persistently infected Uninfected
3 .2x104
log TCD50/ml . of stock ENC virus
~ 16
< 16
7 .5
2,048
2,048
7 .8
*as estimated in uninfected L cells
It seems therefore that L cells persistently infected with tick-borne encephalitis virus are refractory to the action of interferon even against an unrelated virus ; consequently, exoge nous interferon could not cure them from persistent infection . It is not known at what stage of the persistent infection the unresponsiveness to interferon develops in L cells . Some results indicate, however,
that this change is gradual . L cells primari-
ly infected with tick-borne encephalitis vü~us are highly resistant to superinfection with high doses of ELiC virus ; this resistance gradually decreases in course of further passages, until practically no resistance remains . A similar tendency was also observed in interferon formation, but even at high passage levels when shown to be fully refractory to the action of interferon, the cells continue to produce barely detectable quantities of interferon (8 ) . It is not known whether the observed unresponsiveness of persistently infected L cells to interferon represents a more
900
IINRESPONSNENESS TO INTERFERON ACTION
No. 12
general phenomenon occurring also in other cells after a longlasting virus carrier state . It is therefore too early to make any definite generalisations . .Neverthelesa, the results obtained, in contradistinction to findings of other authors, suggest that come relationships might exist which could be of importance . After the excellent work by Isaacs et al (9) it seems very likely that interferon formation represents the control mechanism of the cell over invasion of foreign nucleic acid and,
con
sequently, over foreign genetic information. If a latent viral infection can suppress this mechanism then the same viral infection should also render the cell more sensitive to mutation and transformation . One possible implication of this state could be the greater sensitivity of cells to malignant transformation . It seem$ noteworthy in this respect to mention the recent results of De I~aeyer and De D~seyer-Guignard (10) showing that the carcinogenic substance methylcholanthrene can suppress interferon production . Similarly, an inhibitory effect of actinomycin D on the formation of interferon was observed (11) . Hermodsson (l2) observed an inhibition of the interferon mechanism by infection with parainfluerza-3 virus . The fact that some carcinogenic substances and some viruses cause inhibition of the interferon mechanism has to be taken into account . F~eferences 1.
~` . HEhLE, G. HENLE, F. DEIIJEiAFcDT and V. Y . HEFiGS, J. Exp . D~ed .
110, 525
(1959) "
2.
L. A . GLASGOw and K. HAGEL, J. Exp . ~ed . 11
3.
L. A . GIASGOW and K. HAGEL, Virolow l~, 328
4.
D . STANL~EK, Acta viral . (Prague)
3,
,
509 (1962) . (l960 .
225 (1963) .
No . 12
IINRE3PON3NENE33 TO INTERFERON ACTION
901
5.
J. VI?~EK and D. STANL~EK, Acta virol .
6.
& . R . WA($dER, Virolo~ ~,
?"
C . COCITO, E . DE MAEZER and P . DE SOAR, Life Sciences
(PraRUe)
323 (1961) .
3, 331 (1963) .
l, 753 (15x'2) . 8.
D . STdNL~EK and J. VIT.~EK, in preparation .
9.
A . ISAACS, R. A. COS and Z. ROTES, Lancet
10 .
ü, 113 (1963) "
E . DE MAEYh~t and J. DE MAE]~R-GUIQdARD, Yirolo~y 20,
(1963) . 11 .
E . HELhER, Biochem. J. ~, 18 P
12 .
S . HERI9~DSSON, ViroloAV 20,
(1963) "
333 (1963) "
536