Coordination between host nucleic acid metabolism and citrus exocortis viroid turnover

Virus Research, Elsevier

213

3 (1985) 213-230

VRR 00208

Coordination between host nucleic acid metabolism and citrus exocortis viroid turnover J.J. Lin * and J.S. Semancik Department

ofPlant

Pathology,

University

(Accepted

of California,

**

Riverside,

CA YZ21,

U.S.A

30 May 1985)

Summary

Citrus exocortis viroid (CEV) replication is sustained in tomato cell suspension cultures. Incorporation of 32P by pulse-labelling or continuous feeding of CEV-containing cells demonstrated the viroid to be a persistent and well-regulated component of the host nucleic acid profile. As the suspension cells approached stationary phase and senescence in the growth cycle, the relative proportion of CEV molecules to host RNA increased. Cells were grown in the presence of specific inhibitors to further examine the coordination between host nucleic acid and CEV synthesis. Actinomycin D in low concentrations (1 p&/ml) inhibited about 80% of host rRNA synthesis, but did not affect CEV replication. No differential inhibition could be observed between CEV synthesis and cellular nucleic acid synthesis when CEV-containing suspension cells were exposed to a series of concentrations (10-4-102 pg/ml) of a-amanitin. Throughout the cell growth cycle, the proportion of synthesis of circular and linear forms of CEV was constant. Both circular and linear forms of CEV were infectious. Modified pulse-chase experiments with [ 3H]uridine demonstrated a closely-coordinated equilibrium between the two accumulated forms of viroid molecules and an unexpectedly stable population of linear molecules. citrus exocortrs viroid, tomato circular and linear CEV

* Present

address.. Department

U.S.A. ** To whom reprint

0168-1702/85/$03.30

requests

suspension

of Genetics

culture,

and Development.

actinomycin

Cornell

University,

should be sent.

a 1985 Elsevier

Science Publishers

B.V. (Biomedical

Division)

D,

a-amanitin.

Ithica,

NY 14853,

214 Introduction Citrus exocortis viroid (CEV) has been recognized as a small, infectious, RNA molecule (Semancik and Weathers. 1972). From sequencing studies, it has been established that CEV RNA has a 371 nucleotide primary structure and assumes a covalently closed circular secondary structure with a high degree of intramolecular base-pairing interrupted by regions of single-stranded loops (Visvader et al., 1982; Gross et al.. 1982). Although protoplasts isolated from healthy and viroid-infected leaves have been used to study viroid replication in vivo (Miihlbach and Sanger, 1977, 1979). the short period of protoplast viability and the low yield of viroids from protoplasts limited the application of protoplasts in studying viroid replication and viroid-host interactions. Cultures of tomato suspension cells were established from callus colonies which had been selected for the presence of viroid after being derived from separated callus cells or regenerating protoplasts initiated from tissue of CEV-infected hybrid tomato plants (Marton et al.. 1982). The evidence that viroids could replicate persistently in cultured cells was observed in plant callus suspensions derived not only from CEV-infected cells but also from PSTV-infected tissues (Zelcer et al., 1981). In this report we describe the application of the cell suspension system to investigate viroid-RNA replication and its relationship with host nucleic acid metabolism. CEV in suspension cells appears intimately associated with host nucleic acid metabolism, as determined by kinetic studies as well as from evidence obtained following treatment with inhibitors. The relationship between circular and linear forms of CEV in suspension cells is also discussed.

Materials and Methods Cell suspen.sior~cultures

Stock suspension cultures of CEV-infected tomato callus cell populations were derived by the method of Marton et al., 1982. For experiments, 0.6 or 2 g of cells from such cultures in the stationary growth phase were transferred respectively into 50 or 125 ml Erlenmeyer flasks containing 4 or 25 ml fresh liquid medium of the following composition: Murashige and Skoog salts (Murashige and Skoog, 1962); thiamine-HCl. 1 mg/l; nicotinic acid, 1 mg/l: pyridoxin-HCl, 1 mg/l; i-inositol, 100 mg/l; indoleacetic acid (IAA). 2 mg/l; 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg/l; Nh-y-y-dimethylallylaminopurine (2ip), 1 mg/l; sucrose, 30 g/l, adjusted to pH 5.7 I 0.1. Cultures were agitated on a gyratory shaker at 125 rpm at 28-30°C and exposed to 16 h per day illumination of 0.16 Cal/cm min provided by 20 W F20T12 PL lamps. Nucleic acid prepurations Cells were extracted by a standard

1982. The homogenization

medium

phenol contained

method as described by Marton et al.. 0.8% SDS, 130 mM Tris (pH 8.9) 17

215 mM EDTA, 5% polyvinylpyrollidone and 1 M LiCl. Nucleic acids were precipitated by 3 ~01s. of 95% ethanol in 0.3 M sodium acetate pH 5.5, resuspended and dialyzed overnight against TKM buffer (10 mM Tris/lO mM KCl/O.l mM MgCl,, pH 7.4) and further purified by the methoxyethanol procedure to remove polysaccharides (Bellamy and Ralph, 1968). Purified nucleic acids were fractionated with 2 M LiCI. The supernatant resulting from the salt fractionation procedure was ethanol precipitated. Both the LiCl-precipitated and the ethanol-precipitated LiCl-soluble nucleic acids were resuspended in and dialyzed against TKM buffer and stored at - 20°C. In vivo lubelling of RNA Isotopic phosphorus (32P) (ICN) was added (0.5 mCi) into cell suspension cultures in 50 ml or 125 ml Erlenmeyer flasks under sterile conditions. Cultures were incubated for various time periods before extraction of nucleic acids. As an approach to a pulse-chase type experiment, [5,6-‘Hluridine (0.5 mCi, specific activity 42 Ci/mmol) was added to 50 ml Erlenmeyer flasks of cell suspensions which had attained a linear growth rate. After incubation for 1 h, treated cells were either (1) immediately phenol extracted as previously described, (2) continuously incubated in [‘Hluridine for an additional 4 or 24 h before extraction, or (3) collected, washed 4-5 times with medium containing a 5000-fold excess of non-radioactive uridine to remove exogenous [ ‘Hluridine, and then transferred into fresh medium containing a 5000-fold excess of non-radioactive uridine. These cells (treatment 3) were then incubated in the presence of the excess non-radioactive uridine for 4 or 24 h before the phenol extraction of nucleic acids. Treatment with inhibitors Indicated concentrations of metabolic inhibitors, actinomycin D or cu-amanitin. were added to cells undergoing linear growth for a 1 h pretreatment prior to the addition of 0.5 mCi 32P per flask. Incubation in the presence of radioactive phosphorus was then carried out for 1 h. Analysis of -“P-labelled CE V on polyacrylumide gels Nucleic acids soluble in 2 M LiCl were analyzed by electrophoresis on 5% polyacrylamide slab gels by a modification of the method of Morris and Wright (1975). Samples for a given gel represented equal volumes of extracts derived from the same fresh weight of cells. Alternatively, the spectrophotometric absorbances at 260 nm of the respective 2 M LiCl-soluble nucleic acid extracts were used as the basis for adjusting the aliquot volumes such that all samples monitored on a given gel reflected equivalent initial cell numbers. The slab gels were run at 4°C for 2.5-3 h at 60 mA. The electrophoretic buffer contained 36 mM Tris, 18 mM sodium acetate and 0.9 mM EDTA, pH 7.2. The gels were stained in 0.1 pg/ml ethidium bromide solution for lo-15 min and were observed with a ultraviolet transilluminator. After observation, the gels were either dried onto chromatography paper (3MM) for autoradiography or fixed,in 0.3% hexadecyltrimethyl-ammonium bromide for 1 h before fluorography (Bonner and Laskey, 1974). Quantitation of RNA species was made by planimeter measurements of the areas under peaks of graphic plots printed in response to spectrophotometric scans at 575

216 nm of the relevant regions of either gel autoradiographs or photographic negatives of images of stained gels. Nucleic acids insoluble in 2 M LiCl were analyzed by electrophoresis in 2.6% agarose-polyacrylamide gel slabs. For separation of circular and linear forms of the viroid RNA, gel pieces containing CEV banded on 5% polyacrylamide were sliced out and loaded directly onto 8 M urea, 5% polyacrylamide denaturing gels (Semancik and Harper. 1984). Following electrophoresis, banded circular and linear forms of CEV were excised and electrophoretically eluted from the gel strips. ethanol precipitated. resuspended in TKM buffer, and stored at - 20°C. Infectivity tests The razor-blade slashing inoculation method (Semancik and Weathers. 1972) utilizing 5 plants/ treatment was employed to determine relative infectivity or totalized infected plant days. Inoculated Gynura aurantiaca were examined for symptom expression every 2 days for 40 to 50 days after inoculation.

Results Characteristics of CE V during the growth cycle of suspension cells CEV-containing suspension cells were incubated with “P for 1 h during different stages of the growth cycle. Synthetic rates (cpm/A,,,) of nucleic acids increased as cell growth progressed from exponential stages to late linear stages, then decreased as cells reached stationary stages (Fig. I). An apparent increase in the total CEV content as analyzed by 5% PAGE was observed in extracts from cells at progressive stages in the growth cycle until late stationary stages when total CEV began to decrease (Fig. 2). The rate of synthesis of CEV also appeared to increase as cell growth progressed from the exponential stage to late linear stage. then decreased as cells reached late stationary stages (Fig. 2, top). This pattern was in agreement with synthetic rates of total 2 M LiCI-soluble nucleic acids (Fig. 1). The pattern for synthesis of 5s RNA resembled that of CEV. except that the maximum synthetic activity occurred earlier during the growth cycle. However, while total viroid appeared to accumulate with time, the total amount of 5S RNA remained constant in cells at all stages of growth (Fig. 2, bottom). An equal amount (fresh weight) of cells was taken from cultures at various stages of the growth cycle and incubated with “P for 1 h to monitor the relationship between host nucleic acid synthesis and CEV replication. The incorporation of ‘lP into 2 M LiCl-soluble nucleic acids showed that the cells in the linear stage had the highest rate of synthesis, cells in stationary stage the second highest, and cells in exponential stage had the lowest rate of synthesis (Table 1). Synthesis of DNA as determined by RNase resistant cpm, was constantly high at the exponential stage and at the linear stage, but decreased at the stationary stage. When syntheses of specific cellular RNAs were quantitated from autoradiography of the 5% polyacrylamide gel. 5S RNA, like the 2 M LiCl-soluble nucleic acid pool, had the highest rate of synthesis in cells at the linear stage, followed by cells at exponential stage,

217

0

0

3

6

9 DAYS

12

15

0

0

6 DAYS

I2

18

IN CULTURE

and synthesis of 2 M LiCl-soluble nucleic acids Fig. 1. Growth curve (fresh weight of cells, 0 -0) (cpm/A,,,, 0- - 0) of CEV-containing suspension cells. Nucleic acids were obtained from cells incubated with 32P for 1 h as described in Materials and Methods. Fig. 2. Total amounts of CEV and 5s RNA present at time of extraction (0 -0) and amounts of (0- - 0). The arbitrary unit (A.U.) is a relative value each synthesized during 1 h 32P incubation obtained from planimeter measurement of the area under the peak of the graphic plot printed in response to a spectrophotometric scan at 575 nm of the relevant region either of the photographic negative of the ethidium bromide-stained gel image or of the gel autoradiograph. Top: CEV; bottom: 5s RNA.

then cells at stationary stage. When CEV synthesis was compared with the synthesis of specific nucleic acid species, CEV showed a pattern similar to the synthesis of 7s RNA. In both cases, synthesis as a proportion of total nucleic acid synthesis in the cell appeared to increase as cell growth progressed from the exponential stage to the stationary stage (Fig. 3, Table 1). The rates of synthesis of CEV and cellular nucleic acids were measured when cells were incubated with 32P for continuous periods. Incorporation of 32P into 2 M LiCl-soluble nucleic acids indicated that cells at different stages of growth had different synthetic rates of nucleic acids (Fig. 4). In cells at the exponential stage, synthesis of nucleic acids increased rapidly, reaching saturation after the cells were incubated with 32P for 8 h. Nucleic acid synthesis in cells at the linear stage increased more slowly than that in cells at the exponential stage, and saturation was not reached until cells were exposed to 32P for 35 h. Cells incubated with 32P for 1-48 h during stationary stage showed a slow, but relatively constant increase in synthesis of nucleic acids. Analysis of nucleic acids by 5% polyacrylamide gel and

218 TABLE

1

SYNTHESIS OF NUCLEIC ACIDS STAGES OF THE CELL GROWTH

IN CELLS CYCLE

Stage of cell growth

INCUBATED

DNA h 5S RNA’ 7S RNA’ CEV ’ CEV/DNA’ CEV/5S RNA’ CEV/‘IS RNA’

32P FOR

1 h AT DIFFERENT

cycle

Exponential stage 2 M LiCI-soluble nucleic acids a

WITH

Linear stage

Stationary stage

1.93

4.99

2.91

437 42.6 1.76 0.46 1.05 1 .og 26.1

731 55.6 5.64 1.11 1.52 2.00 19.7

194 31.5 9.06 1.66 8.56 5.21 18.3

’ Expressed as cpm per ng of 2 M LiCl-soluble nucleic acids (N.A.) obtained from phenol extraction as described in Materials and Methods. h DNA (cpm/ng N.A.) obtained after treatment of 2 M LiCI-soluble aliquots containing 2-g ng N.A. with 30 ~1 of bovine pancreatic ribonuclease A (1 pg/ml) for 30 min at 25°C. ’ Arbitrary units (A.U.) were obtained from planimeter measurements of the plot of the scanned gel autoradiograph as described in Materials and Methods, then divided by the amount of 2 M LiCl-soluble nucleic acids (pg N.A.) loaded onto the 5% polyacrylamide gel, resulting in data presented as A.U./ng N.A. ’ (A.U./ng N.A.)x10m3. ’ Percent.

electrophoresis showed that CEV synthesis other cellular nucleic acids (Fig. 5).

increased

similarly

to the synthesis

of

Replication of CEV in the presence of inhibitors Effects of application of inhibitors to suspension cultures were investigated as an approach to elucidate the degree of coordination between viroid and host nucleic acid syntheses. In addition, data obtained from such inhibition studies might assist in implicating the involvement of specific templates and enzyme systems in viroid synthesis. The effect of actinomycin D, an inhibitor of RNA transcription from DNA templates, on viroid synthesis remains controversial, with reports of both specific inhibition (Diener and Smith, 1975; Miihlbach and Sanger, 1979) and no effect on viroid replication (Grill and Semancik, 1980; Flores and Semancik, 1982). The key questions emerging from these studies are at what concentration actinomycin D effects a general toxic reaction rather than a specific inhibitory action and whether or not this concentration range is tissue dependent. If cells were pretreated with a series of actinomycin D concentrations for 1 h before incubation with 32P, the synthesis of large ribosomal RNA decreased rapidly, even with as low a concentration as 0.33 pg/ml (Fig. 6). In contrast, no decrease of 32P incorporation into CEV at up to 1 pg/ml of actinomycin D was observed. When

219

DNA{

CEV+ 7s RNA*

5s RNA-, 4s RNA+

A

B

C Std

CEV+ 7s RNA*

5s RNA+ 4s RNA+ 0

e

16

24

32

TIME Cf ‘*P0,lNC0RP0RATl0N

40

48

(hr)

Fig. 3. Polyacrylamide slab gel (5%) electrophoresis pattern of the 2 M LiCl-soluble nucleic acid fraction obtained after 1 h 32P incorporation into CEV-containing suspension cells as described in Materials and Methods. Top: Ethidium bromide-stained patterns of nucleic acid extracted from equal weights of suspension cells at (A) exponential, (B) linear, or (C) stationary stage of growth compared to a CEV-containing nucleic acid preparation from Gynura aurantiaca (Std). Bottom: Autoradiograph of the same gel after 3-day exposure to X-ray film. Fig. 4. Relative amounts taining suspension cells linear (A(0 -O), and 48 h in the presence stage, 40.7 X lo3 for the

of newly synthesized 2 M LiCl-soluble nucleic acids extracted from CEV-confollowing different time periods of labelling with 32P. Cells at the exponential - -A), or stationary growth stage (0. ‘0) were labelled for 1, 2, 4, 8, 24 of 0.5 mCi 32P. Maximum cpm/A260 were 52.3 X lo3 for cells in the exponential linear stage, and 70.8 X lo3 for the stationary stage.

the syntheses of cellular RNA and CEV were compared, large ribosomal RNA, and 7s RNA were inhibited by 50-80% at 1 pg/ml of actinomycin D, whereas CEV synthesis was not affected (Fig. 6). At actinomycin D concentrations greater than 1

220

CEV-

0

1

2

4

8 24 48

24

8

Std

CEV,-

CEVI-

01

24

48

Std

Fig. 5. Time course of the synthesis of 2 M LiCl-soluble nucleic acids and CEV. Nucleic acids were extracted after 3zP incorporation for different periods of time (h) into CEV-containing suspension cells. Left: Patterns of nucleic acids extracted from suspension cells after incubation for different periods of time (h) as seen with ethidium bromide staining. Bight: Autoradiographs of the same gels after j-day exposures to X-ray film. Top: 2 M LiCl-soluble nucleic acid patterns on 5% polyacrylamide gel. Bottom: Denatured CEV bands resulting from electrophoresis on 5% polyacrylamide plus X M urea of the regions containing CEV excised from the native get above (Top. right).

pg/ml, not only did synthesis of CEV begin to decrease, but also synthesis of DNA was inhibited (Figs. 6, 7). This suggestion that inhibition by actinomycin D of CEV synthesis parallels the inhibition of DNA synthesis might be viewed as confirmation of an anticipated decline in a host dependent process. As such, CEV synthesis could he expected to be inhibited prior to the decrease in the rate of DNA synthesis noted in the presence of greater than 3 pg/ml actinomy~ill D. Therefore, inhibition of nucleic acid synthesis at high concentrations of actinomycin D (> 1 pg/ml) was probably due to the general toxic effects of the inhibitor, and not by specific inhibition of DNA-dependent RNA synthesis. The difference between responses of CEV synthesis versus host cell nucleic acid syntheses at low concentrations compared to inhibition of both types of synthesis at higher concentrations of actinomycin D, supports the proposition that replication of CEV

221

CONCENTRATION

OF

ACTINOMYCIN

D (uglml)

Fig. 6. Inhibition of synthesis of DNA (0 PO), large ribosomal RNA (0~ -0) 7s RNA .A) and CEV (A.---.A) by various concentrations of actinomycin D. Synthesis of DNA was determined by measurement of RNAse A resistant cpm in equal amounts of 2 M LiCl-soluble nucleic acids extracted from cells exposed to the inhibitor; synthesis of large ribosomal RNA was determined by specific activity (cpm/pg nucleic acid) of 2 M LiCl-insoluble nucleic acids; synthesis of 7s RNA and CEV was determined by planimeter measurement of the relative areas of the respective peaks obtained from scanning the autoradiograph of the 5% polyacrylamide gel at 575 nm. (A.

is not directly through DNA template, but allows that de novo synthesis of host genome dependent factors are implicitly necessary to cell viability and, therefore, CEV synthesis. a-Amanitin, a potent concentration-dependent inhibitor of host RNA polymerase II, has been reported to inhibit viroid synthesis in several cell-free systems (Flores and Semancik, 1982; Rackwitz et al., 1981; Semancik and Harper, 1984). Estimation of the effective intracellular and intranuclear concentrations presents a technical difficulty inherent in the application of any inhibitor to intact cell systems. However, when increasing a-amanitin concentrations (10P4-lo* pg/ml) were added to the cell suspension culture medium, no differential inhibition was noted between the synthesis of CEV and cellular RNAs (Fig. 8). When the medium was made to 10m4 M (lo2 pg/ml) in a-amanitin, a concentration which in a cell-free system would be well in excess of the 10-6-10P8 M levels effective for inhibition of RNA polymerase II, the synthesis of CEV as well as all cellular RNAs was inhibited by only about 27755% (Fig. 9). Since the replication of cucumber pale fruit viroid in recently inoculated tomato protoplasts was reported (Miihlbach and Sanger, 1979) to be specifically inhibited by a single concentration of 50 pg/ml of a-amanitin, fundamental distinctions must exist between this protoplast system and the cell suspension cultures utilized in the studies reported here. Properties and relationship of circulur and linear forms of CE V in suspension cells When purified circular and linear forms of CEV were obtained by electrophoretic

222

DNA{ CEV+ 7s RNA+ CEV-’

5s RNA+ 4s RNA+

A A

B C

D E

F G

H

B

C

D

E Std

Std

DNA -i

CEV+

CEV-t 7s RNA-,

5s RNA+ 4s RNA+

Fig. 7. Polyacrylamide slab gel (5%) electrophoresis pattern of 2 M LiCl-soluble nucleic acids extracted after s*P incorporation for 1 h into CEV-containing suspension cells. Top: Ethidium bromide-stained patterns of nucleic acids from cells exposed to (A) 0 pg/ml; (B) 0.33 ug/ml: (C) 1 ug/ml; (D) 3 ug/ml: (E) 9 pg/ml actinomycin D; or from CEV-infected Gynuru aurantiuca used as a standard (Std). Bottom: Autoradiograph of the same gel after exposing the X-ray film for 3 days. Fig. 8. Polyacrylamide slab gel (5%) electrophoresis pattern of 2 M LiCl-soluble nucleic acids obtained from suspension cells after ‘*P incorporation in the presence of cu-amanitin as described in the Materials and Methods, Top: Ethidium bromide-stained patterns of nucleic acids extracted from CEV-containing suspension cells exposed to a-amanitin concentrations of (A) 10m4, (B) 1O-3, (C) lo-*, (D) lo-‘, (E) 10’. (F) 10’. (G) lo*, (H) 0 pg/ml compared to a CEV-containing standard (Std) from Gynum aurunfiaca. Bottom: Autoradiograph of the same gel after exposure to X-ray film for 2 days.

223

a-AMANITIN

CONCENTRATION,

q/ml

Fig. 9. Effects of cy-amanitin on the synthesis of large ribosomal, 7S, 5s and CEV-RNA. Top: Synthesis of rRNA determined by specific activity as in Fig. 6. Bottom: Synthesis of 7S, 5s and CEV-RNA determined by planimeter measurements of scanned autoradiograph bands as described in Fig. 2.

elution from the respective bands excised from denaturing gels and inoculated into Gynuru auruntiacu, both forms induced typical CEV symptoms. Plants infected by circular or linear CEV contained both circular and linear forms when extracts were analyzed after a 3 week infection period. When cells were incubated with 32P for continuous periods, the accumulation of 32P into both circular and linear forms of CEV increased with time parallel to the accumulation in all other cellular nucleic acids contained in the 2 M LiCl supernatant (Fig. 5). Circular forms of CEV appeared to be synthesized at a faster rate than linear forms of CEV (Fig. 10, top), but the ratio between circular and linear forms of CEV remained constant after the 8th h of incubation. These data were observed in CEV-containing cells during exponential and linear stages of cell growth. Cells in the stationary growth stage had different kinetics of nucleic acid synthesis than cells at exponential and linear stages (Fig. 4), but the ratio of incorporation of 32P into circular and linear forms in the stationary stage (Fig. 10, bottom) was similar to the ratio in the exponential and linear stages. If cells at different growth stages were administered 32P for 1 h and then extracted for CEV, the ratio between circular and linear forms of the viroid was constant (Table 2). This indicated the synthetic distribution of circular forms and linear forms in CEV-containing cells was similar, regardless of the stage of cell growth. When CEV-containing cells were incubated for 1 h in medium containing [5,6-3H]uridine, then transferred into medium containing excess non-radioactive uridine for 4 or 24 h, the incorporation into nucleic acids of labelled uridine

224 15c

120 E 3

90

& d t

60

zi 30

120

0 B

16 DURATION

24 OF %PO,

32

40

INCUBATION

46 (hr)

Fig. 10. Kinetic analysis of synthesis of total CEV (A -A) and of circular (O- - -0) and linear (0. . ‘0) forms of CEV. Top: Exponential phase cells. Bottom: Stationary phase cells. Arbitrary units (A.U.) were obtained by planimeter measurements as described in Fig. 2. Total CEV synthesis was determined from the CEV band on the autoradiograph of an initial 5% polyacrylamide gel. Synthesis of circular and linear forms was measured from the two CEV bands on the autoradiograph of the denaturing gel run subsequent to excision of the CEV band from the 5% polyacrylamide gel as described in the Materials and Methods.

administered during the 1 h interval could be traced. In spite of the inherent limitations of a large uridine pool size and slow rates of cell division in plant suspension cell cultfires, this approach was employed in an attempt to follow the fate TABLE

2

SYNTHESIS DIFFERENT WITH 32P

OF CIRCULAR AND LINEAR MOLECULES OF CEV BY SUSPENSION CELLS AT STAGES OF THE CELL GROWTH CYCLE MEASURED AFTER 1 h INCUBATION

Stage of cell growth

Circular

Linear

Circular/Linear

Exponential phase Linear phase Stationary phase

3.40 a 7.28 10.6

3.10 6.93 10.8

1.10 1.05 0.98

a Arbitrary described

units (A.U.) obtained from in Materials and Methods.

planimeter

measurement

of scanned

gel autoradiograph

as

225

35 _

30

_

25 _

--

0

0

6

12

TIME (hr)

16

24

0

/’

5

IO

I’

/’

15

/’

/’

0’

20

,’

I’

/

P

25

TIME (hr)

Fig. Il. Nucleic acid synthesis measured by [“Hluridine incorporation into CEV-containing suspension cells. Top: Incorporation into 2 M LiCl-soluble nucleic acids of cells exposed either continuously to or for 1 h followed by transfer to culture medium containing an excess of [ 3Hluridine (0 -0) non-radioactive uridine (0 - - - 0). Bottom: Incorporation into 25s (A -A) and 18s (A- ~ -A) RNA of cells exposed to [‘H]uridine for 1 h then transferred to culture medium containing an excess of non-radioactive uridine. Data obtained from scintillation counts of the 25S- or 18S-containing bands excised from 2.6% polyacrylamide gel containing 0.5% agarose run at 50 mA for 2.5-3 h at 4°C. Fig. 12. Incorporation of (‘Hluridine into CEV forms extracted from suspension cells exposed to labelled uridine either continuously (circular CEV. 0- - 0; linear CEV, A' .a) or for 1 h followed by transfer to culture medium containing an excess of non-radioactive uridine (circular CEV, O-O: linear CEV, A.-. - .A). Arbitrary units obtained from gel autoradiograph as described in Fig. 2.

of CEV molecules labelled during the 1 h [ 3Hluridine pulse. Radioactive nucleic acids extracted from cells exposed for an hour to [ 3Hluridine followed by longer exposures to excess non-radioactive uridine were compared with nucleic acids extracted from cells cultured continuously with [‘Hluridine. Total radioactivity in the nucleic acids from 1 h-labelled cells increased slowly with time, while isotope was incorporated rapidly into nucleic acids from continuously-labelled

226 cells (Fig. 11, top). Although 25s and 1% ribosomal RNA from pulse-labelled cells increased in radioactivity during the first 4 h in excess non-radioactive uridine, after 24 h exposure to non-radioactive uridine, a partial chase of the [5,6-3H]uridine label was indicated by a decrease in radioactivity detected in these rRNAs (Fig. 11. bottom). One might consider the exposure of [‘Hluridine-treated cells to excess nonradioactive uridine not so much a chase of the unincorporated isotope as a reduction in the pool of labelled precursors to synthesis of nucleic acids, including CEV. Under such circumstances, if the kinetics of isotope incorporation by circular and linear viroid forms were similar, the difference in CEV labelling between continuously and pulse-labelled cells should be a simple reduction in the amounts of isotope accumulated into the two forms in the briefly-labelled cells. Fig. 12 clearly indicates this is not the case. As predicted from previous data (Fig. lo), the apparent synthesis of circular forms exceeded that of linear forms by a factor of 2 in continuous receiving only 1 h exposure to [ 3Hluridine-fed cultures. However, in cultures [‘Hluridine followed by 4-24 h in excess cold uridine, both labelled forms of CEV were essentially equivalent. This could indicate a diminished accumulation of newly-produced circular forms due to a reduction in synthesis or an increase in degradation, an increased accumulation of labelled linear forms, or a combination of these factors.

Discussion A close coordination between host nucleic acids and viroid was observed when these species were monitored for synthesis and accumulation during the growth cycle of CEV-infected suspension cells. The resulting data suggest that a foreign nucleic acid (viroid) could be accepted and processed by these cells as a part of their nucleic acid population. Incorporation of isotopic label by CEV appeared to be remarkably similar to incorporation by 7s RNA throughout the cell growth cycle. This finding was contrary to the suggested absence of closely-linked synthesis of PSTV with any host RNA species in heterogeneous tomato suspension cultures (Zelcer et al., 1981). However, since the 7S RNA appears to be a cytoplasmic component, by evidence of subcellular distribution and by correlation with mammalian systems (Walter and Blobel, 1982). and CEV is generally found to be localized in the host cell nucleus, this relationship may be a coincidental result of similar rates of synthesis and degradation rather than an indication of common origin or function. The apparent accumulation of CEV in stationary phase cells may result from the inherent structural stability of the viroid molecule. The nucleotide sequences of viroids plus the lack of discovery to date of any new protein synthesized by viroid-infected cells tend to preclude the ability of viroids to enzymatically mediate their own replication. Dependence on some already in-place suggested. The persistence of CEV in system(s) of the host cell is, therefore, suspepsion cells may be a result of recognition by the host cell of the viroid as part of the cell’s own snRNA population. The viroid consequently may become subject to

227 the same synthesis and regulatory processes as a cellular snRNA. The viroid-RNA may, therefore, be thought of as yet another snRNA in plants (Krol et al., 1983; Skuzeski and Jendrisak, 1985) albeit exogenously derived, particularly since there are instances when its incorporation into the host nucleic acid profile seems not to result in detriment to the host. In the case of ASV infection of avocado, the symptomless carrier condition resulting from seed transmission of ASV is characterized by a high level of viroid-RNA synthesis accompanied by the absence of any obvious expression of pathogenesis. Furthermore, CEV-containing tomato cells demonstrate enhanced growth properties in culture under high temperature stress when compared to healthy cells, suggesting even a possible beneficial influence contributed by the presence of the viroid-RNA (Marton et al., 1982). Actinomycin D at concentrations below 1 pgg/ml had no effect on CEV synthesis, but inhibited 70-80% of large ribosomal RNA synthesis in CEV-containing suspension cells. When cells were placed in concentrations of actinomycin D higher than 1 pg/ml, not only synthesis of CEV but also synthesis of cellular DNA and RNA was inhibited. Similar results have been observed after treatment of CEV-infected Gynura or PSTV-infected potato sprout leaves (Grill and Semancik, 1980) in which viroid synthesis was not affected by concentrations of actinomycin D up to 10 pg/ml, but viroid synthesis along with host RNA synthesis was inhibited at higher concentrations (lo-20 pg/ml) of actinomycin D. However, in cell-free, nuclei-rich fractions from Gynura leaves, no inhibition of CEV synthesis was observed, even in actinomycin D concentrations up to 20 pg/ml (Flores and Semancik, 1982). In contrast, inhibition of viroid synthesis by actinomycin D has been reported in previous studies, either in vivo (Diener and Smith, 1975; Muhlbach and Sanger, 1979) or in vitro (Takahashi and Diener, 1975). However, only one relatively high concentration of actinomycin D (either 20, 30 or 50 pg/ml) was used in each of these studies and the toxic effects of these concentrations might clearly be expected based on the Gynura leaf systems (Grill and Semancik, 1980) and the sensitivity noted here in CEV-containing suspension cells. Although viroid synthesis inhibition caused by a low concentration of actinomycin D has been mentioned in suspension cultures of PSTV (Palukaitis and Zaitlin, 1983) no data were presented to describe the concentrations of actinomycin D and the inhibitory effects on cellular nucleic acid synthesis. Considering the sensitivity of the cultured suspension cells to actinomycin D, it was most surprising to observe no effect on CEV synthesis in cultures containing a-amanitin greater than 10 pgg/ml, a concentration previously demonstrated to be inhibitory to viroid synthesis in cell-free systems (Flores and Semancik, 1982). Clearly, the problem of uptake into intact cells could pose an obstacle. However, protoplasts exposed to an external concentration of 50 pg/ml of cu-amanitin were reported (Muhlbach and Sanger, 1979) to have displayed a highly selective inhibition of cucumber pale fruit viroid synthesis. Unfortunately, these results could not be duplicated in cell suspension cultures exposed to a range of cY-amanitin concentrations to a maximum of 200 pg/ml, in which only a general suppression of viroid and host RNA syntheses was observed. Administration of a-amanitin to cultured mammalian cells has also produced

22x inconsistent effects (Lindell, 1980). Because tomato protoplasts were isolated prior to inoculation with CPFV (Miihlbach and Stinger, 1979) and then treated with n-amanitin, the inhibition of CPFV synthesis by c*-amanitin might reflect a lack of host factors for the initial synthesis of CPFV or some general impairment, rather than the classical inhibitory action of n-amanitin on RNA polymerase II. The consistent recovery of circular and linear forms of CEV in various tissues, including callus and suspension cultures. introduces questions as to the relationship between these accumulated forms in the replication of viroid-RNA. The apparent ability for induction of disease symptoms by either of the two CEV forms, as well as that reported for both the circular and linear forms of PSTV (Owens et al., 1977) and CSV (Palukaitis and Symons. 1980) indicate the biological equivalence of the two molecular forms of the viroid nucleotide sequence. Clearly, some linear form of polynucleotide must precede ligation of the covalently-closed, circular viroid-RNA. Do the linear precusor molecules comprise the extractable linear populations or only some portion thereof? Following a 1 h pulse of “P, the synthetic ratio of circular to linear form of CEV was constant. Continuous incubation up to 48 h of viroid-containing cells in j2P demonstrated a similar relationship, with a slightly higher ratio of recently synthesized circular to linear forms in log phase versus exponential or stationary phase cells. Experiments designed to determine the fate of CEV forms after a short exposure to [“Hluridine followed by incubation with non-radioactive uridine indicated that (1) the linear precursors of the circular forms probably constitute a small component in the total linear pool, (2) some portion of the linear forms appears to be derived from circular molecules, and (3) an unexpectedly high concentration of stable linear molecules persists. When PSTV-infected plants were incubated for different periods with 32P, the circular form was the major viroid product after short incubation times, but the predominant progeny shifted to the linear form after plants were exposed to “P for longer periods; therefore, it was speculated that linear forms came from circular forms (Hadidi and Diener, 1978). More recently, purified ligases from wheat germ were found to be able to ligate linear forms to circular forms of PSTV in vitro (Branch et al., 1982). Isotope incorporation by circular forms of CEV decreased faster than did incorporation into linear forms in cells incubated for a short time with [5,6-‘Hluridine and then chased in excess non-radioactive uridine. This result supports the possibility that a substantial amount of linear forms may be derived from circular forms. Nevertheless, the linear population may be comprised of subspecies representing precursors of the circular form, products of nicked circles, and possibly some linear species which never become circular molecules because of conformation, compartmentalization. and/or enzymatic constraints. Acknowledgement We wish to recognize by Kathy Harper.

the excellent

technical

assistance

and thoughtful

discussion

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