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Synthesis of TMV RNA at restrictive high temperatures
VIROLOGY
73, 319-326
Synthesis
(1976)
of TMV RNA at Restrictive
High Temperatures
W. 0. DAWSON Department
of Plant Pathology and Cell Interaction Group, Riverside, California 92502 Accepted April
University
of California,
2,1976
The primary effect of high temperatures that restrict tobacco mosaic virus (TMV) multiplication was an immediate inhibition of synthesis and a more gradual degradation of single-stranded TMV RNA. Incorporation of radioactive precursors into doublestranded TMV RNA, replicative form (RF) and replicative intermediate (RI), was affected much less. Immediately after a step-up shift from 25 to 40”, single-stranded TMV-RNA synthesis was almost totally prevented, whereas there was little inhibition of incorporation into RF or RI. However, incorporation of 32P into double-stranded TMV RNA rapidly declined after the synthesis of single-stranded TMV RNA had stopped. At high temperatures that were restrictive for TMV multiplication the function responsible for incorporation of 3zP into single-stranded viral RNA was uncoupled from that responsible for incorporation into viral double-stranded RNA. INTRODUCTION
It has been demonstrated that tobacco mosaic virus (TMV) does not multiply at high temperatures. Yarwood (1952) defined the maximum temperature permitting TMV multiplication in tobacco as 37”. Lebeurier and Hirth (1966) measured the time course of TMV accumulation in tobacco leaf discs incubated at different temperatures. Between 16 and 32”, TMV accumulated to maximal concentrations; but at 34 and 36”, only about one-tenth as much virus accumulated. In an effort to determine what function is sensitive to the high temperature, TMV RNA synthesis is examined at high temperatures that prevent TMV multiplication. It will be demonstrated that the primary effect of the restrictive temperatures is inhibition of single-stranded TMV RNA synthesis. MATERIALS
AND
METHODS
Culture conditions. Young tobacco leaves (Nicotiana tabacum L. var. Xanthi) were “systemically inoculated” at 3” with TMV, strain Ul, using the differential temperature procedure described previously (Dawson et al., 1975). Virus repli-
cation was initiated by moving the plants into a plant growth chamber maintained at 25”. All subsequent experiments were conducted in plant growth chambers with a 14-hr photoperiod of 20,000 lux at the temperature designated in the Results. Labeling procedure. “Systemically inoculated” leaves were detached and the whole leaves were submerged and vacuum infiltrated in 60 @i/ml of H,32P0, in 0.1 mM KPO, buffer, pH. 7.0, and 65 pg/ml of actinomycin D; they were then maintained in that solution on ice for 10 min. The leaves were then removed to petri dishes and incubated at the designated temperature at 20,000 lux. The labeling period was terminated by freezing the tissue at -20”. RNA extraction. Tissue (3-4 g) frozen with liquid nitrogen was powdered with a mortar and pestle. The fine powder was removed to a 50-ml beaker containing 10 ml of 0.01 M Tris-HCl, pH 7.6, 0.05 M NaCl, 1% sodium lauryl sulfate (SLS), and 10 ml of water-saturated phenol containing 10% m-cresol and 0.1% 8-hydroxyquinoline. The solution was stirred 15 min at room temperature. The aqueous phase was recovered aRer centrifugation at 319
Copyright All rights
0 1976 by Academic F’ress, Inc. of reproduction in any form reserved.
320
W. 0. DAWSON
10,000 g for 10 min, made 0.5 M NaCl, and stirred 15 min at room temperature after addition of 10 ml of the above phenol solution. After centrifugation, the aqueous phase was precipitated by the addition of 2 volumes of ethanol and left standing at -20” overnight. The precipitate was dissolved in 2 ml of 0.05 M Tris-HCl, pH 6.65, 0.1 M NaCl, 0.001 M EDTA, 0.1% n-lauroyl sarcosine, and dialyzed against that buffer overnight. This solution was then precipitated with 2 volumes of ethanol, dissolved in 1 .O ml of 0.01 M MgCl, and incubated 1 hr at 37” with 50 pglml of DNAse (RNAse-free), after which the solution was made 2 N LiCl and stored overnight at 5”. The precipitate was collected by centrifugation at 10,000 g for 10 min and was dissolved in 1.0 ml E buffer (0.04 M Tris-acetate, 0.02 M sodium acetate, 0.002 M EDTA, pH 7.8) containing 0.2% SLS and 20% sucrose. This fraction (50 ~1) was analyzed by electrophoresis to examine single-stranded TMV RNA. To analyze RI-core, 0.8 ml of this solution was precipitated with 2 volumes of ethanol, dissolved in 0.5 ml of 0.2 M NaCl, 0.01 M Tris-HCl, pH 7.6, 0.01 M MgCl, and incubated with RNAse A (5 pglml) plus RNAse T, (0.5 pg/ ml) at 37” for 30 min. This fraction was precipitated by addition of 2 volumes of ethanol and dissolved in 0.3 ml of E buffer plus 0.2% SLS and 20% sucrose and 0.2 ml was analyzed by polyacrylamide gel electrophoresis. RF was analyzed by precipitation of the 2 N LiCl supernatant by adding 2 volumes of ethanol and dissolving the precipitate in 0.3 ml of E buffer plus 0.2% SLS and 20% sucrose and 0.2 ml was analyzed by polyacrylamide gel electrophoresis. The homogenization procedure was identical except that frozen tissue was blended in a Virtis homogenizer for 1 min. Gel electrophoresis. Polyacrylamide gel electrophoresis was performed on 2.5% polyacrylamide gels by the procedure of Loening (1967) but with the addition of 0.2% SLS to the electrophoresis buffer. After electrophoresis, the gels were scanned at 260 nm with a Beckman Acta II spectrophotometer equipped with a gel scanner. Determination of radioactiuity. Gels
were sliced into 2-mm slices and put into 10 ml of 2.5 mill 7-amino-1,3-naphthalenedisulfonic acid, and radioactivity was counted in a liquid scintillation counter by Cerenkov radiation (Lauchli, 1969). RESULTS
Synthesis of TMV RNA at High Temperatures Tobacco leaves synchronously infected with TMV by “systemic inoculation” at 3” (Dawson et al., 1975) were shifted to 25,36, or 40” and incubated for 24 hr. The leaves were then incubated with 32P plus actinomycin D for 2 hr at the same temperatures. Incorporation of 32P into progeny single-stranded TMV RNA, replicative form double-stranded RNA (RF), and that portion of the replicative intermediate that is resistant to RNAse (RI-core) was determined (Nilsson-Tillgren, 1970; Jackson et al., 1971). At 25”, 90% of the virusspecific incorporation of 32P was found in TMV RNA, with only 6% in RF and 4% in RI-core (Fig. 1). At 36”, substantially less label was incorporated into TMV RNA; at 40”, none could be detected. Approximately 14% as much TMV RNA was labeled at 36” as at 25”; however, 2.5 times as much RF and 1.6 times as much RI-core was produced at 36” as at 25”. At 36”, proportionally more double-stranded RNA was made with approximately 24% of the virus-specific incorporation occurring in TMV RNA, 51% in RF, and 25% in RI-core. At 40”, there was no evidence of virus synthesis although host RNA synthesis continued with no evidence of excessive breakdown. Viral RNA Synthesis after a Shift to High Temperatures Step-up temperature shift experiments were done to examine the temperature sensitivity of an active, replicating system. Leaves “systemically inoculated” at 3” were incubated at 25” for 24 hr, at which time the infection reached the rapid, linear phase of TMV replication. The leaves then were shifted to 25, 36, 38, 40, or 45”, and RNA was labeled with 32P for 2 hr beginning immediately after the shift. At progressively higher temperatures, there was less incorporation into TMV
TMV
RNA
SYNTHESIS
AT HIGH
321
TEMPERATURES
RI-core
600
400 600
1400
f
36O ss
600-
36O RI-core
400-
3000
4o” RF
2000
4o” 600-
RI-core
400-
600
FRACTION
NUMBER
FIG. 1. Incorporation of 32P into TMV RNA of leaves maintained continuously at 2536, or 40”. “Systemitally inoculated” leaves were incubated 24 hr at constant temperature and then labeled 2 hr at that temperature. RNA was extracted and fractionated into total 2 N LiCl precipitate (SS TMV RNA), 2 N LiCl supernatant (RF), and RNAse resistant 2 N LiCl precipitate (RI-core) fractions, as described in Materials and Methods. Samples consisting of 5, 67, and 53% of TMV RNA, RF, and RI-core, respectively, were analyzed. Each fraction was electrophoresed 3 hr at 7 mA per gel on 2.5% polyacrylamide gels. The tops of the gels are on the left.
RNA (Fig. 2). In relation to the amount of incorporation at 25”, there was only 25% as much incorporation at 38”, 0.6% as much at 40”, and none at 45” (The profiles of the different species of RNA in Fig. 2 represent different amounts of the total RNA samples. The amount of incorporation into each species is normalized in Table 1.) Although SS TMV RNA synthesis was dramatically inhibited at the higher temperatures, incorporation into double-stranded RNA was only slightly affected. At 36”, the incorporation of 32P into RF and RI-core was more than at 25”. At even higher temperatures, incorporation into RF and RIcore was reduced much less than that into TMV RNA. At 40”, for example, TMV RNA incorporated only 0.6% as much radioactivity as it did at 25”; but there was
58% as much incorporation into RF and 76% as much incorporation into RI-core. In one experiment at 50”, a small amount of label was incorporated into RF only (data not shown). It is interesting to look at the ratio of each species of virus RNA labeled at the different temperatures (Table 1). At 25”, 90% of the incorporation of 32P was found in TMV RNA; at 36 and 38”, about one-half of the incorporation was in single strands and one-half in double strands; at 40”, 51% of the incorporation was in RF and 41% in RI-core. All of the label at 45” was found in double-stranded RNA. Thus, in the presence of actinomycin D, the bulk of 32P incorporated into RNA of infected cells at high temperatures was found in viral double-stranded RNA.
322
W. 0. DAWSON r 1400-
1000
25O RF
25’ RI-core
600
400
_I 1400-
36”
\
36O
36O RI-core
600IOOO-
1400-
z
IOOO-
a 0
600-
360 ss
380 3000
-
600
-
36” RI-core
I
400-
200
1400-
40’ ss
400 RF
3000-
40” RI-core
600-
IOOO2000-
400-
600200t\ 450 RF
400
IO
20
30
IO
FRACTION
20
30
45O RI-core
600-
-
‘““kh-__ IO
20
30
NUMBER
FIG. 2. Incorporation of s2P into TMV RNA at high temperatures immediately after a shift from 25”. “Systemically inoculated” leaves incubated 24 hr at 25” were shifted to a higher temperature and labeled 2 hr with 32P beginning immediately after the temperature shift. RNA was extracted, fractionated, and electrophoresed as in Fig. 1. The upper-left graph shows the position of the host ribosomal RNA CM = lo-” daltons).
Kinetics of Incorporation into Viral after Step-up to 40”
RNA
The shift from 25 to 40” provided conditions that almost totally prevented TMV RNA synthesis, while only slightly affect-
ing incorporation of label into RF and RIcore when labeled immediately after the temperature shift. Next, the rate of incorporation of 32P into TMV RNA was examined at different times after the step-up temperature shift. Beginning at 0, l/4,2,4,
TMV
INCORPORATION Temperature during labeling (“)
RNA
OF 32P INTO TMV
Total cpm in TMV TMV
RNA
SYNTHESIS
AT HIGH
TABLE 1 RNA AFTER A STEP-UP
6.7 1.7 5.0 3.8
x x x x 0
RNA”
RF
104 IO’ 101 1W
4.5 6.5 3.4 2.6 0.4
x
x x x x
RIIO3 lo3 lo3 103 103
TEMPERATURE
Percentage of incorporation at 25” TMV
RNA
RF
core
25 36 38 40 45
323
TEMPERATURES
2.7 3.3 2.6 2.1 0.4
x x x x x
Percentage of cpm in each species RI-
“TMV
103 103 101 103 103
25 7 0.6 0
8, and 12 hr after the shift to 40”, tissue was incubated with 32P for 2 hr. The results are shown in Table 2. When the incorporation began immediately after the shift to 40”, TMV RNA incorporation was reduced 99%, while that of RF and RIcore was reduced 42 and 24%, respectively. The TMV RNA synthesis that did occur at 40” was produced during the first few minutes after the shift. When the labeling period began 15 min or more after the temperature step-up, no synthesis of TMV RNA occurred. In another experiment, the RNA was labeled beginning l/2 hr after the temperature shift for a shorter pulse (5 min) to overcome breakdown of TMV RNA that occurs at 40” (see next section): no 32P was incorporated into TMV RNA during the 5-min pulse. The rate of incorporation of 32P into RF and RI-core also decreased rapidly with time after the step-up to 40”. The greatest decline occurred during the first 15 min. Between 0 and 15 min at 40*, the rate of TABLE
Time of initiation of labeling after shift from 25” to 40”’ (hr)
2
-2
(labeled
at 25”)
0 114
a Two-hour
TMV
RNA
6.6 x 10’
385 0 0 0 0 0
2 4 8 12 labeling
RNA AFTER
Total cpm into SiAated
period.
into TMV
RI-
RF
2686 2062 215 114 58 9
4518 2588 712 334 182 52 52
core
19
RNA
RF
core
144 76 56 9
122 95 78 15
a “Systemically inoculated’ leaves incubated 24 hr at 25” were shifted to a higher temperature beginning immediately after the temperature shift. * Total counts per minute in virus-specific peaks in Fig. 2 in excess of that in healthy control and normalized to a 3-g sample
RATE OF 32P INCORPORATION INTO TMV SHIFT FROM 25 to 40”
Shift”
RIcore
90.3 63.4 45.5 7.5 0
6.1 24.3 30.9 51.2 50.0
3.6 12.3 23.6 41.3 50.0
and labeled 2 hr with 32P samples were determined
incorporation into RF decreased 73% and that into RI-core 88%. The rate of each continued to decrease with time until 8 hr, after which the rates remained relatively constant. Although the incorporation into TMV RNA completely stopped at 40”, that into RF or RI-core did not stop completely. However, after 8 hr the rate of incorporation into each double-stranded molecule was approximately 1% of that at 25”. Although incorporation into RF and RI-core rapidly decreased with time at 40”, their ratio of incorporation remained constant at 3 RF to 1 RI-core after the first 15 min. Stability
of TMV RNA at 40”
The above sections demonstrate that 40 inhibits the synthesis of TMV RNA and that most of the synthesis that occurs at 40” does so during the first 15 min after the temperature shift. In fact, all of the singlestranded TMV RNA incorporation occurred during this period. To examine the turnover of label incorporated into the different species of TMV RNA, the kinetics of incorporation in relation to length of labeling period were determined. Since most of the incorporation occurred immediately aRer the shift to 40”, each labeling period began at that time (Fig. 3). Single stranded TMV RNA incorporated more isotope during the 1Bmin label than the 5min label, demonstrating that, although synthesis of TMV RNA stopped by 15 min (Table 21, it continued beyond 5 min. Longer labeling periods demonstrated that TMV RNA broke down. The amount of radioactive label incorporated into single strands decreased with length of label greater than 15 min until after 10 hr no
324
W. 0. DAWSON 3000. ,8’------------*o
RF
/ If 2000~
. /-
.
;\
lation, breaks into a few relatively discrete pieces. It was also demonstrated that although the RI-core extracted with liquid nitrogen is a relatively homogeneous, “RFlike” molecule (Fig. 4A), when it was ex-
\
E a u
\ RI-:ore
i .
1000 nI
Length and temperature of labeling
’
[ \y-\Tss 1 42 HOURS
4 OF LABEL
TABLE 3 TMV RNAAT~OO
BREAKDOWNOF
IO AT 40”
FIG. 3. The incorporation of 52P into TMV RNA at 40” as a function of time of label. “Systemically inoculated” leaves incubated 24 hr at 25” were shifted to 40” and labeled with 32P for GO, l/4, 2, 4, and 10 hr beginning immediately aRer the shift. RNA was fractionated and electrophoresed as in Fig. 1, and the counts in each peak were summed.
incorporation was found in the area of TMV RNA. The amount of incorporation into RF and RI-core reached a maximum at 2 hr, when the synthesis rates of both species had decreased to a low level. The amount of incorporation in RF remained relatively constant afterwards, demonstrating that it was stable at 40”. However, there was some turnover of RI-core at 40”. Table 3 summarizes the results of another experiment to examine the breakdown of TMV RNA at 40”. TMV RNA was labeled with 32P for 2 hr at 25” and then shifted to 40” for 2 additional hours. RNA labeled in this manner was compared to the sum of the incorporation of RNA labeled only at 25” and only at 40”. In two experiments, the amount of TMV RNA that broke down during 2 hr at 40” was 92 and 46%, respectively; RF and RI-core did not break down in this experiment. Resistance ofRI Labeled at 40” to Shear In another communication (Dawson et al., 1976), we have demonstrated that TMV RF consists of two components: one, comprising about lo-20% of the population, is resistant to shear by homogenization in a Virtis homogenizer; another, composed of the about 80-90% of the popu-
Counts perg$;te Expt.
1. 2. 3. 4.
2hrat25 2hrat40” Sumof1+2 2 hr at 25” and 2 hr at 40”
5. &
6.7 4.0 6.7 5.5
1
Expt. 2
lo4 10” lo4 10”
4.7 1.0 4.8 2.6
8.2%
x 100 (o/o)
5 a "
x x x x
in TMV
x x x x
10’ lo” 10” 10’
54.2%
500 400 300 200 100 700
c
600 500 400 300 200 100 L 30 FRAd:lO;ON”MBER FIG. 4. Effect of shear upon TMV RI-core. Tissue as in Fig. 1 was labeled 2 hr at 25” (A, B) or 40” (C). RNA was extracted from tissue frozen with liquid nitrogen and powdered with a mortar and pestle (A) or from tissue homogenized in a Virtis homogenizer (B, C!). The 2 N LiCl precipitate fraction was RNAsetreated and electrophoresed 3 hr at 7 mA per gel on 2.5% polyacrylamide gels.
TMV
RNA
SYNTHESIS
AT HIGH
tracted by Virtis homogenization it broke, leaving few molecules as large as RF (Fig. 4B). However, when RI-core from tissue labeled at 40” was isolated by the Virtis homogenization procedure, a substantial portion of the RI molecules were resistant to shear and electrophoresed as RF-like molecules (Fig. 40. DISCUSSION
Incubation of infected tobacco plants at high temperatures that restrict TMV multiplication markedly altered the profile of viral RNA synthesis. Although TMV multiplied when continuously incubated at 36”, incorporation of 32P into TMV RNA was dramatically reduced while that into RF and RI was not. In plants maintained at 40”, there was no evidence of synthesis of any TMV RNA species. To examine an active replicating system for the functions that are sensitive to high temperatures, step-up temperature shiR experiments were chosen. Immediately after the shift to high temperatures, there was a dramatic decrease in the amount of 39P incorporated into single-stranded TMV RNA, paralleled by little effect upon RF and RI. In leaves shifted to 40” there was almost complete inhibition of incorporation into TMV RNA with only slight reduction in incorporation into RF or RI. This is in contrast to no synthesis of any species of TMV RNA in plants incubated continuously at 40”. Kinetics of incorporation of 32P into TMV RNA at 40” demonstrated that the high temperature had two effects upon TMV RNA. One was that synthesis of TMV RNA was completely blocked at 40”. There was a slight amount of incorporation during the first 15 min after the temperature shift, but afterwards incorporation totally stopped. The second effect was that TMV RNA broke down at 40”. Incorporation of radioisotope into this species of RNA during long labeling periods was less than during shorter labeling periods and TMV RNA synthesized at 25” broke down when incubated at 40”. Poliovirus behaves similarly at 40” (Mikkejeva and Ghendon, 1974). In cells incubated continuously at 40”, only l-2% as much virus was synthesized as at 36”. In cells shifted from 36 to
TEMPERATURES
325
40”, synthesis of progeny single-stranded poliovirus RNA decreased to 7% after 3045 min. Also, the single-stranded RNA of poliovirus broke down at 40”, with only one-half as much incorporation during a lhr labeling pulse as compared to the sum of four successive 15-min pulses. The high temperature “uncoupled” the function that is responsible for incorporation of nzP into TMV RNA from those that incorporate label into RF and RI. Immediately after the shift to 40”, the synthesis of TMV RNA was almost totally inhibited, whereas incorporation into RF and RI was only slightly affected. Within 15 min at 40”, synthesis of TMV RNA stopped. After TMV RNA synthesis stopped, the incorporation rates of RF and RI rapidly decreased, but did not stop. This suggests that there are at least two functions of TMV RNA synthesis that are differentially sensitive to high temperature: (1) a sensitive function that produces progeny single strands, and (2) a more resistant function that allows continued incorporation into double strands. Synthesis of TMV RNA at restrictive temperatures is similar to that reported for a temperature sensitive poliovirus mutant (Cooper et CZZ.,1970). When cells infected with the mutant k-28 were shifted to the restrictive temperature, synthesis of double-stranded RNA of poliovirus continued but synthesis of single-stranded RNA was almost totally inhibited. The RI labeled at 40” did not break down upon shear by Virtis homogenization as much as RI labeled at 25”. There may be a relationship between the resistance of RI to shear and to inhibition of TMV RNA synthesis at 40”. Perhaps the high temperature prevents the initiation and/or termination of new single-stranded RNA molecules, resulting in an RF-like structure with single-stranded tails and enzyme only at the end of the molecule. This molecule might be less susceptible to shear than a molecule with active polymerase all along the molecule. The relation of RF to viral RNA synthesis has not been established and there are some arguments that RF may not be an intermediate of viral RNA replication (Baltimore, 1968). The stability of TMV
326
W. 0. DAWSON
RF at high temperatures in relation to that of RI and TMV RNA suggest that one function of RF may be a survival structure. Temperatures as high as 40” exist in the natural environment of TMV. ACKNOWLEDGMENT This work was supported in part by a grant from the National Science Foundation (GB 43403). REFERENCES BALTIMORE, D. (1968). Inhibition of poliovirus replication by guanidine. In “Medical and Applied Virology” (M. Sanders and E. H. Lennette, eds.), pp. 340-347. Green, St. Louis, Missouri. COOPER, P. D., STANCEK, D., and SUMMERS, D. F. (1970). Synthesis of double-stranded RNA by poliovirus temperature-sensitive mutants. Virology 40, 971-977. DAWSON, W. O., GERMAN, T. L., and SCHLEGEL, D. E. (1976). Homogenization-resistant and -susceptible components of tobacco mosaic virus replicative form RNA. J. Gen. Viral., in press. DAWSON, W. O., S~HLEGEL, D. E., and LUNG, M. C. Y. (1975). Synthesis of tobacco mosaic virus in intact tobacco leaves systemically inoculated by
differential temperature treatment. Virology 65, 565-573. JACKSON, A. O., MITCHELL, D. M., and SIEGEL, A. (1971). Replication of tobacco mosaic virus. I. Isolation and characterization of double-stranded forms of ribonucleic acid. Virology 45, 182-191. UUCHLI, A. (1969). Radioassay for p-emitters in biological materials using Cerenkov radiation. Int. J. Appl. Radiat. Isot. 20, 265-270. LEBEURIER, G., and HIRTH, L. (1966). Effect of elevated temperatures on the development of two strains of tobacco mosaic virus. Virology 29, 385395. LOENING, U. E. (1967). The fractionation of highmolecule-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251257. MIKHEJEVA, A., and GHENDON, Y. (1974). The influence of supraoptimal temperature on poliovirus type 1 Mahoney strain reproduction. Arch. Gesamte Virusforsch. 45, 65-77. NILSSON-TILLGREN, T. (1970). Studies on the biosynthesis of TMV. III. Isolation and characterization of the replicative form and the replicative intermediate RNA. Molec. Gen. Genet. 109, 246-256. YARWOOD, C. E. (1952). Latent period and generation time for two plant viruses. Amer. J. Botany 39, 613-618.
73, 319-326
Synthesis
(1976)
of TMV RNA at Restrictive
High Temperatures
W. 0. DAWSON Department
of Plant Pathology and Cell Interaction Group, Riverside, California 92502 Accepted April
University
of California,
2,1976
The primary effect of high temperatures that restrict tobacco mosaic virus (TMV) multiplication was an immediate inhibition of synthesis and a more gradual degradation of single-stranded TMV RNA. Incorporation of radioactive precursors into doublestranded TMV RNA, replicative form (RF) and replicative intermediate (RI), was affected much less. Immediately after a step-up shift from 25 to 40”, single-stranded TMV-RNA synthesis was almost totally prevented, whereas there was little inhibition of incorporation into RF or RI. However, incorporation of 32P into double-stranded TMV RNA rapidly declined after the synthesis of single-stranded TMV RNA had stopped. At high temperatures that were restrictive for TMV multiplication the function responsible for incorporation of 3zP into single-stranded viral RNA was uncoupled from that responsible for incorporation into viral double-stranded RNA. INTRODUCTION
It has been demonstrated that tobacco mosaic virus (TMV) does not multiply at high temperatures. Yarwood (1952) defined the maximum temperature permitting TMV multiplication in tobacco as 37”. Lebeurier and Hirth (1966) measured the time course of TMV accumulation in tobacco leaf discs incubated at different temperatures. Between 16 and 32”, TMV accumulated to maximal concentrations; but at 34 and 36”, only about one-tenth as much virus accumulated. In an effort to determine what function is sensitive to the high temperature, TMV RNA synthesis is examined at high temperatures that prevent TMV multiplication. It will be demonstrated that the primary effect of the restrictive temperatures is inhibition of single-stranded TMV RNA synthesis. MATERIALS
AND
METHODS
Culture conditions. Young tobacco leaves (Nicotiana tabacum L. var. Xanthi) were “systemically inoculated” at 3” with TMV, strain Ul, using the differential temperature procedure described previously (Dawson et al., 1975). Virus repli-
cation was initiated by moving the plants into a plant growth chamber maintained at 25”. All subsequent experiments were conducted in plant growth chambers with a 14-hr photoperiod of 20,000 lux at the temperature designated in the Results. Labeling procedure. “Systemically inoculated” leaves were detached and the whole leaves were submerged and vacuum infiltrated in 60 @i/ml of H,32P0, in 0.1 mM KPO, buffer, pH. 7.0, and 65 pg/ml of actinomycin D; they were then maintained in that solution on ice for 10 min. The leaves were then removed to petri dishes and incubated at the designated temperature at 20,000 lux. The labeling period was terminated by freezing the tissue at -20”. RNA extraction. Tissue (3-4 g) frozen with liquid nitrogen was powdered with a mortar and pestle. The fine powder was removed to a 50-ml beaker containing 10 ml of 0.01 M Tris-HCl, pH 7.6, 0.05 M NaCl, 1% sodium lauryl sulfate (SLS), and 10 ml of water-saturated phenol containing 10% m-cresol and 0.1% 8-hydroxyquinoline. The solution was stirred 15 min at room temperature. The aqueous phase was recovered aRer centrifugation at 319
Copyright All rights
0 1976 by Academic F’ress, Inc. of reproduction in any form reserved.
320
W. 0. DAWSON
10,000 g for 10 min, made 0.5 M NaCl, and stirred 15 min at room temperature after addition of 10 ml of the above phenol solution. After centrifugation, the aqueous phase was precipitated by the addition of 2 volumes of ethanol and left standing at -20” overnight. The precipitate was dissolved in 2 ml of 0.05 M Tris-HCl, pH 6.65, 0.1 M NaCl, 0.001 M EDTA, 0.1% n-lauroyl sarcosine, and dialyzed against that buffer overnight. This solution was then precipitated with 2 volumes of ethanol, dissolved in 1 .O ml of 0.01 M MgCl, and incubated 1 hr at 37” with 50 pglml of DNAse (RNAse-free), after which the solution was made 2 N LiCl and stored overnight at 5”. The precipitate was collected by centrifugation at 10,000 g for 10 min and was dissolved in 1.0 ml E buffer (0.04 M Tris-acetate, 0.02 M sodium acetate, 0.002 M EDTA, pH 7.8) containing 0.2% SLS and 20% sucrose. This fraction (50 ~1) was analyzed by electrophoresis to examine single-stranded TMV RNA. To analyze RI-core, 0.8 ml of this solution was precipitated with 2 volumes of ethanol, dissolved in 0.5 ml of 0.2 M NaCl, 0.01 M Tris-HCl, pH 7.6, 0.01 M MgCl, and incubated with RNAse A (5 pglml) plus RNAse T, (0.5 pg/ ml) at 37” for 30 min. This fraction was precipitated by addition of 2 volumes of ethanol and dissolved in 0.3 ml of E buffer plus 0.2% SLS and 20% sucrose and 0.2 ml was analyzed by polyacrylamide gel electrophoresis. RF was analyzed by precipitation of the 2 N LiCl supernatant by adding 2 volumes of ethanol and dissolving the precipitate in 0.3 ml of E buffer plus 0.2% SLS and 20% sucrose and 0.2 ml was analyzed by polyacrylamide gel electrophoresis. The homogenization procedure was identical except that frozen tissue was blended in a Virtis homogenizer for 1 min. Gel electrophoresis. Polyacrylamide gel electrophoresis was performed on 2.5% polyacrylamide gels by the procedure of Loening (1967) but with the addition of 0.2% SLS to the electrophoresis buffer. After electrophoresis, the gels were scanned at 260 nm with a Beckman Acta II spectrophotometer equipped with a gel scanner. Determination of radioactiuity. Gels
were sliced into 2-mm slices and put into 10 ml of 2.5 mill 7-amino-1,3-naphthalenedisulfonic acid, and radioactivity was counted in a liquid scintillation counter by Cerenkov radiation (Lauchli, 1969). RESULTS
Synthesis of TMV RNA at High Temperatures Tobacco leaves synchronously infected with TMV by “systemic inoculation” at 3” (Dawson et al., 1975) were shifted to 25,36, or 40” and incubated for 24 hr. The leaves were then incubated with 32P plus actinomycin D for 2 hr at the same temperatures. Incorporation of 32P into progeny single-stranded TMV RNA, replicative form double-stranded RNA (RF), and that portion of the replicative intermediate that is resistant to RNAse (RI-core) was determined (Nilsson-Tillgren, 1970; Jackson et al., 1971). At 25”, 90% of the virusspecific incorporation of 32P was found in TMV RNA, with only 6% in RF and 4% in RI-core (Fig. 1). At 36”, substantially less label was incorporated into TMV RNA; at 40”, none could be detected. Approximately 14% as much TMV RNA was labeled at 36” as at 25”; however, 2.5 times as much RF and 1.6 times as much RI-core was produced at 36” as at 25”. At 36”, proportionally more double-stranded RNA was made with approximately 24% of the virus-specific incorporation occurring in TMV RNA, 51% in RF, and 25% in RI-core. At 40”, there was no evidence of virus synthesis although host RNA synthesis continued with no evidence of excessive breakdown. Viral RNA Synthesis after a Shift to High Temperatures Step-up temperature shift experiments were done to examine the temperature sensitivity of an active, replicating system. Leaves “systemically inoculated” at 3” were incubated at 25” for 24 hr, at which time the infection reached the rapid, linear phase of TMV replication. The leaves then were shifted to 25, 36, 38, 40, or 45”, and RNA was labeled with 32P for 2 hr beginning immediately after the shift. At progressively higher temperatures, there was less incorporation into TMV
TMV
RNA
SYNTHESIS
AT HIGH
321
TEMPERATURES
RI-core
600
400 600
1400
f
36O ss
600-
36O RI-core
400-
3000
4o” RF
2000
4o” 600-
RI-core
400-
600
FRACTION
NUMBER
FIG. 1. Incorporation of 32P into TMV RNA of leaves maintained continuously at 2536, or 40”. “Systemitally inoculated” leaves were incubated 24 hr at constant temperature and then labeled 2 hr at that temperature. RNA was extracted and fractionated into total 2 N LiCl precipitate (SS TMV RNA), 2 N LiCl supernatant (RF), and RNAse resistant 2 N LiCl precipitate (RI-core) fractions, as described in Materials and Methods. Samples consisting of 5, 67, and 53% of TMV RNA, RF, and RI-core, respectively, were analyzed. Each fraction was electrophoresed 3 hr at 7 mA per gel on 2.5% polyacrylamide gels. The tops of the gels are on the left.
RNA (Fig. 2). In relation to the amount of incorporation at 25”, there was only 25% as much incorporation at 38”, 0.6% as much at 40”, and none at 45” (The profiles of the different species of RNA in Fig. 2 represent different amounts of the total RNA samples. The amount of incorporation into each species is normalized in Table 1.) Although SS TMV RNA synthesis was dramatically inhibited at the higher temperatures, incorporation into double-stranded RNA was only slightly affected. At 36”, the incorporation of 32P into RF and RI-core was more than at 25”. At even higher temperatures, incorporation into RF and RIcore was reduced much less than that into TMV RNA. At 40”, for example, TMV RNA incorporated only 0.6% as much radioactivity as it did at 25”; but there was
58% as much incorporation into RF and 76% as much incorporation into RI-core. In one experiment at 50”, a small amount of label was incorporated into RF only (data not shown). It is interesting to look at the ratio of each species of virus RNA labeled at the different temperatures (Table 1). At 25”, 90% of the incorporation of 32P was found in TMV RNA; at 36 and 38”, about one-half of the incorporation was in single strands and one-half in double strands; at 40”, 51% of the incorporation was in RF and 41% in RI-core. All of the label at 45” was found in double-stranded RNA. Thus, in the presence of actinomycin D, the bulk of 32P incorporated into RNA of infected cells at high temperatures was found in viral double-stranded RNA.
322
W. 0. DAWSON r 1400-
1000
25O RF
25’ RI-core
600
400
_I 1400-
36”
\
36O
36O RI-core
600IOOO-
1400-
z
IOOO-
a 0
600-
360 ss
380 3000
-
600
-
36” RI-core
I
400-
200
1400-
40’ ss
400 RF
3000-
40” RI-core
600-
IOOO2000-
400-
600200t\ 450 RF
400
IO
20
30
IO
FRACTION
20
30
45O RI-core
600-
-
‘““kh-__ IO
20
30
NUMBER
FIG. 2. Incorporation of s2P into TMV RNA at high temperatures immediately after a shift from 25”. “Systemically inoculated” leaves incubated 24 hr at 25” were shifted to a higher temperature and labeled 2 hr with 32P beginning immediately after the temperature shift. RNA was extracted, fractionated, and electrophoresed as in Fig. 1. The upper-left graph shows the position of the host ribosomal RNA CM = lo-” daltons).
Kinetics of Incorporation into Viral after Step-up to 40”
RNA
The shift from 25 to 40” provided conditions that almost totally prevented TMV RNA synthesis, while only slightly affect-
ing incorporation of label into RF and RIcore when labeled immediately after the temperature shift. Next, the rate of incorporation of 32P into TMV RNA was examined at different times after the step-up temperature shift. Beginning at 0, l/4,2,4,
TMV
INCORPORATION Temperature during labeling (“)
RNA
OF 32P INTO TMV
Total cpm in TMV TMV
RNA
SYNTHESIS
AT HIGH
TABLE 1 RNA AFTER A STEP-UP
6.7 1.7 5.0 3.8
x x x x 0
RNA”
RF
104 IO’ 101 1W
4.5 6.5 3.4 2.6 0.4
x
x x x x
RIIO3 lo3 lo3 103 103
TEMPERATURE
Percentage of incorporation at 25” TMV
RNA
RF
core
25 36 38 40 45
323
TEMPERATURES
2.7 3.3 2.6 2.1 0.4
x x x x x
Percentage of cpm in each species RI-
“TMV
103 103 101 103 103
25 7 0.6 0
8, and 12 hr after the shift to 40”, tissue was incubated with 32P for 2 hr. The results are shown in Table 2. When the incorporation began immediately after the shift to 40”, TMV RNA incorporation was reduced 99%, while that of RF and RIcore was reduced 42 and 24%, respectively. The TMV RNA synthesis that did occur at 40” was produced during the first few minutes after the shift. When the labeling period began 15 min or more after the temperature step-up, no synthesis of TMV RNA occurred. In another experiment, the RNA was labeled beginning l/2 hr after the temperature shift for a shorter pulse (5 min) to overcome breakdown of TMV RNA that occurs at 40” (see next section): no 32P was incorporated into TMV RNA during the 5-min pulse. The rate of incorporation of 32P into RF and RI-core also decreased rapidly with time after the step-up to 40”. The greatest decline occurred during the first 15 min. Between 0 and 15 min at 40*, the rate of TABLE
Time of initiation of labeling after shift from 25” to 40”’ (hr)
2
-2
(labeled
at 25”)
0 114
a Two-hour
TMV
RNA
6.6 x 10’
385 0 0 0 0 0
2 4 8 12 labeling
RNA AFTER
Total cpm into SiAated
period.
into TMV
RI-
RF
2686 2062 215 114 58 9
4518 2588 712 334 182 52 52
core
19
RNA
RF
core
144 76 56 9
122 95 78 15
a “Systemically inoculated’ leaves incubated 24 hr at 25” were shifted to a higher temperature beginning immediately after the temperature shift. * Total counts per minute in virus-specific peaks in Fig. 2 in excess of that in healthy control and normalized to a 3-g sample
RATE OF 32P INCORPORATION INTO TMV SHIFT FROM 25 to 40”
Shift”
RIcore
90.3 63.4 45.5 7.5 0
6.1 24.3 30.9 51.2 50.0
3.6 12.3 23.6 41.3 50.0
and labeled 2 hr with 32P samples were determined
incorporation into RF decreased 73% and that into RI-core 88%. The rate of each continued to decrease with time until 8 hr, after which the rates remained relatively constant. Although the incorporation into TMV RNA completely stopped at 40”, that into RF or RI-core did not stop completely. However, after 8 hr the rate of incorporation into each double-stranded molecule was approximately 1% of that at 25”. Although incorporation into RF and RI-core rapidly decreased with time at 40”, their ratio of incorporation remained constant at 3 RF to 1 RI-core after the first 15 min. Stability
of TMV RNA at 40”
The above sections demonstrate that 40 inhibits the synthesis of TMV RNA and that most of the synthesis that occurs at 40” does so during the first 15 min after the temperature shift. In fact, all of the singlestranded TMV RNA incorporation occurred during this period. To examine the turnover of label incorporated into the different species of TMV RNA, the kinetics of incorporation in relation to length of labeling period were determined. Since most of the incorporation occurred immediately aRer the shift to 40”, each labeling period began at that time (Fig. 3). Single stranded TMV RNA incorporated more isotope during the 1Bmin label than the 5min label, demonstrating that, although synthesis of TMV RNA stopped by 15 min (Table 21, it continued beyond 5 min. Longer labeling periods demonstrated that TMV RNA broke down. The amount of radioactive label incorporated into single strands decreased with length of label greater than 15 min until after 10 hr no
324
W. 0. DAWSON 3000. ,8’------------*o
RF
/ If 2000~
. /-
.
;\
lation, breaks into a few relatively discrete pieces. It was also demonstrated that although the RI-core extracted with liquid nitrogen is a relatively homogeneous, “RFlike” molecule (Fig. 4A), when it was ex-
\
E a u
\ RI-:ore
i .
1000 nI
Length and temperature of labeling
’
[ \y-\Tss 1 42 HOURS
4 OF LABEL
TABLE 3 TMV RNAAT~OO
BREAKDOWNOF
IO AT 40”
FIG. 3. The incorporation of 52P into TMV RNA at 40” as a function of time of label. “Systemically inoculated” leaves incubated 24 hr at 25” were shifted to 40” and labeled with 32P for GO, l/4, 2, 4, and 10 hr beginning immediately aRer the shift. RNA was fractionated and electrophoresed as in Fig. 1, and the counts in each peak were summed.
incorporation was found in the area of TMV RNA. The amount of incorporation into RF and RI-core reached a maximum at 2 hr, when the synthesis rates of both species had decreased to a low level. The amount of incorporation in RF remained relatively constant afterwards, demonstrating that it was stable at 40”. However, there was some turnover of RI-core at 40”. Table 3 summarizes the results of another experiment to examine the breakdown of TMV RNA at 40”. TMV RNA was labeled with 32P for 2 hr at 25” and then shifted to 40” for 2 additional hours. RNA labeled in this manner was compared to the sum of the incorporation of RNA labeled only at 25” and only at 40”. In two experiments, the amount of TMV RNA that broke down during 2 hr at 40” was 92 and 46%, respectively; RF and RI-core did not break down in this experiment. Resistance ofRI Labeled at 40” to Shear In another communication (Dawson et al., 1976), we have demonstrated that TMV RF consists of two components: one, comprising about lo-20% of the population, is resistant to shear by homogenization in a Virtis homogenizer; another, composed of the about 80-90% of the popu-
Counts perg$;te Expt.
1. 2. 3. 4.
2hrat25 2hrat40” Sumof1+2 2 hr at 25” and 2 hr at 40”
5. &
6.7 4.0 6.7 5.5
1
Expt. 2
lo4 10” lo4 10”
4.7 1.0 4.8 2.6
8.2%
x 100 (o/o)
5 a "
x x x x
in TMV
x x x x
10’ lo” 10” 10’
54.2%
500 400 300 200 100 700
c
600 500 400 300 200 100 L 30 FRAd:lO;ON”MBER FIG. 4. Effect of shear upon TMV RI-core. Tissue as in Fig. 1 was labeled 2 hr at 25” (A, B) or 40” (C). RNA was extracted from tissue frozen with liquid nitrogen and powdered with a mortar and pestle (A) or from tissue homogenized in a Virtis homogenizer (B, C!). The 2 N LiCl precipitate fraction was RNAsetreated and electrophoresed 3 hr at 7 mA per gel on 2.5% polyacrylamide gels.
TMV
RNA
SYNTHESIS
AT HIGH
tracted by Virtis homogenization it broke, leaving few molecules as large as RF (Fig. 4B). However, when RI-core from tissue labeled at 40” was isolated by the Virtis homogenization procedure, a substantial portion of the RI molecules were resistant to shear and electrophoresed as RF-like molecules (Fig. 40. DISCUSSION
Incubation of infected tobacco plants at high temperatures that restrict TMV multiplication markedly altered the profile of viral RNA synthesis. Although TMV multiplied when continuously incubated at 36”, incorporation of 32P into TMV RNA was dramatically reduced while that into RF and RI was not. In plants maintained at 40”, there was no evidence of synthesis of any TMV RNA species. To examine an active replicating system for the functions that are sensitive to high temperatures, step-up temperature shiR experiments were chosen. Immediately after the shift to high temperatures, there was a dramatic decrease in the amount of 39P incorporated into single-stranded TMV RNA, paralleled by little effect upon RF and RI. In leaves shifted to 40” there was almost complete inhibition of incorporation into TMV RNA with only slight reduction in incorporation into RF or RI. This is in contrast to no synthesis of any species of TMV RNA in plants incubated continuously at 40”. Kinetics of incorporation of 32P into TMV RNA at 40” demonstrated that the high temperature had two effects upon TMV RNA. One was that synthesis of TMV RNA was completely blocked at 40”. There was a slight amount of incorporation during the first 15 min after the temperature shift, but afterwards incorporation totally stopped. The second effect was that TMV RNA broke down at 40”. Incorporation of radioisotope into this species of RNA during long labeling periods was less than during shorter labeling periods and TMV RNA synthesized at 25” broke down when incubated at 40”. Poliovirus behaves similarly at 40” (Mikkejeva and Ghendon, 1974). In cells incubated continuously at 40”, only l-2% as much virus was synthesized as at 36”. In cells shifted from 36 to
TEMPERATURES
325
40”, synthesis of progeny single-stranded poliovirus RNA decreased to 7% after 3045 min. Also, the single-stranded RNA of poliovirus broke down at 40”, with only one-half as much incorporation during a lhr labeling pulse as compared to the sum of four successive 15-min pulses. The high temperature “uncoupled” the function that is responsible for incorporation of nzP into TMV RNA from those that incorporate label into RF and RI. Immediately after the shift to 40”, the synthesis of TMV RNA was almost totally inhibited, whereas incorporation into RF and RI was only slightly affected. Within 15 min at 40”, synthesis of TMV RNA stopped. After TMV RNA synthesis stopped, the incorporation rates of RF and RI rapidly decreased, but did not stop. This suggests that there are at least two functions of TMV RNA synthesis that are differentially sensitive to high temperature: (1) a sensitive function that produces progeny single strands, and (2) a more resistant function that allows continued incorporation into double strands. Synthesis of TMV RNA at restrictive temperatures is similar to that reported for a temperature sensitive poliovirus mutant (Cooper et CZZ.,1970). When cells infected with the mutant k-28 were shifted to the restrictive temperature, synthesis of double-stranded RNA of poliovirus continued but synthesis of single-stranded RNA was almost totally inhibited. The RI labeled at 40” did not break down upon shear by Virtis homogenization as much as RI labeled at 25”. There may be a relationship between the resistance of RI to shear and to inhibition of TMV RNA synthesis at 40”. Perhaps the high temperature prevents the initiation and/or termination of new single-stranded RNA molecules, resulting in an RF-like structure with single-stranded tails and enzyme only at the end of the molecule. This molecule might be less susceptible to shear than a molecule with active polymerase all along the molecule. The relation of RF to viral RNA synthesis has not been established and there are some arguments that RF may not be an intermediate of viral RNA replication (Baltimore, 1968). The stability of TMV
326
W. 0. DAWSON
RF at high temperatures in relation to that of RI and TMV RNA suggest that one function of RF may be a survival structure. Temperatures as high as 40” exist in the natural environment of TMV. ACKNOWLEDGMENT This work was supported in part by a grant from the National Science Foundation (GB 43403). REFERENCES BALTIMORE, D. (1968). Inhibition of poliovirus replication by guanidine. In “Medical and Applied Virology” (M. Sanders and E. H. Lennette, eds.), pp. 340-347. Green, St. Louis, Missouri. COOPER, P. D., STANCEK, D., and SUMMERS, D. F. (1970). Synthesis of double-stranded RNA by poliovirus temperature-sensitive mutants. Virology 40, 971-977. DAWSON, W. O., GERMAN, T. L., and SCHLEGEL, D. E. (1976). Homogenization-resistant and -susceptible components of tobacco mosaic virus replicative form RNA. J. Gen. Viral., in press. DAWSON, W. O., S~HLEGEL, D. E., and LUNG, M. C. Y. (1975). Synthesis of tobacco mosaic virus in intact tobacco leaves systemically inoculated by
differential temperature treatment. Virology 65, 565-573. JACKSON, A. O., MITCHELL, D. M., and SIEGEL, A. (1971). Replication of tobacco mosaic virus. I. Isolation and characterization of double-stranded forms of ribonucleic acid. Virology 45, 182-191. UUCHLI, A. (1969). Radioassay for p-emitters in biological materials using Cerenkov radiation. Int. J. Appl. Radiat. Isot. 20, 265-270. LEBEURIER, G., and HIRTH, L. (1966). Effect of elevated temperatures on the development of two strains of tobacco mosaic virus. Virology 29, 385395. LOENING, U. E. (1967). The fractionation of highmolecule-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251257. MIKHEJEVA, A., and GHENDON, Y. (1974). The influence of supraoptimal temperature on poliovirus type 1 Mahoney strain reproduction. Arch. Gesamte Virusforsch. 45, 65-77. NILSSON-TILLGREN, T. (1970). Studies on the biosynthesis of TMV. III. Isolation and characterization of the replicative form and the replicative intermediate RNA. Molec. Gen. Genet. 109, 246-256. YARWOOD, C. E. (1952). Latent period and generation time for two plant viruses. Amer. J. Botany 39, 613-618.