JouRNAL OF VIROLOGY, Jan. 1976, p. 68-73
Vol. 17, No. 1 Printed in U.S.A.
Copyright 0 1976 American Society for Microbiology
Comparison of RNA Polymerase Associated With Newcastle Disease Virus and a Temperature-Sensitive Mutant of Newcastle Disease Virus Isolated From Persistently Infected L Cells TREVOR L. STANWICKI* AND J. V. HALLUM2 Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana 70112 Received for publication 16 July 1975
An in vitro comparison was made of the RNA polymerase activity associated with Newcastle disease virus (NDVo} and three clones of the temperature-sensitive mutant (NDVpi) isolated from persistently infected L cells. Less polymerase activity was associated with the NDVpi clones. Also, compared to NDVo, an increase in incubation temperature from 32 to 37 or 42 C resulted in a marked decrease in polymerase activity for the temperature-sensitive mutants which coincided with their inability to replicate at 42 C.
Thacore and Youngner (12) established a persistent infection of L (Lpi) cells with the Herts strain of Newcastle disease virus (NDV). These Lpi cells continually produce the mutant virus (NDVpi) which differs from the wild-type virus (NDVo) in that, among other phenotypic properties, it is temperature sensitive, forms smaller plaques on chicken embryo fibroblasts, and has a decreased ability to elute from chicken red blood cells (8, 12, 13, 14). Subsequently Preble and Youngner (9), using an indirect method to measure the RNA polymerase activity in NDVpi-infected cells at 42 C, found that clones of the temperature-sensitive mutants (NDVpi) had defects associated with RNA synthesis at the nonpermissive temperature. They suggested that selection of temperature-sensitive mutants may play a role in the establishment or maintenance of the persistent infection in Lpi cells (10). Recently in this laboratory an RNAdependent DNA polymerase was found in preparations of NDVpi but not NDVo (2). In this study three clones of NDVpi and one clone of NDVo, which displayed characteristic plaque morphology, temperature sensitivity, and the ability to elute from chicken red blood cells (RBC), were selected and assayed for RNA polymerase activity at 32, 37, and 42 C. Prepa-
rations of the NDVpi clones contained less polymerase activity than the NDVo clone. Also, the RNA polymerase activity associated with the NDVpi clones was more heat labile than that of the NDVo preparations. MATERIALS AND METHODS Cell cultures. Uninfected L cells and Lpi cells persistently infected with NDVpi were obtained from J. S. Youngner, University of Pittsburg Medical School, and subcultured as previously described (12). Baby hamster kidney-21, clone 13 (BHK) cells obtained from GIBCO (Grand Island Biological Co., N.Y.) were cultured in Eagle minimal essential medium supplemented with 10% fetal calf serum (Grand Island Biological Co.) for cell growth and 2% fetal calf serum for cell maintenance. Viruses. The Herts strain of NDVo was obtained from J. S. Youngner. Stock cultures of NDVo and NDVpi were propagated in BHK cells, harvested when cytopathic effect was observed, and stored at -70 C. Viral assay. A plaque assay on BHK cells was used to assay NDVo and NDVpi infectivity. Monolayers of BHK cells in 30-ml plastic tissue culture flasks (Falcon Plastics, Oxnard, Calif.) were inoculated with 10-fold dilutions of virus (0.1 ml). After viral adsorption for 1 h at 37 C, the inoculated monolayers were overlaid with 1.5% carboxymethyl cellulose in culture medium supplemented with 1% serum and incubated at 37 C until plaques appeared (approximately 2 days for NDVo, 3 days for NDVpi). The monolayers were gently washed with phosphate-buffered saline (pH 7.2) to remove the semisolid overlay, stained and fixed for 5 min with 1% crystal violet in 200i methanol, and examined for the number of plaque-forming units per milliliter.
' Present address: Department of Pediatrics, Division of Infectious Diseases and Immunology, Emory University School of Medicine, 69 Butler Street, S.E., Atlanta, Ga. 30303. 2Present address: Department of Microbiology and Immunology, University of Oregon Health Science Center, Portland, Ore. 97201.
68
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RNA POLYMERASE ACTIVITY OF NDV MUTANTS
Cloning NDVo and NDVpi. A modification of the procedure described by Thacore and Youngner (12) was used to clone NDVo and NDVpi. Both NDVo and NDVpi were passed five times in BHK cells. Monolayers of BHK cells in plastic petri dishes (60 by 15 mm; Falcon Plastics) were then inoculated with approximately 10 PFU of NDVo or NDVpi. After 1 h of adsorption at 37X C, the adsorbed virus was removed from the monolayers with three PBS washings, and a methyl cellulose overlay was added. The methyl cellulose over well isolated plaques was removed and transferred to BHK cells in roller tubes containing 2.0 ml of maintenance medium. Viral clones were harvested from those cultures displaying cytopathic effect and examined for infectivity, characteristic plaque morphology, temperature sensitivity, and ability to elute from chicken RBC as previously described for NDVo and NDVpi (8, 12, 13, 14). The distinctive plaque morphology reported by Thacore and Youngner (12) for NDVo and NDVpi on chicken embryo cell cultures was also observed on BHK cells. One NDVo clone and three NDVpi clones (designated NDVpi-A, -B, and -C) were randomly selected, propagated in BHK cells, harvested in approximately 10 liters of culture fluid, and concentrated and purified as previously described (2). RNA polymerase assay. The reaction mixture for the transcriptase was similar to that described by Huang et al. (3). Disruption of NDVo and NDVpi proteins was accomplished with 0.4%/ (vol/vol) Triton X-100. The activity of [3H]GTP (0.001 gmol) was 1,200 counts/min per gmol. The incubation temperature and time intervals for the reaction mixtures are noted in the legends and footnotes. The reactions were terminated in an ice bath and assayed for acidprecipitable [3H ]GMP by the addition of 0.1 M sodium pyrophosphate, carrier yeast RNA (100 Mg), and trichloroacetic acid, followed by filtration through Selectron membrane filters (Schleicher and Schuell, Inc., Keene, N. H.); they were then counted in a Beckman liquid scintillation spectrometer with 2,5-diphenyloxazole- 1,4-bis-2-(5-phenyloxazolyl) benzene-toluene scintillation fluid. Qualitatively similar results were obtained when 0.1% or 0.05% (vol/vol) of Triton X-100 was used instead of 0.4% (vol/vol) in the polymerase assay with NDVo and NDVpi and NDVpi-B at 32 or 37 C; however, the activity at 42 C was significantly reduced with these lower concentrations of Triton X-100. Concentrations greater than 0.4% produced a slight inhibitory effect on the activity at the experimental temperatures used. All polymerase assays were performed on viral preparations immediately after concentration and purification. When purified NDVpi and NDVo clones were stored at -70 C, or at 4 C for 48 h, the polymerase activity was undetectable at all assay temperatures for the NDVpi preparations, and at 42 C for NDVo. The method of Lowry et al. (5) was used to determine viral protein. Bovine plasma albumin was used as a calibration standard. Complementarity of polymerase product to virion RNA. For hybridization experiments, RNA from
69
NDVo and NDVpi was extracted and purified as described by Duesberg and Robinson (1) and previously reported for this laboratory (2). The labeled products from either NDVo (7.18 x 103 counts/min) or NDVpi (4.55 x 103 counts/min) were mixed with 15 gg of virion RNA and annealed as described by Huang et al. (3). Samples were then chilled to 4 C and divided into two equal portions, one of which was treated with 50 Ag of RNase A for 30 min at 22 C. All samples were acid precipitated and assayed for [3lH ]GMP. Virion RNA was omitted from the controls. Reagents. Reagents were obtained from the following sources: [3H ]GTP from New England Nuclear, Boston, Mass.; RNase, DNase, unlabeled nucleoside triphosphates (ATP, CTP, UTP), Triton X-100, bovine albumin (crystalline form), actinomycin D, and yeast RNA from Sigma Chemical Co., St. Louis, Mo.; and carboxymethyl cellulose (4,000 centipoise) from Fisher Scientific Co., Pittsburg, Pa. All other chemicals were reagent grade.
RESULTS Characterization of cloned viruses. After concentration and purification, the NDVo and NDVpi clones were examined for their characteristic temperature sensitivity, hemagglutination from chicken RBC, and plaque morphology. The NDVo clone readily eluted from chicken RBC and gave similar viral yields at 37 and 42 C, whereas, with the NDVpi clones, less than 20% of the virus eluted from RBC and the viral yield at 42 C was appreciably impaired (Table 1). Concurrently at 37 C all the plaques produced by the NDVo clone were greater than 1.5 mm; at the same temperature the NDVpi clones produced plaques less than 1 mm. Kinetics of polymerase activity at 32, 37, and 42 C. A polymerase assay was performed at 32, 37, and 42 C on NDVo and NDVpi preparations. Optimal polymerase activities for all the viral preparations occurred after 60 min at 32 C, TABLE 1. Characterization of cloned viruses Viral
clone NDVo NDVpi-A NDVpi-B
NDVpi-C
PFU/mla 37 C 42 C 2.3 x 108 3.4 x 107 8.4 x 10, 2.7 x 10,
7.8 2.3 1.2 2.8
x 107 x 102 x 102 x 103
Hemagglutina- Plaque diamc tion elution '7( > 2 %7 < 1 mm (%) b mm 74.3 18.2 17.4
100 0 0
0 100 100
19.4
0
100
aBHK cells were inoculated with 100 PFU of the viral clones and incubated at either 37 or 42 C for 24 h. The released virus was harvested and assayed. bViral clones were adsorbed to chicken RBC for 1 h in an ice bath, eluted for 1 h at 37 C, and assayed for the percentage of infectious virus eluted. cThe diameter of the plaques produced by the viral clones was determined. The percentage of plaques characteristic of NDVo or NDVpi was recorded.
70
STANWICK AND HALLUM
J. VIROL.
with NDVo displaying the greatest activity (Fig. 1). An increase in incubation temperature to 37 C resulted in a marked decrease in activity for the temperature-sensitive mutants. Also, a decrease in GMP incorporation was observed for all viruses after 60 min at 37 C. After 6 min at 42 C the activity for the temperature-sensitive mutants was approximately 74 to 90% less than the activity after 60 min at 32 C. This decrease in polymerase activity coincided with the inability to replicate at 42 C. On the other hand, the activity of NDVo only decreased by 24% for the comparable time and temperature. No polymerase activity was detected in concentrated culture fluid from uninoculated BHK cells. Kinetics of polymerase activity at 42 C. So
that the thermolability of the temperature-sen50
sitive mutants could be confirmed in a different manner, the polymerase activity for all four viral clones was examined at 42 C. Compared to the activity for NDVo, a significant decline in the activities for the NDVpi preparations was observed (Fig. 2). The specific activity for NDVo was approximately four times that of the temperature-sensitive mutants. Several assays were performed at 42 C with varying amounts of NDVo and NDVpi-B so that it could be determined whether other factor(s) in the system might be involved in the apparent thermolability of the polymerase activity (Fig. 3). NDVpi-B, displaying the greatest thermolability, was chosen as a prototype for the temperature-sensitive mutants. The decrease in activity observed when equivalent activities of NDVo and NDVpi-B were incubated together
NDV. 32C
40
37C
//
30-
42C
z
0
fI
10
NDVpi-8
NOV pi -C
32C
-j
4
9
32C 7-
6
5
/742C
I
15
30
45
60
90
TIME (MIN)
FIG. 1. Kinetics of RNA polymerase activity for NDVo and three clones of NDVpi at 32, 37, and 42 C. The reaction mixture in a total volume of 0.1 ml consisted of the following components: 5 jmol of Tris (pH 7.3), 0.4 Amol of magnesium acetate, 0.3 pmol of dithiothreitol, 10 Mmol of NaCl, 0.07Mmol each of ATP, UTP, and CTP; 0.001 jsmol of [3HJGTP (1,200 counts/min per umol), 0.4% (vol/vol) of Triton X-100, and 4 $&g of viral protein. Samples were removed at the indicated time intervals, and the trichloroacetic acid-precipitable radioactivity was measured.
71
RNA POLYMERASE ACTIVITY OF NDV MUTANTS
VOL. 17, 1976
depended on the presence of CTP, UTP, Triton X-100, and dithiothreitol. The polymerase activity was sensitive to RNase but not to DNase or actinomycin D. When viral preparations were centrifuged to equilibrium in a potassium tartrate gradient, the polymerase activity coincided with the viral infectivity for both NDVo and NDVpi-B at respective buoyant densities of 1.20 g/cm3 and 1.21 g/cm3. The only detectable hemagglutination activity in the gradient coincided with the infectious bands. U
41
W 10
0
I, I
a
0
I
Ia
30
_
60
TIME AT 42 C (MIN) FIG. 2. Incubation of RNA polymerase at 42 C. The reaction mixture (0.3 ml) used for incubation of the polymerase consisted of the following: 33.3 pmol of Tris-hydrochloride (pH 7.3), 2.7 Amol of magnesium acetate, 50.6 Amol of NaCl, 0.7.umol of dithiothreitol, 0.88% Triton X-100, and 2.7 Ag of viral protein. At the indicated times, 0.045-ml volumes were removed from the reaction mixture, and ATP, CTP, UTP, and [3H]GTP were added to a total volume of 0.1 ml and the final concentrations per 0.1 ml as described in Fig. 1. The polymerase mixture was incubated at 32 C for 60 min and assayed for the incorporation of GMP. The polymerase activity at the various time intervals is presented in relation to the activity at time zero. The radioactivity of a reaction sample without virions (less than 14% of the complete system) was subtracted from all the experimental values. Symbols: 0, NDVo; 0, NDVpi-A; A, NDVpi-B; x, NDVpi-C.
(reaction iv) approximated that observed with NDVo alone (reaction i), suggesting that the factor(s) responsible for the thermolability of
NDVpi-B activity had no affect on an excess of polymerase activity. Furthermore, the addition of an equal amount of heat-treated NDVpi-B did not affect the activity of NDVpi-B alone (reaction v). Characterization of polymerase assay and the product. Enzymatic activity of reaction mixtures containing either NDVo or NDVpi
0
30
60
TIME AT 42 C (MIN) FIG. 3. Assays to detect the existence of a possible inhibitor of the RNA-polymerase activity at 42 C. Polymerase reactions were performed as described in Fig. 2. Six reaction mixtures containing the following amounts of NDVo and NDVpi-B were assayed for polymerase activity at the specified times: (i) NDVo, 6.67 p&g (0); (ii) NDVpi-B, 26.67 pg (0); (iii) NDVo, 26.67 pg (x); (iv) NDVo, 6.67 ug, and NDVpi, 26.67 jug, incubated together (0); (v) addition of untreated NDVpi-B, 0.4 jig, to the 35 C assay with NDVpi-B, 26.67 jig, after 42 C incubation (A); (vi) addition of untreated NDVo, 0.1 lig, to the 35 C assay with NDVo, 0.9 jig, after 42 C incubation (A). The radioactivity of a reaction system without virions (less than 10% of the complete mixtures) was subtracted from all the experimental values.
72
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J. VIROL.
Hybridization experiments demonstrated that the polymerase products from NDVo or NDVpi-B preparations annealed RNA from either viral preparation (Table 2). As reported by others, a high degree of self-annealing was also observed in these experiments (3, 7, 11).
DISCUSSION Direct measurement of transcriptase activity associated with three clones of NDVpi indicates that the temperature sensitivity of these mutants compared to NDVo is due to the thermolability of the viral-associated polymerase activity. Using an indirect measurement of viralspecific RNA synthesis in chicken fibroblasts infected with five NDVpi clones, Preble and Youngner (9) observed that the temperaturesensitive mutants appeared to fall into two groups, which they suggested may be due to two separate defects in the RNA polymerase. When they transferred their infected cultures from 37 to 42 C, RNA synthesis rapidly ceased with three of the clones; with the other two, RNA synthesis continued several hours after the transfer. With the technique used in our study we were unable to detect any difference in activities among the three selected NDVpi clones. Further evidence for the extreme lability of the polymerase activity of NDVpi compared to NDVo is that polymerase activity could not be detected when purified preparations of NDVpi were stored at 4 C or - 70 C for more than 48 h. Meager and Burke (6) reported that the polymerase activity of NDV was lost after storage at 4 C and restored by subsequent incubation at 37 C. We were able to reproduce this phenomenon only with the NDVo clone, and not the TABLE 2. Hybridization of polymerase products with RNA from NDVo or NDVpi Source of polymerase
product0
Annealed with RNA,
RNase
resistance
NDVo
None NDVpi NDVo
24 99 97
NDVpi
None NDVpi
28 96 102
NDVo
aStandard polymerase reaction mixtures containing virions of NDVo or NDVpi were terminated after 60 min at 32 C. b Polymerase products of NDVo and NDVpi were tested for annealing to RNA from either NDVo or NDVpi or annealed to no RNA (None).
NDVpi clone (Stanwick and Hallum, unpublished data). A decline in GMP incorporation was observed after 60 min at 32 and 37 C with the NDVpi
clones and at 37 and 42 C with the NDVo clone. The decline was still present when the [3H ]GMP concentrations were increased. Huang et al. (4), studying four strains other than the Herts strain of NDV, also detected an unexplained decline in the incorporation of GMP after the first 30 min at 37 C but not at 32 C. At this time, a reasonable explanation for this observation would be the degree to which NDVo and NDVpi polymerase activities are thermolabile. That less polymerase activity is associated with NDVpi than NDVo when both viral preparations are propagated at 37 C is another parameter distinguishing the two viruses; however, the possibility that the polymerase associated with NDVpi may be partially inactivated prior to harvest must also be considered. At this time it is unknown whether there is any correlation between the decreased RNA polymerase activity and the presence of RNA-dependent DNA polymerase activity in preparations of NDVpi, and whether they have any significance with regard to the persistence of NDVpi in L cells. ACKNOWLEDGMENTS This investigation was supported by the John A. Hartford Foundation, Inc., New York, N.Y.
LITERATURE CITED 1. Duesberg, R. H., and W. H. Robinson. 1965. Isolation of the nucleic acid of Newcastle disease virus. Proc. Natl. Acad. Sci. U.S.A. 54:794-800. 2. Furman, P. A., and J. V. Hallum. 1973. RNA-dependent DNA polymerase activity in preparations of a mutant of Newcastle disease virus arising from persistently infected L cells. J. Virol. 12:548-555. 3. Huang, A. S., D. Baltimore, and M. A. Bratt. 1971. Ribonucleic acid polymerase in virions of Newcastle disease virus: comparison with the vesicular stomatitis virus polymerase. J. Virol. 7:389-394. 4. Huang, A. S., D. Baltimore, and M. Stampfer. 1970. Ribonucleic acid synthesis of vesicular stomatitis virus. III. Multiple complementary messenger RNA molecules. Virology 42:946-957. 5. Lowry, 0. H., J. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 6. Meager, A., and D. C. Burke. 1973. Studies of the structural basis of the RNA polymerase activity of Newcastle disease virus particles. J. Gen. Virol. 18:305-317. 7. Porter, A., and D. W. Kingsbury. 1970. Complementary RNA's in paramyxoviruses and paramyxovirus-infected cells. Nature (London) 228:1196-1197. 8. Preble, 0. T., and J. S. Youngner. 1973. Temperaturesensitive mutants isolated from L cells persistently infected with Newcastle disease virus. J. Virol. 9:200-206.
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17, 1976
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9. Preble, 0. T., and J. S. Youngner. 1973. Temperaturesensitive defect of mutants isolated from L cells persistently infected with Newcastle disease virus. J. Virol. 12:472-480. 10. Preble, 0. T., and J. S. Youngner. 1973. Selection of temperature-sensitive mutants during persistent infection: role in maintenance of persistent Newcastle disease virus infections of L cells. J. Virol. 12:481-491. 11. Robinson, W. S. 1970. Self-annealing of subgroups 2 myxovirus RNA's. Nature (London) 225:994-995. 12. Thacore, H., and J. S. Youngner. 1969. Cells persistently infected with Newcastle disease virus. I. Properties of
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mutants isolated from persistently infected L cells. J.
Virol. 4:244-251. 13. Thacore, H. R., and J. S. Youngner. 1970. Cells persistently infected with Newcastle disease virus. II. Ribonucleic acid and protein synthesis in cells infected with mutants isolated from persistently infected L cells. J. Virol. 6:42-48. 14. Thacore, H. R., and J. S. Youngner. 1971. Cells persistently infected with Newcastle disease virus. III. Thermal stability of hemagglutinin and neuraminidase of a mutant isolated from persistently infected L cells. J. Virol. 7:53-58.
Vol. 17, No. 1 Printed in U.S.A.
Copyright 0 1976 American Society for Microbiology
Comparison of RNA Polymerase Associated With Newcastle Disease Virus and a Temperature-Sensitive Mutant of Newcastle Disease Virus Isolated From Persistently Infected L Cells TREVOR L. STANWICKI* AND J. V. HALLUM2 Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana 70112 Received for publication 16 July 1975
An in vitro comparison was made of the RNA polymerase activity associated with Newcastle disease virus (NDVo} and three clones of the temperature-sensitive mutant (NDVpi) isolated from persistently infected L cells. Less polymerase activity was associated with the NDVpi clones. Also, compared to NDVo, an increase in incubation temperature from 32 to 37 or 42 C resulted in a marked decrease in polymerase activity for the temperature-sensitive mutants which coincided with their inability to replicate at 42 C.
Thacore and Youngner (12) established a persistent infection of L (Lpi) cells with the Herts strain of Newcastle disease virus (NDV). These Lpi cells continually produce the mutant virus (NDVpi) which differs from the wild-type virus (NDVo) in that, among other phenotypic properties, it is temperature sensitive, forms smaller plaques on chicken embryo fibroblasts, and has a decreased ability to elute from chicken red blood cells (8, 12, 13, 14). Subsequently Preble and Youngner (9), using an indirect method to measure the RNA polymerase activity in NDVpi-infected cells at 42 C, found that clones of the temperature-sensitive mutants (NDVpi) had defects associated with RNA synthesis at the nonpermissive temperature. They suggested that selection of temperature-sensitive mutants may play a role in the establishment or maintenance of the persistent infection in Lpi cells (10). Recently in this laboratory an RNAdependent DNA polymerase was found in preparations of NDVpi but not NDVo (2). In this study three clones of NDVpi and one clone of NDVo, which displayed characteristic plaque morphology, temperature sensitivity, and the ability to elute from chicken red blood cells (RBC), were selected and assayed for RNA polymerase activity at 32, 37, and 42 C. Prepa-
rations of the NDVpi clones contained less polymerase activity than the NDVo clone. Also, the RNA polymerase activity associated with the NDVpi clones was more heat labile than that of the NDVo preparations. MATERIALS AND METHODS Cell cultures. Uninfected L cells and Lpi cells persistently infected with NDVpi were obtained from J. S. Youngner, University of Pittsburg Medical School, and subcultured as previously described (12). Baby hamster kidney-21, clone 13 (BHK) cells obtained from GIBCO (Grand Island Biological Co., N.Y.) were cultured in Eagle minimal essential medium supplemented with 10% fetal calf serum (Grand Island Biological Co.) for cell growth and 2% fetal calf serum for cell maintenance. Viruses. The Herts strain of NDVo was obtained from J. S. Youngner. Stock cultures of NDVo and NDVpi were propagated in BHK cells, harvested when cytopathic effect was observed, and stored at -70 C. Viral assay. A plaque assay on BHK cells was used to assay NDVo and NDVpi infectivity. Monolayers of BHK cells in 30-ml plastic tissue culture flasks (Falcon Plastics, Oxnard, Calif.) were inoculated with 10-fold dilutions of virus (0.1 ml). After viral adsorption for 1 h at 37 C, the inoculated monolayers were overlaid with 1.5% carboxymethyl cellulose in culture medium supplemented with 1% serum and incubated at 37 C until plaques appeared (approximately 2 days for NDVo, 3 days for NDVpi). The monolayers were gently washed with phosphate-buffered saline (pH 7.2) to remove the semisolid overlay, stained and fixed for 5 min with 1% crystal violet in 200i methanol, and examined for the number of plaque-forming units per milliliter.
' Present address: Department of Pediatrics, Division of Infectious Diseases and Immunology, Emory University School of Medicine, 69 Butler Street, S.E., Atlanta, Ga. 30303. 2Present address: Department of Microbiology and Immunology, University of Oregon Health Science Center, Portland, Ore. 97201.
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RNA POLYMERASE ACTIVITY OF NDV MUTANTS
Cloning NDVo and NDVpi. A modification of the procedure described by Thacore and Youngner (12) was used to clone NDVo and NDVpi. Both NDVo and NDVpi were passed five times in BHK cells. Monolayers of BHK cells in plastic petri dishes (60 by 15 mm; Falcon Plastics) were then inoculated with approximately 10 PFU of NDVo or NDVpi. After 1 h of adsorption at 37X C, the adsorbed virus was removed from the monolayers with three PBS washings, and a methyl cellulose overlay was added. The methyl cellulose over well isolated plaques was removed and transferred to BHK cells in roller tubes containing 2.0 ml of maintenance medium. Viral clones were harvested from those cultures displaying cytopathic effect and examined for infectivity, characteristic plaque morphology, temperature sensitivity, and ability to elute from chicken RBC as previously described for NDVo and NDVpi (8, 12, 13, 14). The distinctive plaque morphology reported by Thacore and Youngner (12) for NDVo and NDVpi on chicken embryo cell cultures was also observed on BHK cells. One NDVo clone and three NDVpi clones (designated NDVpi-A, -B, and -C) were randomly selected, propagated in BHK cells, harvested in approximately 10 liters of culture fluid, and concentrated and purified as previously described (2). RNA polymerase assay. The reaction mixture for the transcriptase was similar to that described by Huang et al. (3). Disruption of NDVo and NDVpi proteins was accomplished with 0.4%/ (vol/vol) Triton X-100. The activity of [3H]GTP (0.001 gmol) was 1,200 counts/min per gmol. The incubation temperature and time intervals for the reaction mixtures are noted in the legends and footnotes. The reactions were terminated in an ice bath and assayed for acidprecipitable [3H ]GMP by the addition of 0.1 M sodium pyrophosphate, carrier yeast RNA (100 Mg), and trichloroacetic acid, followed by filtration through Selectron membrane filters (Schleicher and Schuell, Inc., Keene, N. H.); they were then counted in a Beckman liquid scintillation spectrometer with 2,5-diphenyloxazole- 1,4-bis-2-(5-phenyloxazolyl) benzene-toluene scintillation fluid. Qualitatively similar results were obtained when 0.1% or 0.05% (vol/vol) of Triton X-100 was used instead of 0.4% (vol/vol) in the polymerase assay with NDVo and NDVpi and NDVpi-B at 32 or 37 C; however, the activity at 42 C was significantly reduced with these lower concentrations of Triton X-100. Concentrations greater than 0.4% produced a slight inhibitory effect on the activity at the experimental temperatures used. All polymerase assays were performed on viral preparations immediately after concentration and purification. When purified NDVpi and NDVo clones were stored at -70 C, or at 4 C for 48 h, the polymerase activity was undetectable at all assay temperatures for the NDVpi preparations, and at 42 C for NDVo. The method of Lowry et al. (5) was used to determine viral protein. Bovine plasma albumin was used as a calibration standard. Complementarity of polymerase product to virion RNA. For hybridization experiments, RNA from
69
NDVo and NDVpi was extracted and purified as described by Duesberg and Robinson (1) and previously reported for this laboratory (2). The labeled products from either NDVo (7.18 x 103 counts/min) or NDVpi (4.55 x 103 counts/min) were mixed with 15 gg of virion RNA and annealed as described by Huang et al. (3). Samples were then chilled to 4 C and divided into two equal portions, one of which was treated with 50 Ag of RNase A for 30 min at 22 C. All samples were acid precipitated and assayed for [3lH ]GMP. Virion RNA was omitted from the controls. Reagents. Reagents were obtained from the following sources: [3H ]GTP from New England Nuclear, Boston, Mass.; RNase, DNase, unlabeled nucleoside triphosphates (ATP, CTP, UTP), Triton X-100, bovine albumin (crystalline form), actinomycin D, and yeast RNA from Sigma Chemical Co., St. Louis, Mo.; and carboxymethyl cellulose (4,000 centipoise) from Fisher Scientific Co., Pittsburg, Pa. All other chemicals were reagent grade.
RESULTS Characterization of cloned viruses. After concentration and purification, the NDVo and NDVpi clones were examined for their characteristic temperature sensitivity, hemagglutination from chicken RBC, and plaque morphology. The NDVo clone readily eluted from chicken RBC and gave similar viral yields at 37 and 42 C, whereas, with the NDVpi clones, less than 20% of the virus eluted from RBC and the viral yield at 42 C was appreciably impaired (Table 1). Concurrently at 37 C all the plaques produced by the NDVo clone were greater than 1.5 mm; at the same temperature the NDVpi clones produced plaques less than 1 mm. Kinetics of polymerase activity at 32, 37, and 42 C. A polymerase assay was performed at 32, 37, and 42 C on NDVo and NDVpi preparations. Optimal polymerase activities for all the viral preparations occurred after 60 min at 32 C, TABLE 1. Characterization of cloned viruses Viral
clone NDVo NDVpi-A NDVpi-B
NDVpi-C
PFU/mla 37 C 42 C 2.3 x 108 3.4 x 107 8.4 x 10, 2.7 x 10,
7.8 2.3 1.2 2.8
x 107 x 102 x 102 x 103
Hemagglutina- Plaque diamc tion elution '7( > 2 %7 < 1 mm (%) b mm 74.3 18.2 17.4
100 0 0
0 100 100
19.4
0
100
aBHK cells were inoculated with 100 PFU of the viral clones and incubated at either 37 or 42 C for 24 h. The released virus was harvested and assayed. bViral clones were adsorbed to chicken RBC for 1 h in an ice bath, eluted for 1 h at 37 C, and assayed for the percentage of infectious virus eluted. cThe diameter of the plaques produced by the viral clones was determined. The percentage of plaques characteristic of NDVo or NDVpi was recorded.
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with NDVo displaying the greatest activity (Fig. 1). An increase in incubation temperature to 37 C resulted in a marked decrease in activity for the temperature-sensitive mutants. Also, a decrease in GMP incorporation was observed for all viruses after 60 min at 37 C. After 6 min at 42 C the activity for the temperature-sensitive mutants was approximately 74 to 90% less than the activity after 60 min at 32 C. This decrease in polymerase activity coincided with the inability to replicate at 42 C. On the other hand, the activity of NDVo only decreased by 24% for the comparable time and temperature. No polymerase activity was detected in concentrated culture fluid from uninoculated BHK cells. Kinetics of polymerase activity at 42 C. So
that the thermolability of the temperature-sen50
sitive mutants could be confirmed in a different manner, the polymerase activity for all four viral clones was examined at 42 C. Compared to the activity for NDVo, a significant decline in the activities for the NDVpi preparations was observed (Fig. 2). The specific activity for NDVo was approximately four times that of the temperature-sensitive mutants. Several assays were performed at 42 C with varying amounts of NDVo and NDVpi-B so that it could be determined whether other factor(s) in the system might be involved in the apparent thermolability of the polymerase activity (Fig. 3). NDVpi-B, displaying the greatest thermolability, was chosen as a prototype for the temperature-sensitive mutants. The decrease in activity observed when equivalent activities of NDVo and NDVpi-B were incubated together
NDV. 32C
40
37C
//
30-
42C
z
0
fI
10
NDVpi-8
NOV pi -C
32C
-j
4
9
32C 7-
6
5
/742C
I
15
30
45
60
90
TIME (MIN)
FIG. 1. Kinetics of RNA polymerase activity for NDVo and three clones of NDVpi at 32, 37, and 42 C. The reaction mixture in a total volume of 0.1 ml consisted of the following components: 5 jmol of Tris (pH 7.3), 0.4 Amol of magnesium acetate, 0.3 pmol of dithiothreitol, 10 Mmol of NaCl, 0.07Mmol each of ATP, UTP, and CTP; 0.001 jsmol of [3HJGTP (1,200 counts/min per umol), 0.4% (vol/vol) of Triton X-100, and 4 $&g of viral protein. Samples were removed at the indicated time intervals, and the trichloroacetic acid-precipitable radioactivity was measured.
71
RNA POLYMERASE ACTIVITY OF NDV MUTANTS
VOL. 17, 1976
depended on the presence of CTP, UTP, Triton X-100, and dithiothreitol. The polymerase activity was sensitive to RNase but not to DNase or actinomycin D. When viral preparations were centrifuged to equilibrium in a potassium tartrate gradient, the polymerase activity coincided with the viral infectivity for both NDVo and NDVpi-B at respective buoyant densities of 1.20 g/cm3 and 1.21 g/cm3. The only detectable hemagglutination activity in the gradient coincided with the infectious bands. U
41
W 10
0
I, I
a
0
I
Ia
30
_
60
TIME AT 42 C (MIN) FIG. 2. Incubation of RNA polymerase at 42 C. The reaction mixture (0.3 ml) used for incubation of the polymerase consisted of the following: 33.3 pmol of Tris-hydrochloride (pH 7.3), 2.7 Amol of magnesium acetate, 50.6 Amol of NaCl, 0.7.umol of dithiothreitol, 0.88% Triton X-100, and 2.7 Ag of viral protein. At the indicated times, 0.045-ml volumes were removed from the reaction mixture, and ATP, CTP, UTP, and [3H]GTP were added to a total volume of 0.1 ml and the final concentrations per 0.1 ml as described in Fig. 1. The polymerase mixture was incubated at 32 C for 60 min and assayed for the incorporation of GMP. The polymerase activity at the various time intervals is presented in relation to the activity at time zero. The radioactivity of a reaction sample without virions (less than 14% of the complete system) was subtracted from all the experimental values. Symbols: 0, NDVo; 0, NDVpi-A; A, NDVpi-B; x, NDVpi-C.
(reaction iv) approximated that observed with NDVo alone (reaction i), suggesting that the factor(s) responsible for the thermolability of
NDVpi-B activity had no affect on an excess of polymerase activity. Furthermore, the addition of an equal amount of heat-treated NDVpi-B did not affect the activity of NDVpi-B alone (reaction v). Characterization of polymerase assay and the product. Enzymatic activity of reaction mixtures containing either NDVo or NDVpi
0
30
60
TIME AT 42 C (MIN) FIG. 3. Assays to detect the existence of a possible inhibitor of the RNA-polymerase activity at 42 C. Polymerase reactions were performed as described in Fig. 2. Six reaction mixtures containing the following amounts of NDVo and NDVpi-B were assayed for polymerase activity at the specified times: (i) NDVo, 6.67 p&g (0); (ii) NDVpi-B, 26.67 pg (0); (iii) NDVo, 26.67 pg (x); (iv) NDVo, 6.67 ug, and NDVpi, 26.67 jug, incubated together (0); (v) addition of untreated NDVpi-B, 0.4 jig, to the 35 C assay with NDVpi-B, 26.67 jig, after 42 C incubation (A); (vi) addition of untreated NDVo, 0.1 lig, to the 35 C assay with NDVo, 0.9 jig, after 42 C incubation (A). The radioactivity of a reaction system without virions (less than 10% of the complete mixtures) was subtracted from all the experimental values.
72
STANWICK AND HALLUM
J. VIROL.
Hybridization experiments demonstrated that the polymerase products from NDVo or NDVpi-B preparations annealed RNA from either viral preparation (Table 2). As reported by others, a high degree of self-annealing was also observed in these experiments (3, 7, 11).
DISCUSSION Direct measurement of transcriptase activity associated with three clones of NDVpi indicates that the temperature sensitivity of these mutants compared to NDVo is due to the thermolability of the viral-associated polymerase activity. Using an indirect measurement of viralspecific RNA synthesis in chicken fibroblasts infected with five NDVpi clones, Preble and Youngner (9) observed that the temperaturesensitive mutants appeared to fall into two groups, which they suggested may be due to two separate defects in the RNA polymerase. When they transferred their infected cultures from 37 to 42 C, RNA synthesis rapidly ceased with three of the clones; with the other two, RNA synthesis continued several hours after the transfer. With the technique used in our study we were unable to detect any difference in activities among the three selected NDVpi clones. Further evidence for the extreme lability of the polymerase activity of NDVpi compared to NDVo is that polymerase activity could not be detected when purified preparations of NDVpi were stored at 4 C or - 70 C for more than 48 h. Meager and Burke (6) reported that the polymerase activity of NDV was lost after storage at 4 C and restored by subsequent incubation at 37 C. We were able to reproduce this phenomenon only with the NDVo clone, and not the TABLE 2. Hybridization of polymerase products with RNA from NDVo or NDVpi Source of polymerase
product0
Annealed with RNA,
RNase
resistance
NDVo
None NDVpi NDVo
24 99 97
NDVpi
None NDVpi
28 96 102
NDVo
aStandard polymerase reaction mixtures containing virions of NDVo or NDVpi were terminated after 60 min at 32 C. b Polymerase products of NDVo and NDVpi were tested for annealing to RNA from either NDVo or NDVpi or annealed to no RNA (None).
NDVpi clone (Stanwick and Hallum, unpublished data). A decline in GMP incorporation was observed after 60 min at 32 and 37 C with the NDVpi
clones and at 37 and 42 C with the NDVo clone. The decline was still present when the [3H ]GMP concentrations were increased. Huang et al. (4), studying four strains other than the Herts strain of NDV, also detected an unexplained decline in the incorporation of GMP after the first 30 min at 37 C but not at 32 C. At this time, a reasonable explanation for this observation would be the degree to which NDVo and NDVpi polymerase activities are thermolabile. That less polymerase activity is associated with NDVpi than NDVo when both viral preparations are propagated at 37 C is another parameter distinguishing the two viruses; however, the possibility that the polymerase associated with NDVpi may be partially inactivated prior to harvest must also be considered. At this time it is unknown whether there is any correlation between the decreased RNA polymerase activity and the presence of RNA-dependent DNA polymerase activity in preparations of NDVpi, and whether they have any significance with regard to the persistence of NDVpi in L cells. ACKNOWLEDGMENTS This investigation was supported by the John A. Hartford Foundation, Inc., New York, N.Y.
LITERATURE CITED 1. Duesberg, R. H., and W. H. Robinson. 1965. Isolation of the nucleic acid of Newcastle disease virus. Proc. Natl. Acad. Sci. U.S.A. 54:794-800. 2. Furman, P. A., and J. V. Hallum. 1973. RNA-dependent DNA polymerase activity in preparations of a mutant of Newcastle disease virus arising from persistently infected L cells. J. Virol. 12:548-555. 3. Huang, A. S., D. Baltimore, and M. A. Bratt. 1971. Ribonucleic acid polymerase in virions of Newcastle disease virus: comparison with the vesicular stomatitis virus polymerase. J. Virol. 7:389-394. 4. Huang, A. S., D. Baltimore, and M. Stampfer. 1970. Ribonucleic acid synthesis of vesicular stomatitis virus. III. Multiple complementary messenger RNA molecules. Virology 42:946-957. 5. Lowry, 0. H., J. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 6. Meager, A., and D. C. Burke. 1973. Studies of the structural basis of the RNA polymerase activity of Newcastle disease virus particles. J. Gen. Virol. 18:305-317. 7. Porter, A., and D. W. Kingsbury. 1970. Complementary RNA's in paramyxoviruses and paramyxovirus-infected cells. Nature (London) 228:1196-1197. 8. Preble, 0. T., and J. S. Youngner. 1973. Temperaturesensitive mutants isolated from L cells persistently infected with Newcastle disease virus. J. Virol. 9:200-206.
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RNA POLYMERASE ACTIVITY OF NDV MUTANTS
9. Preble, 0. T., and J. S. Youngner. 1973. Temperaturesensitive defect of mutants isolated from L cells persistently infected with Newcastle disease virus. J. Virol. 12:472-480. 10. Preble, 0. T., and J. S. Youngner. 1973. Selection of temperature-sensitive mutants during persistent infection: role in maintenance of persistent Newcastle disease virus infections of L cells. J. Virol. 12:481-491. 11. Robinson, W. S. 1970. Self-annealing of subgroups 2 myxovirus RNA's. Nature (London) 225:994-995. 12. Thacore, H., and J. S. Youngner. 1969. Cells persistently infected with Newcastle disease virus. I. Properties of
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mutants isolated from persistently infected L cells. J.
Virol. 4:244-251. 13. Thacore, H. R., and J. S. Youngner. 1970. Cells persistently infected with Newcastle disease virus. II. Ribonucleic acid and protein synthesis in cells infected with mutants isolated from persistently infected L cells. J. Virol. 6:42-48. 14. Thacore, H. R., and J. S. Youngner. 1971. Cells persistently infected with Newcastle disease virus. III. Thermal stability of hemagglutinin and neuraminidase of a mutant isolated from persistently infected L cells. J. Virol. 7:53-58.
