VIROLOGY
67.576-587
(1975)
Isolation
and Characterization Mutants
of Fowl Plague Virus
C. SCHOLTISSEK Znstitut
of Temperature-Sensitive
AND
ANN L. BOWLES
fiir Virologie, Justus Liebig-Uniuersitht,
Giessen, Germany
Accepted June 2, 1975
Twenty-five temperature-sensitive (ts) mutants of fowl plague virus have been isolated and tentatively characterized. Twenty-three of them can be arranged into six recombination groups. The classification of the two other ts mutants is still uncertain. RNA-negative mutants have been found in four recombination groups. One group harbors ts mutants with a lesion in the hemagglutinin gene, another group consists of mutants with a lesion in the neuraminidase gene. One group seems to have a maturation defect. Cells infected with still another group retain the RNA polymerase-template complex within the nucleus. The biological properties of the ts mutants of the various recombination groups suggest that at least some of the gene products might have more than one function. INTRODUCTION
Although temperature-sensitive (ts) mutants of influenza viruses have been isolated in several laboratories (Simpson and Hirst, 1968; Mackenzie, 1970; Mills and Chanock, 1971; Sugiura et al., 1972; Ueda, 1972; Mackenzie and Dimmock, 1973; Markushin and Ghendon, 1973; Scholtissek et al., 1974; Palese et al., 1974), the exact number of complementation and recombination groups is still a matter of discussion. By genetic methods, Hirst (1973) recently classified his ts mutants and arranged them into eight groups; the biochemical basis of the classification, however, is not yet established. Ghendon et al. (1973) found that, of five ts mutants of fowl plague virus belonging to different complementation groups, four had defects in viral RNA synthesis. A clear correlation between complementation groups and function of viral gene products is rendered difficult insofar as little is known concerning regulation processes during virus production and the extent to which one gene product depends on the function of another. On the other hand, studies on such problems might be facilitated if genetically well characterized ts mutants are availa-
ble. With these kinds of problems in mind we isolated and characterized a number of ts mutants of an avian influenza A virus (fowl plague). The genome of influenza viruses consists of and functions in pieces (Pons and Hirst, 1968; Duesberg, 1968; Lewandowski et al., 1971; Skehel, 1971; Scholtissek and Rott, 1964). Thus by double infection with two different influenza A viruses new strains might emerge by reassortment of the corresponding RNA pieces. This kind of reassortment will be called here recombination, although this type of recombination is different from the classical one in which breakage and reunion of the nucleic acid molecules is involved (Hirst, 1962). The nomenclature for the polypeptides that are found in influenza-infected cells and that are assumed to be viral gene products is adopted from Kilbourne et al., (1972). The following influenza polypeptides were found in influenza particles: The P protein(s) (possibly two proteins, P, and P,), which are assumed to be the viral RNA polymerase(s); the unsplit hemagglutinin (HA) and/or the two hemagglutinin split products (HA, and HA,); the nucleocapsid protein (NP), the neuraminidase, and the
576 Cbpyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
TS MUTANTS
matrix protein (M). In infected cells an additional nonstructural protein (NS) is found. Thus between six and seven gene products are already known. MATERIALS
AND
METHODS
Virus strains and tissue cultures. A large-plaque variant of fowl plague virus, strain Restock, which was further propagated in embryonated eggs, served as wildtype virus. Primary cultures of chick embryos were used for the single-cycle experiments and for the plaque tests as described ‘recently (Scholtissek et al., 1974). The chemical mutagenesis by 5-fluorouracil and the isolation of ts mutants was performed as described by Simpson and Hirst (1968). The permissive temperature used was 33”, the nonpermissive one, 40”. An exact description of the procedures used in these studies for isolation and propagation of ts mutants has been reported recently (Scholtissek et al., 1974).
OF FPV
577
or the resuspended nuclear fraction was used for the incubation with [‘H]GTP, 10 pg/ml of actinomycin D, and cofactors at 32” as described (Scholtissek, 1969). The incorporation of labeled GMP into virusspecific RNA is taken as a measure of viral RNA polymerase activity. Under these conditions only complementary RNA is synthesized (Scholtissek, 1969). Determination of virus-specific RNA synthesized in vivo. Newly synthesized
virus-specific RNA was determined by labeling infected cells with [‘Hluridine and specific hybridization of the isolated RNA with a surplus of either nonlabeled virion RNA or nonlabeled complementary RNA as described recently (Scholtissek and Rott, 1970; Scholtissek et al., 1974). Determination
of various viral activities.
Infectivity was determined by the plaque assay in primary chick embryo cells (Zimmermann and Schafer, 1960). The hemagglutinin (HA) was titrated according to Recombination and complementation Davenport et al. (1960) after homogenizabetween ts mutants. Primary chick embryo tion of the infected cells and removal of cell cells were either singly or doubly infected debris by low speed centrifugation. Viral neuraminidase was determined according with egg fluids of ts mutants with a multiplicity of 3-5 PFU/cell at 33”. After two to Drzeniek et al. (1966) with bovine sialowashings with phosphate-buffered saline, lactose as substrate. The amount of neuracells were treated with antiserum against minidase which liberated 1 pmol of sialic fowl plague virus in phosphate-buffered acid from the substrate per minute at 37” saline for 15 min at room temperature in was defined as one enzyme unit. Synthesis of viral proteins. The synthesis order to remove residual virus. After two further washings the cultures were incu- of viral proteins was followed by labeling bated either at 33 or 40” for eight hr. The infected cells with 10 &i/ml of [sH]leu40” incubator was located in a warm room tine, [‘Hlvaline and [sH]tyrosine, each for at 40’ in order to avoid temperature shifts 1 hr starting at the indicated times after during further manipulations. Eight hours infection. In a few experiments cultures after infection the cells were processed. infected with wild-type virus were labeled Determination of the virus-specific RNA with 5 &i/culture of ‘C-labeled protein polymerase. At the indicated times after hydrolysate. These samples were used for infection the cells were washed twice with a coelectrophoresis with SH-labeled samples. buffer containing 0.01 M Tris-HCl, pH 7.4, Samples of 100 ~1 were used for gel elec0.01 A4 KCl, 1.5 mA4 MgC12, and 1 mM trophoresis. Proteins were dissociated and 2-mercaptoethanol and scraped off the sur- separated by electrophoresis on 10% acrylface. After homogenization of the swollen amide gels as described by Caliguiri et al. cells in a tight-fitting Dounce homogenizer (1969). The slicing and processing of gels (1 ml/culture) the nuclei were removed by for the determination of radioactivity by centrifugation for 10 min at 2000 g. The liquid scintillation has been reported by nuclei were washed once with the same Klenk et al. (1970). buffer and were resuspended in the original Chemicals and isotopes. [5-SH]uridine volume of this buffer. The first supernatant (29 Wmmol), L-[4,5-sH]leucine (1.0 Ci/
578
SCHOLTISSEK
mmol), L- [2,3-*H]valine (1.5 Ci/mmol), L[3,5-JH]tyrosine (1.0 Ci/mmol), U-“Clabeled protein hydrolysate (57 mCi/ matom), and [8-‘H]GTP (1.0 Ci/mmol), were obtained from the Radiochemical Centre, Amersham, England.
AND BOWLES
mutants were leaky in that they produced tiny plaques after prolonged incubation at 40”. These mutants are marked by the letter a in the table.
2. Complementation and Recombination Between the Various ts Mutants RESULTS Recombination frequencies and com1. Plaque Titers at 33 and 40” plementation levels in pairwise crosses between the various ts mutants were deterAfter three cycles of plaque purification the individually isolated plaques of the ts mined according to Sugiura et al. (1972). mutants of fowl plague virus were injected There was a broad variation in the results into embryonated eggs, which were then between individual experiments as has also incubated at 33”. After 2 days, egg fluids been noticed by others (Simpson and with a positive HA titer were either stored Hirst, 1968; Sugiura et al., 1972). Comat -70” or were used immediately for plementation levels between 5 and 1000 (as further experiments. The plaque titers of defined by Suguira et al. (1972)) were the egg fluids of isolated ts mutants at 33 found. If the recombination frequency was and 40” are listed in Table 1. Some of the below 0.1% the ts mutants were regarded as belonging to the same group. In such cases TABLE 1 the complementation level was always PFU TITER OF TS MUTANTS DETERMINED AT below 1. In Table 2 the recombination 33 AND 40" frequencies are listed. In other experiments cells were either No. of ts PFUhl of egg fluid PFU mutant assayed at 400/33” singly or doubly infected and incubated at ~ (-log,,) 40”, and the plaque test on the virus yield 33” 40” was also performed at 40”. A difference in 1.1 x 102 3 2.1 x 108 6.3 the plaque yield between doubly- and sin8 3.5 x 108 2.24 x 10’” 5.2 gly-infected cells by a factor of 100 or more 18 2.75 x 10’ >6.4 6.7 6.8 6.8 however, has to be expected, since in these 210 1.3 x 107 < 10” >6.1 cases two genes of that mutant had to be 227 1.0 x 10’ >6.0 6.2 In most cases in which the recombina6.8 tion frequency was determined quantita288 1.1 x 106 8.0 x 103”.b 2.1 tively, the short recombination test (in290 2.0 x 106 5.0 x 10’“. b 6.7 fected cells incubated only at 40”, plaque 293 3.2 x 10’ 6.5 test only at 40’) was also performed. In no Wild type 2.9 x 10’ 4.5 x 10’ -0.8 case was there a discrepancy between the results of the two tests. 0 Tiny plaques after prolonged incubation. b Heterogeneous plaque morphology. There is as yet no explanation for the
TS MUTANTS
appearance of irregular and tiny plaques which were found during recombination between ts 18 and the ts mutants 117, 132, 206, 293, 290, 236, 90, and 93. There was, however, a clearcut recombination between the ts mutants 3 and 115 and ts 18. We have been unable to passage at 40” any of the tiny plaques obtained in relatively high yields during recombination between ts 18 and ts 90, 93 or 236. Thus it is not clear whether real recombination has occurred between ts 18 and the ts mutants mentioned. There are several mutants of group I (ts 254, ts 8, ts 121) which do not recombine with ts 18 but do so readily with ts 90, ts 93, or ts 236. The ts 18 mutant has another unusual property: Although it is not a leaky mutant, it produces in a single cycle at 40” a normal yield of virus, forming plaques at 33” but not at 40”. This yield is comparable to that found during incubation at 33’. The titers of hemagglutinin formed at both temperatures also are identical. Therefore the complementation levels between ts 18 and all the other mutants could not be determined. According to Table 2, 23 of the 25 ts mutants can be classified into six recombination groups as summarized in Table 3. Mutants ts 18 and 236 show an uncertain recombination to each other and to mutants of recombination group III (only tiny plaques at 40”). Therefore from these data it cannot be concluded whether ts 18 and ts 236 belong to group III or represent other group(s). Several of the ts strains are double mutants, in many cases one tight and one leaky. For example, recombination between ts 113 and ts 196 led to a recombinant which at 40” forms very tiny plaques after 2 days. No wild-type virus was found. Recombination between ts 227 and ts 196 led to a mixture of plaques of two different sizes: About 50% were wild type and the rest were of the tiny variety, as mentioned above. Some of the tiny plaques were picked and replated. These recombinants did not show any recombination with ts 113; there was, however, recombination to wild type with, e.g., ts 90. Thus ts 196 is a double mutant belonging to group II (tight)
579
OF FPV
and group IV (leaky). Correspondingly, ts 206 and ts 293 have been found to be double mutants belonging to group I (tight) and group V (small, turbid plaques = leaky); ts 8 belongs to group I and group IV, and ts 71 to group II and group VI. 3. Biochemical
Properties
of ts Mutants
a) Viral RNA polymer-use. Ts 3 has already been characterized in detail for viral RNA synthesis and production of RNA polymerase (Scholtissek et al., 1974). In Table 4, data on the induction of viral RNA polymerase by the ts mutants are summarized. Such mutants that produce at 40” less than 5% of the enzyme activity observed after incubation at 33” are regarded as polymerase negative. Some ts mutants (290, 121, 71) induced intermediate polymerase activities. According to Table 1 these mutants are leaky, possibly because of leakiness of the polymerase activity. The heat stability in vitro of the polymerase produced at the permissive temperature has been studied with ts 263, ts 293, and ts 18. With all three mutants the heat stability of the enzyme was comparable to that of the wild type. When chick embryo cells infected with ts 90 or ts 93 were analyzed for polymerase activity, significant enzyme activity could not be detected in the cytoplasmic extracts either after incubation at 33” or at 40”. At 33”, polymerase activity, however, accumulated in the nuclear fraction. Corresponding data for ts 90 are shown in Table 5. At the nonpermissive temperature enzyme activity in the nuclear fraction of ts 90-or ts 93-infected cells was also reduced by about 80%. b) Synthesis of viral RNA in vivo. Since the determination of virus-specific RNA is very time- and material-consuming, only one or a few ts mutants of each group were studied (Table 6). At 40” or after a shift from 33 to 40” viral RNA synthesis is inhibited in cells infected with ts 132, ts 263, and ts 236, as has been described for ts 3 (Scholtissek et al., 1974). In the case of ts 263 the heat stability in viuo of the RNA polymerase synthesizing complementary RNA has been studied,
580
SCHOLTISSEK
AND BOWLES TAB RECOMBINATION FREQUENCY(70) AND RECOMBINATION
No. of ts mutant 3 115 117 132 206 254 293 8 121 290 263 196 71 236 90 93 113 210 288 227 79 19 81 283 18
3
115
117
67.576-587
(1975)
Isolation
and Characterization Mutants
of Fowl Plague Virus
C. SCHOLTISSEK Znstitut
of Temperature-Sensitive
AND
ANN L. BOWLES
fiir Virologie, Justus Liebig-Uniuersitht,
Giessen, Germany
Accepted June 2, 1975
Twenty-five temperature-sensitive (ts) mutants of fowl plague virus have been isolated and tentatively characterized. Twenty-three of them can be arranged into six recombination groups. The classification of the two other ts mutants is still uncertain. RNA-negative mutants have been found in four recombination groups. One group harbors ts mutants with a lesion in the hemagglutinin gene, another group consists of mutants with a lesion in the neuraminidase gene. One group seems to have a maturation defect. Cells infected with still another group retain the RNA polymerase-template complex within the nucleus. The biological properties of the ts mutants of the various recombination groups suggest that at least some of the gene products might have more than one function. INTRODUCTION
Although temperature-sensitive (ts) mutants of influenza viruses have been isolated in several laboratories (Simpson and Hirst, 1968; Mackenzie, 1970; Mills and Chanock, 1971; Sugiura et al., 1972; Ueda, 1972; Mackenzie and Dimmock, 1973; Markushin and Ghendon, 1973; Scholtissek et al., 1974; Palese et al., 1974), the exact number of complementation and recombination groups is still a matter of discussion. By genetic methods, Hirst (1973) recently classified his ts mutants and arranged them into eight groups; the biochemical basis of the classification, however, is not yet established. Ghendon et al. (1973) found that, of five ts mutants of fowl plague virus belonging to different complementation groups, four had defects in viral RNA synthesis. A clear correlation between complementation groups and function of viral gene products is rendered difficult insofar as little is known concerning regulation processes during virus production and the extent to which one gene product depends on the function of another. On the other hand, studies on such problems might be facilitated if genetically well characterized ts mutants are availa-
ble. With these kinds of problems in mind we isolated and characterized a number of ts mutants of an avian influenza A virus (fowl plague). The genome of influenza viruses consists of and functions in pieces (Pons and Hirst, 1968; Duesberg, 1968; Lewandowski et al., 1971; Skehel, 1971; Scholtissek and Rott, 1964). Thus by double infection with two different influenza A viruses new strains might emerge by reassortment of the corresponding RNA pieces. This kind of reassortment will be called here recombination, although this type of recombination is different from the classical one in which breakage and reunion of the nucleic acid molecules is involved (Hirst, 1962). The nomenclature for the polypeptides that are found in influenza-infected cells and that are assumed to be viral gene products is adopted from Kilbourne et al., (1972). The following influenza polypeptides were found in influenza particles: The P protein(s) (possibly two proteins, P, and P,), which are assumed to be the viral RNA polymerase(s); the unsplit hemagglutinin (HA) and/or the two hemagglutinin split products (HA, and HA,); the nucleocapsid protein (NP), the neuraminidase, and the
576 Cbpyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
TS MUTANTS
matrix protein (M). In infected cells an additional nonstructural protein (NS) is found. Thus between six and seven gene products are already known. MATERIALS
AND
METHODS
Virus strains and tissue cultures. A large-plaque variant of fowl plague virus, strain Restock, which was further propagated in embryonated eggs, served as wildtype virus. Primary cultures of chick embryos were used for the single-cycle experiments and for the plaque tests as described ‘recently (Scholtissek et al., 1974). The chemical mutagenesis by 5-fluorouracil and the isolation of ts mutants was performed as described by Simpson and Hirst (1968). The permissive temperature used was 33”, the nonpermissive one, 40”. An exact description of the procedures used in these studies for isolation and propagation of ts mutants has been reported recently (Scholtissek et al., 1974).
OF FPV
577
or the resuspended nuclear fraction was used for the incubation with [‘H]GTP, 10 pg/ml of actinomycin D, and cofactors at 32” as described (Scholtissek, 1969). The incorporation of labeled GMP into virusspecific RNA is taken as a measure of viral RNA polymerase activity. Under these conditions only complementary RNA is synthesized (Scholtissek, 1969). Determination of virus-specific RNA synthesized in vivo. Newly synthesized
virus-specific RNA was determined by labeling infected cells with [‘Hluridine and specific hybridization of the isolated RNA with a surplus of either nonlabeled virion RNA or nonlabeled complementary RNA as described recently (Scholtissek and Rott, 1970; Scholtissek et al., 1974). Determination
of various viral activities.
Infectivity was determined by the plaque assay in primary chick embryo cells (Zimmermann and Schafer, 1960). The hemagglutinin (HA) was titrated according to Recombination and complementation Davenport et al. (1960) after homogenizabetween ts mutants. Primary chick embryo tion of the infected cells and removal of cell cells were either singly or doubly infected debris by low speed centrifugation. Viral neuraminidase was determined according with egg fluids of ts mutants with a multiplicity of 3-5 PFU/cell at 33”. After two to Drzeniek et al. (1966) with bovine sialowashings with phosphate-buffered saline, lactose as substrate. The amount of neuracells were treated with antiserum against minidase which liberated 1 pmol of sialic fowl plague virus in phosphate-buffered acid from the substrate per minute at 37” saline for 15 min at room temperature in was defined as one enzyme unit. Synthesis of viral proteins. The synthesis order to remove residual virus. After two further washings the cultures were incu- of viral proteins was followed by labeling bated either at 33 or 40” for eight hr. The infected cells with 10 &i/ml of [sH]leu40” incubator was located in a warm room tine, [‘Hlvaline and [sH]tyrosine, each for at 40’ in order to avoid temperature shifts 1 hr starting at the indicated times after during further manipulations. Eight hours infection. In a few experiments cultures after infection the cells were processed. infected with wild-type virus were labeled Determination of the virus-specific RNA with 5 &i/culture of ‘C-labeled protein polymerase. At the indicated times after hydrolysate. These samples were used for infection the cells were washed twice with a coelectrophoresis with SH-labeled samples. buffer containing 0.01 M Tris-HCl, pH 7.4, Samples of 100 ~1 were used for gel elec0.01 A4 KCl, 1.5 mA4 MgC12, and 1 mM trophoresis. Proteins were dissociated and 2-mercaptoethanol and scraped off the sur- separated by electrophoresis on 10% acrylface. After homogenization of the swollen amide gels as described by Caliguiri et al. cells in a tight-fitting Dounce homogenizer (1969). The slicing and processing of gels (1 ml/culture) the nuclei were removed by for the determination of radioactivity by centrifugation for 10 min at 2000 g. The liquid scintillation has been reported by nuclei were washed once with the same Klenk et al. (1970). buffer and were resuspended in the original Chemicals and isotopes. [5-SH]uridine volume of this buffer. The first supernatant (29 Wmmol), L-[4,5-sH]leucine (1.0 Ci/
578
SCHOLTISSEK
mmol), L- [2,3-*H]valine (1.5 Ci/mmol), L[3,5-JH]tyrosine (1.0 Ci/mmol), U-“Clabeled protein hydrolysate (57 mCi/ matom), and [8-‘H]GTP (1.0 Ci/mmol), were obtained from the Radiochemical Centre, Amersham, England.
AND BOWLES
mutants were leaky in that they produced tiny plaques after prolonged incubation at 40”. These mutants are marked by the letter a in the table.
2. Complementation and Recombination Between the Various ts Mutants RESULTS Recombination frequencies and com1. Plaque Titers at 33 and 40” plementation levels in pairwise crosses between the various ts mutants were deterAfter three cycles of plaque purification the individually isolated plaques of the ts mined according to Sugiura et al. (1972). mutants of fowl plague virus were injected There was a broad variation in the results into embryonated eggs, which were then between individual experiments as has also incubated at 33”. After 2 days, egg fluids been noticed by others (Simpson and with a positive HA titer were either stored Hirst, 1968; Sugiura et al., 1972). Comat -70” or were used immediately for plementation levels between 5 and 1000 (as further experiments. The plaque titers of defined by Suguira et al. (1972)) were the egg fluids of isolated ts mutants at 33 found. If the recombination frequency was and 40” are listed in Table 1. Some of the below 0.1% the ts mutants were regarded as belonging to the same group. In such cases TABLE 1 the complementation level was always PFU TITER OF TS MUTANTS DETERMINED AT below 1. In Table 2 the recombination 33 AND 40" frequencies are listed. In other experiments cells were either No. of ts PFUhl of egg fluid PFU mutant assayed at 400/33” singly or doubly infected and incubated at ~ (-log,,) 40”, and the plaque test on the virus yield 33” 40” was also performed at 40”. A difference in 1.1 x 102 3 2.1 x 108 6.3 the plaque yield between doubly- and sin8 3.5 x 108 2.24 x 10’” 5.2 gly-infected cells by a factor of 100 or more 18 2.75 x 10’ >6.4 6.7 6.8 6.8 however, has to be expected, since in these 210 1.3 x 107 < 10” >6.1 cases two genes of that mutant had to be 227 1.0 x 10’ >6.0 6.2 In most cases in which the recombina6.8 tion frequency was determined quantita288 1.1 x 106 8.0 x 103”.b 2.1 tively, the short recombination test (in290 2.0 x 106 5.0 x 10’“. b 6.7 fected cells incubated only at 40”, plaque 293 3.2 x 10’ 6.5 test only at 40’) was also performed. In no Wild type 2.9 x 10’ 4.5 x 10’ -0.8 case was there a discrepancy between the results of the two tests. 0 Tiny plaques after prolonged incubation. b Heterogeneous plaque morphology. There is as yet no explanation for the
TS MUTANTS
appearance of irregular and tiny plaques which were found during recombination between ts 18 and the ts mutants 117, 132, 206, 293, 290, 236, 90, and 93. There was, however, a clearcut recombination between the ts mutants 3 and 115 and ts 18. We have been unable to passage at 40” any of the tiny plaques obtained in relatively high yields during recombination between ts 18 and ts 90, 93 or 236. Thus it is not clear whether real recombination has occurred between ts 18 and the ts mutants mentioned. There are several mutants of group I (ts 254, ts 8, ts 121) which do not recombine with ts 18 but do so readily with ts 90, ts 93, or ts 236. The ts 18 mutant has another unusual property: Although it is not a leaky mutant, it produces in a single cycle at 40” a normal yield of virus, forming plaques at 33” but not at 40”. This yield is comparable to that found during incubation at 33’. The titers of hemagglutinin formed at both temperatures also are identical. Therefore the complementation levels between ts 18 and all the other mutants could not be determined. According to Table 2, 23 of the 25 ts mutants can be classified into six recombination groups as summarized in Table 3. Mutants ts 18 and 236 show an uncertain recombination to each other and to mutants of recombination group III (only tiny plaques at 40”). Therefore from these data it cannot be concluded whether ts 18 and ts 236 belong to group III or represent other group(s). Several of the ts strains are double mutants, in many cases one tight and one leaky. For example, recombination between ts 113 and ts 196 led to a recombinant which at 40” forms very tiny plaques after 2 days. No wild-type virus was found. Recombination between ts 227 and ts 196 led to a mixture of plaques of two different sizes: About 50% were wild type and the rest were of the tiny variety, as mentioned above. Some of the tiny plaques were picked and replated. These recombinants did not show any recombination with ts 113; there was, however, recombination to wild type with, e.g., ts 90. Thus ts 196 is a double mutant belonging to group II (tight)
579
OF FPV
and group IV (leaky). Correspondingly, ts 206 and ts 293 have been found to be double mutants belonging to group I (tight) and group V (small, turbid plaques = leaky); ts 8 belongs to group I and group IV, and ts 71 to group II and group VI. 3. Biochemical
Properties
of ts Mutants
a) Viral RNA polymer-use. Ts 3 has already been characterized in detail for viral RNA synthesis and production of RNA polymerase (Scholtissek et al., 1974). In Table 4, data on the induction of viral RNA polymerase by the ts mutants are summarized. Such mutants that produce at 40” less than 5% of the enzyme activity observed after incubation at 33” are regarded as polymerase negative. Some ts mutants (290, 121, 71) induced intermediate polymerase activities. According to Table 1 these mutants are leaky, possibly because of leakiness of the polymerase activity. The heat stability in vitro of the polymerase produced at the permissive temperature has been studied with ts 263, ts 293, and ts 18. With all three mutants the heat stability of the enzyme was comparable to that of the wild type. When chick embryo cells infected with ts 90 or ts 93 were analyzed for polymerase activity, significant enzyme activity could not be detected in the cytoplasmic extracts either after incubation at 33” or at 40”. At 33”, polymerase activity, however, accumulated in the nuclear fraction. Corresponding data for ts 90 are shown in Table 5. At the nonpermissive temperature enzyme activity in the nuclear fraction of ts 90-or ts 93-infected cells was also reduced by about 80%. b) Synthesis of viral RNA in vivo. Since the determination of virus-specific RNA is very time- and material-consuming, only one or a few ts mutants of each group were studied (Table 6). At 40” or after a shift from 33 to 40” viral RNA synthesis is inhibited in cells infected with ts 132, ts 263, and ts 236, as has been described for ts 3 (Scholtissek et al., 1974). In the case of ts 263 the heat stability in viuo of the RNA polymerase synthesizing complementary RNA has been studied,
580
SCHOLTISSEK
AND BOWLES TAB RECOMBINATION FREQUENCY(70) AND RECOMBINATION
No. of ts mutant 3 115 117 132 206 254 293 8 121 290 263 196 71 236 90 93 113 210 288 227 79 19 81 283 18
3
115
117
