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Inability of measles virus temperature-sensitive mutants to induce interferon
VIROLOGY
76, 409-415
Inability
(1977)
of Measles
Virus Temperature-Sensitive Interferon
JENNIFER Department
of Microbiology,
McKIMM
AND
FRED
The Milton S. Hershey Medical Center, College of Medicine, Hershey, Pennsylvania Accepted
September
Mutants
to induce
RAPP’ The Pennsylvania 17033
State
University,
7,1976
The interferon (IF)-inducing ability of temperature-sensitive mutants of measles virus, and a revertant selected from one of these, was tested in BSC-1 and green monkey kidney cells. Under conditions shown to yield significant levels of interferon with the parental virus, no detectable levels of IF were induced with the mutants or the revertant under conditions permissive or nonpermissive for virus replication. Input multiplicity affected the level of induction with the parental virus, but there was no correlation between virus yields and the amount of IF produced. Lack of induction could not be explained by low virus yields for the mutants or revertant, as these were comparable to those of the parental virus achieved in the permissive system. Complementation between the various groups of mutants and the revertant failed to induce IF under either permissive or nonpermissive conditions. The possibility of positive control over induction was examined by coinfecting with the inducing parental virus and the noninducers; no such control was demonstrated, for levels produced were similar to that obtained with the parental virus alone. These results suggest that a function which is required for interferon induction is either lost during mutagenesis or is absent in otherwise functional measles virions.
permissive conditions. Edmonston and Schwarz strains of measles virus have demonstrated the ability to induce interferon synthesis in vitro (Ho and Enders, 1959; de Maeyer and Enders, 1961, 1965; Enders, 1962; Anderson and Atherton, 1964; Mirchamsy and Rapp, 1969). This property of the wild-type, therefore, was used to investigate these measles virus ts mutants and the revertant in an effort to determine a correlation between virus phenotype and the ability to induce interferon.
INTRODUCTION
Previous reports from this laboratory have described the isolation and properties of temperature-sensitive (ts) mutants of measles virus (Haspel et al., 1975a,b). A recent report has also described the isolation and characterization of a t.s+ revertant from one of the three complementation groups (Breschkin et al., 1976). Temperature-sensitive mutants of reovirus (Lai and Joklik, 1973), Semliki Forest virus (Lomniczi and Burke, 1970), and Sindbis virus (Lockart et al., 1968; Atkins and Lancashire, 1976) have been used in attempts to understand the mechanism by which virus-induced interferon synthesis occurs. These mutants, which have all been shown to induce interferon (IF) at permissive temperatures, apparently vary in their capacity to induce IF under non’ Author addressed.
to whom
requests
for reprints
should
MATERIALS
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Cells and media. BSC-1 cells, originally obtained from R. Dulbecco (Salk Institute), were grown in Eagle’s medium as described by Haspel et al. (1975b). Green monkey kidney (GMK) cells (Flow Laboratories) were nassed once in Eagle’s medium before -use in petri dish cultures. Growth medium for both cells was medium
be 409
Copyright All rights
AND
410
McKIMM
199 supplemented with 10% fetal calf serum (FCS), 10% tryptose phosphate broth (TPB), and 2 mg/ml of NaHC03. Cultures were incubated in a humidified atmosphere of 5% COZ. For induction and assay of interferon, maintenance medium consisted of medium 199 with 2% FCS and 2% TPB. All media contained 1 pg/ml of fungizone, 5 U/ml of mycostatin, 100 U/ml of penicillin, and 100 pg/ml of streptomycin. Viruses. Derivation of the parental CC virus (Haspel et al., 1973) and properties of the mutants selected from CC and Schwarz viruses have been described previously (Haspel et al., 1975b). Isolation and characterization of the ts+ revertant G-9 of ts G-3 have recently been reported by Breschkin et al. (1976). Edmonston virus and its mutants were generously made available by F. Payne (University of Michigan). Vesicular stomatitis virus (VSV; Indiana strain) was used for interferon assays. Induction of interferon. Cells were grown to confluency and aged overnight. Following removal of culture fluid, the cells were infected with virus at the desired multiplicity. Virus was adsorbed for 90 min at room temperature. Without removing the inoculum, 2.5 ml of maintenance medium was added and the cells were incubated at the required temperature. Following the appropriate time for development of cytopathic effect (CPE), the fluids were removed, neutralized with antiserum, and stored at 4” overnight or until assayed. Cells were suspended in 2.5 ml of medium, sonicated for 45 set, and quick-frozen in a dry ice-alcohol bath and stored at -70”. Samples were sonicated briefly prior to assay. Assay of interferon. To determine the interferon titer, 2 ml of serial twofold dilutions in maintenance medium was inoculated onto confluent monolayers of BSC-1 cells in petri dishes (60 mm). After overnight incubation, the medium was removed and each culture was challenged with approximately 70 PFU of VSV. After a 1-hr adsorption period at room temperature, approximately 6 ml of overlay was added (Rapp, 1964) and the cells were incu-
AND
RAPP
bated for 24 hr at 37”. A second overlay containing 0.02% neutral red was then added, and plaques were counted after overnight incubation. One IF unit, PRDSO, was defined as the reciprocal of the highest dilution that produced a 50% reduction in the number of plaques in the control dishes. An internal standard was included in each assay. Yields are expressed as the total from each 60-mm dish. Complementation. Combinations of sonicated virus were incubated at room temperatures for 30 min in the presence of 10 pglml of poly-L-ornithine (mol wt > 160,000). The treated viruses were inoculated onto confluent monolayers of BSC-1 cells previously washed with Tris at an input multiplicity of 0.1 to 1. RESULTS
Interferon Virus
Induction
by Parental
Measles
Since input multiplicity is known to influence the yield of interferon (Lai and Joklik, 1973; Atkins et al., 1974), the effect of this variable on BSC-1 cells infected with the parental strain was determined. Fluids and cells were harvested after 55 hr at 33” when all cultures showed some cytopathology. Results are given in Table 1. TABLE
1
EFFECT OF INPUT MULTIPLICITY OF PARENTAL MEASLES (CC) VIRUS ON INTERFERON YIELDSO m.o.i.b 0.01 0.1 1.0
IF yield PRD,’ 20 80-160 160-320
in
Virus yield (PFU/mBd 4 x 105 5 x 105 4 x 104
a Data represent results from two separate experiments. In both, duplicate cultures were infected with the required multiplicities. Interferon yields were determined by serial twofold dilutions of the fluids. Each dilution was tested in duplicate. Virus yields were also titrated in duplicate. b Multiplicity of infection. c One PRD,, is defined as the reciprocal of the highest dilution producing a 50% reduction in the number of plaques in control dishes. Interferon yields are expressed as total from each infected 60mm dish. d Cells were suspended in 2.5 ml of medium for determination of virus yields.
MEASLES
ts MUTANTS
Interferon was induced at an input multiplicity as low as 0.01. Because low titers of stocks prevented higher routine input, an input of 0.1 was used in all subsequent experiments. The interferon was stable at pH 2. Yields were the same, independent of the method of neutralizing the inducing virus, i.e., by dropping the pH to 2, irradiating with ultraviolet light, or adding antiserum. The interferon is stable at neutral pH for months when stored at 4”. For these reasons, the interfering substance was considered to be interferon. The time course, associated CPE, and interferon induction were followed to determine whether there is any correlation between these events, as has been reported by Atkins and Lancashire (1976) with Sindbis mutants. The parental CC virus was assayed at 12 hourly intervals at 33”. Results are shown in Table 2. Once CPE was apparent, interferon was detectable in the medium. Maximum production of interferon was achieved by the time the cells demonstrated approximately 50% CPE. The interferon levels were then stable for at least a further 24 to 36 hr. Duplicate cultures were infected at a multiplicity of infection (m.o.i.1 of 0.1 with a member of a complementation group or the revertant, and were then incubated at 33, 37, and 39”. Fluids were harvested at 24, 48, and 72 hr, and were titrated in duplicate for interferon production. Some TABLE RESULTS Hours 12 24 36 48 60 72
OF THE TIME
COURSE
AND
CPE was observed within 48 hr in all cultures incubated at 33 and 37”, except those inoculated with tsT. Following 72 hr of incubation at 33 and 37”, the tsT-inoculated cells also exhibited CPE. At all times and temperatures, levels of interferon induced by the mutants or revertant were below the sensitivity of the assay system. These results are in contrast to previously reported observations with temperaturesensitive mutants of Sindbis virus (Lockart et al., 1968; Atkins et al., 1974; Atkins and Lancashire, 1976), Semliki Forest virus (Lomniczi and Burke, 1970), and reovirus (Lai and Joklik, 1973). Although these mutants varied in their ability to induce interferon at nonpermissive temperatures, they all induced interferon under conditions permissive for virus replication. These measles mutants thus exhibit an unusual property not previously described for any other ts mutants. Yield
of Virus and Relation to Interferon Induction The possibility that yields of virus were insufficient to induce detectable levels of interferon was next investigated. Lai and Joklik (1973) discovered a correlation between the amount of interferon induced and virus yields for reovirus, but no such relation was found for Sindbis virus (Atkins and Lancashire, 1976). Results, which agree with those of Atkins and Lancashire, are presented in Table 3. There is no ap2
AND ASSOCIATED CYTOPATHOLOGY COMPARED FOR THE PARENTAL VIRUS”,~ CPE’
- No visible CPE - No visible CPE + 25% Small syncytia ++ (25-50% CPE) ++ (25-50% CPE) +++ Cells starting slough (>75% CPE)
IF yield
to
411
INTERFERON
in PRD,,” ‘3 <5 20 40-80 40-80 40-80
TO INTERFERON
PRODUCTION
Virus yields (PFU/mlY 1.7 9.0 7.0 5.0 2.0 4.0
n Two cultures were taken for assay of interferon and virus yields at each time interval. titrated in duplicate for determining both interferon and virus yields. An input multiplicity b Results using parental virus at 33”. e Cytopathic effect. d Interferon yields are expressed as total from each infected 60-mm dish. c Cells were suspended in 2.5 ml of medium for determination of virus yields.
x x x x x x
lo’ 10s 10” lo” 106 10”
Samples were of 0.1 was used.
412
McKIMM
AND TABLE
OF VIRUS
YIELDS
AND
RELATION
RAPP 3
TO INTERFERON
Strain
33" IF Parental Schwarz tST
(CC)
tsc
tsG-3 Revertant
INDUCTION’.
*
Yields
G-9
37"
39
Virus
IF
Virus
40-80 320 c2 <2 <2 <2
4 x 106 9 x 106
20-40
5 x 106
10-20
6 x lo6
<2 <2 <2 <2
1 x 106 1 x 106 7 x 106 6 x 10’
1 x 4 x 5 x 6x
IF
20-40 20-40 <2 <2 c2 c2
103 105 106 lo5
a Interferon yields expressed as PRD,, from each 60-mm plate. Under permissive are comparable for the mutants, revertant, and parental virus, but no interferon mutants or revertant. * Duplicate cultures were infected at an input multiplicity of 0.1, approximately harvested when cells showed approximately 50% CPE and were titrated in duplicate suspended in 2.5 ml of medium and assayed in duplicate for virus yields.
parent relationship between virus yield and interferon induction. Yields of the mutants and revertant under permissive conditions were comparable to yields of wild-type virus (see also Tables 1 and 2), but no interferon was detected for these mutants or for the revertant. Mutants T, C, and G-3 represent three different complementation groups (Haspel et al., 1975b). To determine whether this is a host celldependent phenomenon, 14 of the attenuated group 1 mutants, two from group 2, and the group 3 mutant and its revertant were tested for their ability to induce IF in GMK cells. Included in these mutants are some which are known to be leaky and/or which have a high reversion frequency. Results are presented in Table 4. As in BSC-1 cells, no detectable levels of interferon were induced. Mutants with high reversion frequencies or leaky mutants have been shown to induce IF under nonpermissive conditions with both reovirus (Lai and Joklik, 1973) and Sindbis virus (Lomniczi and Burke, 1970). In our experiments, IF was not induced, even under permissive conditions. Three mutants (Edmonston 154/798/841) (Bergholz et al., 1975) obtained from F. Payne were also negative under both permissive and nonpermissive conditions. Complementation
Analysis
To determine whether more than one virus gene is involved in induction, combi-
3
OF INTERFBRON
MUTANTS
IN GMK
Mutants CC (parental) T
C G-3 G-9
B, H, J, K, L, M, P, Q, R,
2 x 106 5 x
106 10’ 104 102 106
conditions, virus yields was detected with the
TABLE INDUCTION
Virus
x lo4 PFU. Fluids for IF yields. Cells
were were
4 BY MEASLES
VIRUS
CELL@~
33"
39"
40-80 <5 <5 <5 <5 <5
40-80 <5 <5 <5 <5 <5
<5
<5
40 <5
40-80 <5
40 <5
40-80 <5
'I', U N Schwarz A Edmonston 154/798/841
(parental)
(parental)
a Interferon yields expressed as PRD,, from each 60-mm dish of green monkey kidney cells tested in BSC-1 cells. Each grouping represents the parental strain and mutants derived from it. b Duplicate cultures were infected with an input multiplicity of 0.1, approximately 3 x lo4 PFU. Fluids were removed when cells demonstrated approximately 50% CPE and were titrated in duplicate for interferon yields.
nations of mutants from each of the three groups and the revertant were inoculated onto BSC-1 cells and the fluids were tested for IF induction. Mutants T and G-3 and revertant G-9 were inoculated with an approximate m.o.i. of 1 at 39” and 0.5 at 33”. Because of low titers of tsC stocks, this mutant was tested only at 33” in order that
MEASLES
ts MUTANTS
complementation could occur during subsequent rounds of replication. The observation that the mutants are mutually complementary (Haspel et al., 1975b) was confirmed in this study. Little CPE was seen under nonpermissive conditions in cells infected with a single mutant. Multinucleated syncytia were observed with the G-31 T combination at 39”, and virus yields were lOOO-fold higher than yields obtained with each mutant alone. With none of the combinations was interferon detected under either permissive or nonpermissive conditions. Complementation Noninducer
between an Inducer
and
The possibility of positive control over interferon induction by the measles mutants and revertant was investigated by coinfecting cells with the inducing parental strain and with the noninducing mutants and revertant. An input multiplicity of approximately 1 was used for all but the group 2 mutant C, in which case only 0.1 could be used. In the combinations CC/T, CC/G-3, and CC/G-g, levels of interferon induction (i.e., approximately 80 units) at 33” were similar to that induced by the parental virus alone (Table 5). An insignificant twofold decrease was observed with the CC/C combination. [This may be due to a high number of defectives in the tsC stock (-2OO:l by hemagglutination)l. Thus, it appears that noninducing mutants lack positive control over interferon induction. DISCUSSION
The results presented here have demonstrated two interesting points: (1) no detectable levels of IF are induced by mutants of measles virus in BSC-1 or GMK cells under permissive or nonpermissive conditions; and (2) the ts+ revertant, which otherwise demonstrates characteristics similar to the parental strain (Breschkin et al., 1976), does not induce interferon in BSC-1 or GMK cells. For the parental virus, induction of IF was dependent on both time of incubation and multiplicity of infection. Infection for various times and multiplicities which had
AND
413
INTERFERON TABLE
INTERFERON PRODUCTION MEASLES VIRUS MUTANTS
WITH COMBINATIONS OF AND PARENTAL CC VIRUS
0
33”
Virus
combina-
5
IF yields
as PRD,
IF yieldz’as
timso
PRD,, ____~
T C G-3 TIC T/G-3 C/G-3 G-9/G-3
80-160 <5 <5 <5 <5 <5 <5 <5
80-160 <5 <5 <5 NDh <5 ND <5
CC/T cc/c CC/G-3 CC/G-9
80-160 40-80 80-160 80-160
ND ND ND ND
cc
-~ o Combinations of viruses were incubated with poly-L-ornithine and inoculated onto cultures in 60mm BSC-1 plates. Fluids were harvested when cells showed approximately 50% CPE. (Maximum levels are induced by the CC parental virus by this time.) * Not done.
been shown to induce significant levels by the parental strain failed to produce any detectable interferon with the mutants or revertant. A correlation between virus yields and interferon production was found for reovirus and its ts mutants (Lai and Joklik, 1973). Atkins et aE. (1974), however, were unable to show this relationship with Sindbis virus and its ts mutants. No correlation between the virus yields and interferon production was found for the parental measles virus or its mutants. Yields of virus under permissive conditions were comparable for all, but only the parental virus induced interferon. That this is not a host cell-dependent phenomenon was demonstrated by testing in GMK cells 14 group 1 mutants, all available group 2 and 3 mutants, and a group 3 revertant. Several of the mutants are known to be leaky or to have fairly high reversion frequencies. Interferon induction under nonpermissive conditions for reovirus (Lai and Joklik, 1973) and Sindbis virus (Lomniczi and Burke, 1970) ts mutants may occur because of a leaky mutation or high reversion frequency. Under nonpermissive conditions and sufficient time for the production of wild-type
414
McKIMM
revertants, or under permissive conditions, none of these measles mutants induced interferon. Complementation between reovirus mutants under restrictive conditions was shown to increase approximately lo-fold the level of interferon produced (Lai and Joklik, 1973). When complementation tests were carried out between the three groups and the revertant measles virus, complementation occurred; IF was not produced, however, even when assayed under conditions permissive for the replication of the mutants. Another paramyxovirus, SV-5, has been shown to inhibit interferon induction and action in primary rhesus monkey kidney cells and in the MDBK line of bovine kidney cells (Choppin and Compans, 1975). No interferon is produced in SV-5 infection of these cells. In addition, super-infection of SV&infected cells with a virus normally able to induce interferon fails to yield IF. However, defective SV-5 particles are able to induce IF and have no effect on the action of preformed IF. Choppin and Compans suggest that this inhibition is due to some form of positive control by the SV-5 genome, and that this is lost by the defective virions. Coinfection with the inducing parental measles virus and noninducing mutants and revertant had no effect on induction by the parental virus. Levels of interferon achieved in cultures infected with combinations of these viruses were similar to those obtained when the parental virus alone was used. Thus, we conclude that the mutants have no repressor effect on the induction of IF. The original parental CC virus, from which all mutants except tsA were selected, was isolated by cocultivation with BSC-1 cells from hamster cells latently infected with passaged Schwarz vaccine strain. It could be argued that such a passage history resulted in some change in the virus, and that the ts mutants may have been selected from noninducers even before mutagenesis. This characteristic would therefore not reappear, as with G-9, upon reversion to the wild-type. However, tsA was derived directly from Schwarz virus passed three times in Vero cells, and
AND
RAPP
preliminary evidence indicates that mutants derived from Edmonston vaccine strain share the properties of these CCand Schwarz-derived mutants. This unusual property is therefore not unique to the CC-derived mutants. Although much attention has been focused on the mechanism by which virusinduced interferon synthesis occurs, little is known concerning the role of the virus genotype. Double-stranded RNA was thought to play an important role in the induction process (de Clercq and Merigan, 1970; Kleinschmidt, 1972). However, use of ts mutants of Sindbis virus (Lockart et al., 1968; Atkins et al., 1974; Atkins and Lancashire, 1976), and Semliki Forest virus (Lomniczi and Burke, 1970) has failed to show a consistent relationship between the amount of double-stranded RNA produced and the amount of interferon induced. Lai and Joklik (1973) found that induction with reovirus ts mutants correlated only with some late function resulting in the formation of intact virions. Under conditions permissive for the production of infectious progeny, the mutants and revertant must produce some double-stranded RNA. Since interferon is not induced, other factors must be important. This agrees with results obtained from a Newcastle disease virus mutant. Thacore and Youngner (1970) found no correlation for this virus between virus RNA synthesis and the induction of interferon synthesis. Genetic studies are now underway to detect possible differences in the parental strains which may have been present even prior to mutagenesis. This implies that virus stocks contain infectious virions with the ability to induce interferon in addition to other virions without this property. Preliminary results (Rapp and McKimm, unpublished observations) seem to support this concept. The results already obtained suggest that interferon induction is at least partially under the genetic control of the inducing virus. ACKNOWLEDGMENTS Preliminary experiments leading to this study were conducted by Dr. P. Knight. Discussions with Dr. A. Breschkin and Dr. E. Morgan were very
MEASLES
ts MUTANTS
helpful. The technical assistance of Barbara Walmer is greatly appreciated. This investigation was supported by Grant Number AI 12203, awarded by the National Institute of Allergy and Infectious Diseases, DHEW. J. M. is a recipient of a postgraduate fellowship from the Australian-American Educational Foundation.
REFERENCES C. D., and ATHERTON, J. G. (19641. Effect of actinomycin D on measles virus growth and interferon production. Nature (London) 203, 671. ATKINS, G. J., and LANCASHIRE, C. L. (1976). The induction of interferon by temperature-sensitive mutants of Sindbis virus: Its relationship to double-stranded RNA synthesis and cytopathic effect. J. Gen. Viral. 30, 157-165. ATKINS, G. J., JOHNSTON, M. D., WESTMACOTT, L. M., and BURKE, P. C. (1974). Induction of interferon in chick cells by temperature-sensitive mutants of Sindbis virus. J. Gen. Virol. 25, 381-390. BERCHOLZ, C. M., KILEY, M. P., and PAYNE, F. E. (19751. Isolation and characterization of temperature-sensitive mutants of measles virus. J. Viral. 16, 192-202. BRESCHKIN, A. M., HASPEL, M. V., and RAPP, F. (1976). Neurovirulence and induction of hydrocephalus with parental, mutant, and revertant strains of measles virus. J. Virol. 18, 809-811. CHOPPIN, P. W., and COMPANS, R. W. (1975). Reproduction of paramyxo-viruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. W. Wagner, eds.), pp. 95-178. Academic Press, New York. DE CLERCQ, E., and MERIGAN, T. C. (19701. Current concepts of interferon and interferon induction. Annu. Rev. Med. 21, 17-46. DE MAEYER, E., and ENDERS, J. F. (19611. An interferon appearing in cell cultures infected with measles virus. Proc. Sot. Exp. Biol. Med. 107,573578. DE MAEYER, E., and ENDERS, J. F. (19651. Growth characteristics, interferon production and plaque formation with different lines of Edmonston meaANDERSON,
AND
INTERFERON
415
sles virus. Arch. Gesamte Virusforsch. 16, 151160. ENDERS, J. F. (19621. Measles virus. Amer. J. Dis. Child. 103, 282-287. HASPEL, M. V., DUFF, R., and RAPP, F. (1975ai. Experimental measles encephalitis: A genetic analysis. Znfec. Immun. 12, 787-790. HASPEL, M. V., DUFF, R., and RAPP, F. (1975b). Isolation and preliminary characterization of temperature-sensitive mutants of measles virus. J. Virol. 16, 1000-1009. HASPEL, M. V., KNIGHT, P. R., DUFF, R. G., and RAPP, F. (1973). Activation of a latent measles virus infection in hamster cells. J. Virol. 12, 690695. Ho, M., and ENDERS, J. F. (19591. Further studies on an inhibition of viral activity appearing in infected cell cultures and its role in chronic viral infections. Virology 1, 446-477. KLEINSCHMIIYP, W. J. (19721. Biochemistry of interferon and its inducers. Annu. Rev. Biochem. 41, 517-542. LAI, M. T., and JOKLIK, W. K. (19731. The induction of interferon by temperature-sensitive mutants of reovirus, UV-irradiated reovirus, and subviral reovirus particles. Virology 51, 191-204. LOCKART, R. Z., JR., BAYLISS, N. L., TOY, S. T., and YIN, F. H. (1968). Viral events necessary for the induction of interferon in chick embryo cells. J. Viral. 2, 962-965. LOMNICZI, B., and BURKE, D. C. (19701. Interferon production by temperature-sensitive mutants of Semliki Forest virus. J. Gen. Virol. 8, 55-68. MIRCHAMSY, H., and RAPP, F. (1969). Role of interferon in replication of virulent and attenuated strains of measles virus. J. Gen. Virol. 4, 513-522. RAPP, F. (1964). Plaque differentiation and replication of virulent and attenuated strains of measles virus. J. Bacterial. 88, 1448-1458. THACORE, M., and YOUNGNER, J. S. (1970). Cells persistently infected with Newcastle disease virus. II. Ribonucleic and protein synthesis in cells infected with mutants isolated from persistently infected L cells. J. Viral. 6, 42-48.
76, 409-415
Inability
(1977)
of Measles
Virus Temperature-Sensitive Interferon
JENNIFER Department
of Microbiology,
McKIMM
AND
FRED
The Milton S. Hershey Medical Center, College of Medicine, Hershey, Pennsylvania Accepted
September
Mutants
to induce
RAPP’ The Pennsylvania 17033
State
University,
7,1976
The interferon (IF)-inducing ability of temperature-sensitive mutants of measles virus, and a revertant selected from one of these, was tested in BSC-1 and green monkey kidney cells. Under conditions shown to yield significant levels of interferon with the parental virus, no detectable levels of IF were induced with the mutants or the revertant under conditions permissive or nonpermissive for virus replication. Input multiplicity affected the level of induction with the parental virus, but there was no correlation between virus yields and the amount of IF produced. Lack of induction could not be explained by low virus yields for the mutants or revertant, as these were comparable to those of the parental virus achieved in the permissive system. Complementation between the various groups of mutants and the revertant failed to induce IF under either permissive or nonpermissive conditions. The possibility of positive control over induction was examined by coinfecting with the inducing parental virus and the noninducers; no such control was demonstrated, for levels produced were similar to that obtained with the parental virus alone. These results suggest that a function which is required for interferon induction is either lost during mutagenesis or is absent in otherwise functional measles virions.
permissive conditions. Edmonston and Schwarz strains of measles virus have demonstrated the ability to induce interferon synthesis in vitro (Ho and Enders, 1959; de Maeyer and Enders, 1961, 1965; Enders, 1962; Anderson and Atherton, 1964; Mirchamsy and Rapp, 1969). This property of the wild-type, therefore, was used to investigate these measles virus ts mutants and the revertant in an effort to determine a correlation between virus phenotype and the ability to induce interferon.
INTRODUCTION
Previous reports from this laboratory have described the isolation and properties of temperature-sensitive (ts) mutants of measles virus (Haspel et al., 1975a,b). A recent report has also described the isolation and characterization of a t.s+ revertant from one of the three complementation groups (Breschkin et al., 1976). Temperature-sensitive mutants of reovirus (Lai and Joklik, 1973), Semliki Forest virus (Lomniczi and Burke, 1970), and Sindbis virus (Lockart et al., 1968; Atkins and Lancashire, 1976) have been used in attempts to understand the mechanism by which virus-induced interferon synthesis occurs. These mutants, which have all been shown to induce interferon (IF) at permissive temperatures, apparently vary in their capacity to induce IF under non’ Author addressed.
to whom
requests
for reprints
should
MATERIALS
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Cells and media. BSC-1 cells, originally obtained from R. Dulbecco (Salk Institute), were grown in Eagle’s medium as described by Haspel et al. (1975b). Green monkey kidney (GMK) cells (Flow Laboratories) were nassed once in Eagle’s medium before -use in petri dish cultures. Growth medium for both cells was medium
be 409
Copyright All rights
AND
410
McKIMM
199 supplemented with 10% fetal calf serum (FCS), 10% tryptose phosphate broth (TPB), and 2 mg/ml of NaHC03. Cultures were incubated in a humidified atmosphere of 5% COZ. For induction and assay of interferon, maintenance medium consisted of medium 199 with 2% FCS and 2% TPB. All media contained 1 pg/ml of fungizone, 5 U/ml of mycostatin, 100 U/ml of penicillin, and 100 pg/ml of streptomycin. Viruses. Derivation of the parental CC virus (Haspel et al., 1973) and properties of the mutants selected from CC and Schwarz viruses have been described previously (Haspel et al., 1975b). Isolation and characterization of the ts+ revertant G-9 of ts G-3 have recently been reported by Breschkin et al. (1976). Edmonston virus and its mutants were generously made available by F. Payne (University of Michigan). Vesicular stomatitis virus (VSV; Indiana strain) was used for interferon assays. Induction of interferon. Cells were grown to confluency and aged overnight. Following removal of culture fluid, the cells were infected with virus at the desired multiplicity. Virus was adsorbed for 90 min at room temperature. Without removing the inoculum, 2.5 ml of maintenance medium was added and the cells were incubated at the required temperature. Following the appropriate time for development of cytopathic effect (CPE), the fluids were removed, neutralized with antiserum, and stored at 4” overnight or until assayed. Cells were suspended in 2.5 ml of medium, sonicated for 45 set, and quick-frozen in a dry ice-alcohol bath and stored at -70”. Samples were sonicated briefly prior to assay. Assay of interferon. To determine the interferon titer, 2 ml of serial twofold dilutions in maintenance medium was inoculated onto confluent monolayers of BSC-1 cells in petri dishes (60 mm). After overnight incubation, the medium was removed and each culture was challenged with approximately 70 PFU of VSV. After a 1-hr adsorption period at room temperature, approximately 6 ml of overlay was added (Rapp, 1964) and the cells were incu-
AND
RAPP
bated for 24 hr at 37”. A second overlay containing 0.02% neutral red was then added, and plaques were counted after overnight incubation. One IF unit, PRDSO, was defined as the reciprocal of the highest dilution that produced a 50% reduction in the number of plaques in the control dishes. An internal standard was included in each assay. Yields are expressed as the total from each 60-mm dish. Complementation. Combinations of sonicated virus were incubated at room temperatures for 30 min in the presence of 10 pglml of poly-L-ornithine (mol wt > 160,000). The treated viruses were inoculated onto confluent monolayers of BSC-1 cells previously washed with Tris at an input multiplicity of 0.1 to 1. RESULTS
Interferon Virus
Induction
by Parental
Measles
Since input multiplicity is known to influence the yield of interferon (Lai and Joklik, 1973; Atkins et al., 1974), the effect of this variable on BSC-1 cells infected with the parental strain was determined. Fluids and cells were harvested after 55 hr at 33” when all cultures showed some cytopathology. Results are given in Table 1. TABLE
1
EFFECT OF INPUT MULTIPLICITY OF PARENTAL MEASLES (CC) VIRUS ON INTERFERON YIELDSO m.o.i.b 0.01 0.1 1.0
IF yield PRD,’ 20 80-160 160-320
in
Virus yield (PFU/mBd 4 x 105 5 x 105 4 x 104
a Data represent results from two separate experiments. In both, duplicate cultures were infected with the required multiplicities. Interferon yields were determined by serial twofold dilutions of the fluids. Each dilution was tested in duplicate. Virus yields were also titrated in duplicate. b Multiplicity of infection. c One PRD,, is defined as the reciprocal of the highest dilution producing a 50% reduction in the number of plaques in control dishes. Interferon yields are expressed as total from each infected 60mm dish. d Cells were suspended in 2.5 ml of medium for determination of virus yields.
MEASLES
ts MUTANTS
Interferon was induced at an input multiplicity as low as 0.01. Because low titers of stocks prevented higher routine input, an input of 0.1 was used in all subsequent experiments. The interferon was stable at pH 2. Yields were the same, independent of the method of neutralizing the inducing virus, i.e., by dropping the pH to 2, irradiating with ultraviolet light, or adding antiserum. The interferon is stable at neutral pH for months when stored at 4”. For these reasons, the interfering substance was considered to be interferon. The time course, associated CPE, and interferon induction were followed to determine whether there is any correlation between these events, as has been reported by Atkins and Lancashire (1976) with Sindbis mutants. The parental CC virus was assayed at 12 hourly intervals at 33”. Results are shown in Table 2. Once CPE was apparent, interferon was detectable in the medium. Maximum production of interferon was achieved by the time the cells demonstrated approximately 50% CPE. The interferon levels were then stable for at least a further 24 to 36 hr. Duplicate cultures were infected at a multiplicity of infection (m.o.i.1 of 0.1 with a member of a complementation group or the revertant, and were then incubated at 33, 37, and 39”. Fluids were harvested at 24, 48, and 72 hr, and were titrated in duplicate for interferon production. Some TABLE RESULTS Hours 12 24 36 48 60 72
OF THE TIME
COURSE
AND
CPE was observed within 48 hr in all cultures incubated at 33 and 37”, except those inoculated with tsT. Following 72 hr of incubation at 33 and 37”, the tsT-inoculated cells also exhibited CPE. At all times and temperatures, levels of interferon induced by the mutants or revertant were below the sensitivity of the assay system. These results are in contrast to previously reported observations with temperaturesensitive mutants of Sindbis virus (Lockart et al., 1968; Atkins et al., 1974; Atkins and Lancashire, 1976), Semliki Forest virus (Lomniczi and Burke, 1970), and reovirus (Lai and Joklik, 1973). Although these mutants varied in their ability to induce interferon at nonpermissive temperatures, they all induced interferon under conditions permissive for virus replication. These measles mutants thus exhibit an unusual property not previously described for any other ts mutants. Yield
of Virus and Relation to Interferon Induction The possibility that yields of virus were insufficient to induce detectable levels of interferon was next investigated. Lai and Joklik (1973) discovered a correlation between the amount of interferon induced and virus yields for reovirus, but no such relation was found for Sindbis virus (Atkins and Lancashire, 1976). Results, which agree with those of Atkins and Lancashire, are presented in Table 3. There is no ap2
AND ASSOCIATED CYTOPATHOLOGY COMPARED FOR THE PARENTAL VIRUS”,~ CPE’
- No visible CPE - No visible CPE + 25% Small syncytia ++ (25-50% CPE) ++ (25-50% CPE) +++ Cells starting slough (>75% CPE)
IF yield
to
411
INTERFERON
in PRD,,” ‘3 <5 20 40-80 40-80 40-80
TO INTERFERON
PRODUCTION
Virus yields (PFU/mlY 1.7 9.0 7.0 5.0 2.0 4.0
n Two cultures were taken for assay of interferon and virus yields at each time interval. titrated in duplicate for determining both interferon and virus yields. An input multiplicity b Results using parental virus at 33”. e Cytopathic effect. d Interferon yields are expressed as total from each infected 60-mm dish. c Cells were suspended in 2.5 ml of medium for determination of virus yields.
x x x x x x
lo’ 10s 10” lo” 106 10”
Samples were of 0.1 was used.
412
McKIMM
AND TABLE
OF VIRUS
YIELDS
AND
RELATION
RAPP 3
TO INTERFERON
Strain
33" IF Parental Schwarz tST
(CC)
tsc
tsG-3 Revertant
INDUCTION’.
*
Yields
G-9
37"
39
Virus
IF
Virus
40-80 320 c2 <2 <2 <2
4 x 106 9 x 106
20-40
5 x 106
10-20
6 x lo6
<2 <2 <2 <2
1 x 106 1 x 106 7 x 106 6 x 10’
1 x 4 x 5 x 6x
IF
20-40 20-40 <2 <2 c2 c2
103 105 106 lo5
a Interferon yields expressed as PRD,, from each 60-mm plate. Under permissive are comparable for the mutants, revertant, and parental virus, but no interferon mutants or revertant. * Duplicate cultures were infected at an input multiplicity of 0.1, approximately harvested when cells showed approximately 50% CPE and were titrated in duplicate suspended in 2.5 ml of medium and assayed in duplicate for virus yields.
parent relationship between virus yield and interferon induction. Yields of the mutants and revertant under permissive conditions were comparable to yields of wild-type virus (see also Tables 1 and 2), but no interferon was detected for these mutants or for the revertant. Mutants T, C, and G-3 represent three different complementation groups (Haspel et al., 1975b). To determine whether this is a host celldependent phenomenon, 14 of the attenuated group 1 mutants, two from group 2, and the group 3 mutant and its revertant were tested for their ability to induce IF in GMK cells. Included in these mutants are some which are known to be leaky and/or which have a high reversion frequency. Results are presented in Table 4. As in BSC-1 cells, no detectable levels of interferon were induced. Mutants with high reversion frequencies or leaky mutants have been shown to induce IF under nonpermissive conditions with both reovirus (Lai and Joklik, 1973) and Sindbis virus (Lomniczi and Burke, 1970). In our experiments, IF was not induced, even under permissive conditions. Three mutants (Edmonston 154/798/841) (Bergholz et al., 1975) obtained from F. Payne were also negative under both permissive and nonpermissive conditions. Complementation
Analysis
To determine whether more than one virus gene is involved in induction, combi-
3
OF INTERFBRON
MUTANTS
IN GMK
Mutants CC (parental) T
C G-3 G-9
B, H, J, K, L, M, P, Q, R,
2 x 106 5 x
106 10’ 104 102 106
conditions, virus yields was detected with the
TABLE INDUCTION
Virus
x lo4 PFU. Fluids for IF yields. Cells
were were
4 BY MEASLES
VIRUS
CELL@~
33"
39"
40-80 <5 <5 <5 <5 <5
40-80 <5 <5 <5 <5 <5
<5
<5
40 <5
40-80 <5
40 <5
40-80 <5
'I', U N Schwarz A Edmonston 154/798/841
(parental)
(parental)
a Interferon yields expressed as PRD,, from each 60-mm dish of green monkey kidney cells tested in BSC-1 cells. Each grouping represents the parental strain and mutants derived from it. b Duplicate cultures were infected with an input multiplicity of 0.1, approximately 3 x lo4 PFU. Fluids were removed when cells demonstrated approximately 50% CPE and were titrated in duplicate for interferon yields.
nations of mutants from each of the three groups and the revertant were inoculated onto BSC-1 cells and the fluids were tested for IF induction. Mutants T and G-3 and revertant G-9 were inoculated with an approximate m.o.i. of 1 at 39” and 0.5 at 33”. Because of low titers of tsC stocks, this mutant was tested only at 33” in order that
MEASLES
ts MUTANTS
complementation could occur during subsequent rounds of replication. The observation that the mutants are mutually complementary (Haspel et al., 1975b) was confirmed in this study. Little CPE was seen under nonpermissive conditions in cells infected with a single mutant. Multinucleated syncytia were observed with the G-31 T combination at 39”, and virus yields were lOOO-fold higher than yields obtained with each mutant alone. With none of the combinations was interferon detected under either permissive or nonpermissive conditions. Complementation Noninducer
between an Inducer
and
The possibility of positive control over interferon induction by the measles mutants and revertant was investigated by coinfecting cells with the inducing parental strain and with the noninducing mutants and revertant. An input multiplicity of approximately 1 was used for all but the group 2 mutant C, in which case only 0.1 could be used. In the combinations CC/T, CC/G-3, and CC/G-g, levels of interferon induction (i.e., approximately 80 units) at 33” were similar to that induced by the parental virus alone (Table 5). An insignificant twofold decrease was observed with the CC/C combination. [This may be due to a high number of defectives in the tsC stock (-2OO:l by hemagglutination)l. Thus, it appears that noninducing mutants lack positive control over interferon induction. DISCUSSION
The results presented here have demonstrated two interesting points: (1) no detectable levels of IF are induced by mutants of measles virus in BSC-1 or GMK cells under permissive or nonpermissive conditions; and (2) the ts+ revertant, which otherwise demonstrates characteristics similar to the parental strain (Breschkin et al., 1976), does not induce interferon in BSC-1 or GMK cells. For the parental virus, induction of IF was dependent on both time of incubation and multiplicity of infection. Infection for various times and multiplicities which had
AND
413
INTERFERON TABLE
INTERFERON PRODUCTION MEASLES VIRUS MUTANTS
WITH COMBINATIONS OF AND PARENTAL CC VIRUS
0
33”
Virus
combina-
5
IF yields
as PRD,
IF yieldz’as
timso
PRD,, ____~
T C G-3 TIC T/G-3 C/G-3 G-9/G-3
80-160 <5 <5 <5 <5 <5 <5 <5
80-160 <5 <5 <5 NDh <5 ND <5
CC/T cc/c CC/G-3 CC/G-9
80-160 40-80 80-160 80-160
ND ND ND ND
cc
-~ o Combinations of viruses were incubated with poly-L-ornithine and inoculated onto cultures in 60mm BSC-1 plates. Fluids were harvested when cells showed approximately 50% CPE. (Maximum levels are induced by the CC parental virus by this time.) * Not done.
been shown to induce significant levels by the parental strain failed to produce any detectable interferon with the mutants or revertant. A correlation between virus yields and interferon production was found for reovirus and its ts mutants (Lai and Joklik, 1973). Atkins et aE. (1974), however, were unable to show this relationship with Sindbis virus and its ts mutants. No correlation between the virus yields and interferon production was found for the parental measles virus or its mutants. Yields of virus under permissive conditions were comparable for all, but only the parental virus induced interferon. That this is not a host cell-dependent phenomenon was demonstrated by testing in GMK cells 14 group 1 mutants, all available group 2 and 3 mutants, and a group 3 revertant. Several of the mutants are known to be leaky or to have fairly high reversion frequencies. Interferon induction under nonpermissive conditions for reovirus (Lai and Joklik, 1973) and Sindbis virus (Lomniczi and Burke, 1970) ts mutants may occur because of a leaky mutation or high reversion frequency. Under nonpermissive conditions and sufficient time for the production of wild-type
414
McKIMM
revertants, or under permissive conditions, none of these measles mutants induced interferon. Complementation between reovirus mutants under restrictive conditions was shown to increase approximately lo-fold the level of interferon produced (Lai and Joklik, 1973). When complementation tests were carried out between the three groups and the revertant measles virus, complementation occurred; IF was not produced, however, even when assayed under conditions permissive for the replication of the mutants. Another paramyxovirus, SV-5, has been shown to inhibit interferon induction and action in primary rhesus monkey kidney cells and in the MDBK line of bovine kidney cells (Choppin and Compans, 1975). No interferon is produced in SV-5 infection of these cells. In addition, super-infection of SV&infected cells with a virus normally able to induce interferon fails to yield IF. However, defective SV-5 particles are able to induce IF and have no effect on the action of preformed IF. Choppin and Compans suggest that this inhibition is due to some form of positive control by the SV-5 genome, and that this is lost by the defective virions. Coinfection with the inducing parental measles virus and noninducing mutants and revertant had no effect on induction by the parental virus. Levels of interferon achieved in cultures infected with combinations of these viruses were similar to those obtained when the parental virus alone was used. Thus, we conclude that the mutants have no repressor effect on the induction of IF. The original parental CC virus, from which all mutants except tsA were selected, was isolated by cocultivation with BSC-1 cells from hamster cells latently infected with passaged Schwarz vaccine strain. It could be argued that such a passage history resulted in some change in the virus, and that the ts mutants may have been selected from noninducers even before mutagenesis. This characteristic would therefore not reappear, as with G-9, upon reversion to the wild-type. However, tsA was derived directly from Schwarz virus passed three times in Vero cells, and
AND
RAPP
preliminary evidence indicates that mutants derived from Edmonston vaccine strain share the properties of these CCand Schwarz-derived mutants. This unusual property is therefore not unique to the CC-derived mutants. Although much attention has been focused on the mechanism by which virusinduced interferon synthesis occurs, little is known concerning the role of the virus genotype. Double-stranded RNA was thought to play an important role in the induction process (de Clercq and Merigan, 1970; Kleinschmidt, 1972). However, use of ts mutants of Sindbis virus (Lockart et al., 1968; Atkins et al., 1974; Atkins and Lancashire, 1976), and Semliki Forest virus (Lomniczi and Burke, 1970) has failed to show a consistent relationship between the amount of double-stranded RNA produced and the amount of interferon induced. Lai and Joklik (1973) found that induction with reovirus ts mutants correlated only with some late function resulting in the formation of intact virions. Under conditions permissive for the production of infectious progeny, the mutants and revertant must produce some double-stranded RNA. Since interferon is not induced, other factors must be important. This agrees with results obtained from a Newcastle disease virus mutant. Thacore and Youngner (1970) found no correlation for this virus between virus RNA synthesis and the induction of interferon synthesis. Genetic studies are now underway to detect possible differences in the parental strains which may have been present even prior to mutagenesis. This implies that virus stocks contain infectious virions with the ability to induce interferon in addition to other virions without this property. Preliminary results (Rapp and McKimm, unpublished observations) seem to support this concept. The results already obtained suggest that interferon induction is at least partially under the genetic control of the inducing virus. ACKNOWLEDGMENTS Preliminary experiments leading to this study were conducted by Dr. P. Knight. Discussions with Dr. A. Breschkin and Dr. E. Morgan were very
MEASLES
ts MUTANTS
helpful. The technical assistance of Barbara Walmer is greatly appreciated. This investigation was supported by Grant Number AI 12203, awarded by the National Institute of Allergy and Infectious Diseases, DHEW. J. M. is a recipient of a postgraduate fellowship from the Australian-American Educational Foundation.
REFERENCES C. D., and ATHERTON, J. G. (19641. Effect of actinomycin D on measles virus growth and interferon production. Nature (London) 203, 671. ATKINS, G. J., and LANCASHIRE, C. L. (1976). The induction of interferon by temperature-sensitive mutants of Sindbis virus: Its relationship to double-stranded RNA synthesis and cytopathic effect. J. Gen. Viral. 30, 157-165. ATKINS, G. J., JOHNSTON, M. D., WESTMACOTT, L. M., and BURKE, P. C. (1974). Induction of interferon in chick cells by temperature-sensitive mutants of Sindbis virus. J. Gen. Virol. 25, 381-390. BERCHOLZ, C. M., KILEY, M. P., and PAYNE, F. E. (19751. Isolation and characterization of temperature-sensitive mutants of measles virus. J. Viral. 16, 192-202. BRESCHKIN, A. M., HASPEL, M. V., and RAPP, F. (1976). Neurovirulence and induction of hydrocephalus with parental, mutant, and revertant strains of measles virus. J. Virol. 18, 809-811. CHOPPIN, P. W., and COMPANS, R. W. (1975). Reproduction of paramyxo-viruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. W. Wagner, eds.), pp. 95-178. Academic Press, New York. DE CLERCQ, E., and MERIGAN, T. C. (19701. Current concepts of interferon and interferon induction. Annu. Rev. Med. 21, 17-46. DE MAEYER, E., and ENDERS, J. F. (19611. An interferon appearing in cell cultures infected with measles virus. Proc. Sot. Exp. Biol. Med. 107,573578. DE MAEYER, E., and ENDERS, J. F. (19651. Growth characteristics, interferon production and plaque formation with different lines of Edmonston meaANDERSON,
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sles virus. Arch. Gesamte Virusforsch. 16, 151160. ENDERS, J. F. (19621. Measles virus. Amer. J. Dis. Child. 103, 282-287. HASPEL, M. V., DUFF, R., and RAPP, F. (1975ai. Experimental measles encephalitis: A genetic analysis. Znfec. Immun. 12, 787-790. HASPEL, M. V., DUFF, R., and RAPP, F. (1975b). Isolation and preliminary characterization of temperature-sensitive mutants of measles virus. J. Virol. 16, 1000-1009. HASPEL, M. V., KNIGHT, P. R., DUFF, R. G., and RAPP, F. (1973). Activation of a latent measles virus infection in hamster cells. J. Virol. 12, 690695. Ho, M., and ENDERS, J. F. (19591. Further studies on an inhibition of viral activity appearing in infected cell cultures and its role in chronic viral infections. Virology 1, 446-477. KLEINSCHMIIYP, W. J. (19721. Biochemistry of interferon and its inducers. Annu. Rev. Biochem. 41, 517-542. LAI, M. T., and JOKLIK, W. K. (19731. The induction of interferon by temperature-sensitive mutants of reovirus, UV-irradiated reovirus, and subviral reovirus particles. Virology 51, 191-204. LOCKART, R. Z., JR., BAYLISS, N. L., TOY, S. T., and YIN, F. H. (1968). Viral events necessary for the induction of interferon in chick embryo cells. J. Viral. 2, 962-965. LOMNICZI, B., and BURKE, D. C. (19701. Interferon production by temperature-sensitive mutants of Semliki Forest virus. J. Gen. Virol. 8, 55-68. MIRCHAMSY, H., and RAPP, F. (1969). Role of interferon in replication of virulent and attenuated strains of measles virus. J. Gen. Virol. 4, 513-522. RAPP, F. (1964). Plaque differentiation and replication of virulent and attenuated strains of measles virus. J. Bacterial. 88, 1448-1458. THACORE, M., and YOUNGNER, J. S. (1970). Cells persistently infected with Newcastle disease virus. II. Ribonucleic and protein synthesis in cells infected with mutants isolated from persistently infected L cells. J. Viral. 6, 42-48.