Cucumber Mosaic Virus Replication in Cowpea Protoplasts - CiteSeerX

723

J. gen. Virol. (I979), 45, 723-736 Printed in Great Britain

Cucumber Mosaic Virus Replication in Cowpea Protoplasts: Time Course of Virus, Coat Protein and R N A Synthesis By T H O M A S J. G O N D A *

A N D R O B E R T H. S Y M O N S

Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5ooi (Accepted 23 July I979) SUMMARY

The time course of the synthesis of cucumber mosaic virus (CMV), CMV coat protein and the four C M V RNAs has been followed in cowpea protoplasts. CMV synthesis was estimated by analytical sucrose gradient centrifugation of extracts from protoplasts and by infectivity assay; coat protein synthesis was determined by incorporation of radioactive amino acids and analytical polyacrylamide slab gel electrophoresis, while the synthesis of CMV RNAs was determined by hybridization analysis with labelled specific complementary DNA (cDNA) probes. There was a co-ordinate synthesis of CMV, CMV coat protein and virus RNA up to 5o h p.i. whereas the rate of coat protein synthesis reached a peak at about 15 h p.i. Coat protein was the only virus-induced protein detected. Results on the relative synthesis of RNAs 3 and 4 indicated that RNA 4 was derived by nucleolytic cleavage of RNA 3 and not by transcription of a negative RNA 4 strand. INTRODUCTION

Purified virions of cucumber mosaic virus (CMV) contain four major RNA species (designated RNAs I to 4, in order of decreasing mol. wt.), of which the largest three are required for infectivity (Peden & Symons, i973 ; Lot et al. ~974)- The coat protein is genetically specified by RNA 3 (Habili & Francki, I974), and is the only functional gene product of CMV identified to date although it seems likely that there are virus-coded components in the CMV-induced RNA replicase found in infected plants (Peden et aL r972; Clark et al. I974; Kumarasamy & Symons, I979b). In vitro translation studies have indicated that there are at least four virus gene products (Schwinghamer & Symons, I977). In order to characterize the in vivo gene products of CMV, we have initiated a study of the synthesis of virus RNA and virus-coded proteins in CMV-infected protoplasts of cowpea (Vigna unguiculata). CMV-infected tobacco protoplasts have been used by Takanami et al. (I977) to study the synthesis of single- and double-strand CMV RNAs, while the infection by CMV of protoplasts of cucumber and of Vigna sesquepedalis has been reported by Coutts & Wood 0976) and Koike et al. (I977), respectively. We report here conditions for the infection of cowpea protoplasts by the Q-strain of CMV (Q-CMV) and results on the time course of virus, coat protein and virus RNA synthesis. * Present address: Department of Microbiology, University of California, San Francisco, California 94143.

oo22-I317/79/oooo-377! $02.00 ~ I979 SGM

724

T.J.

GONDA

AND

R. H. S Y M O N S

METHODS

Virus. The Q-strain of CMV (Francki et aL 1966) was purified from tobacco plants (Nicotiana tabacum cv. White Burley) as described by Peden & Symons (1973). Brome mosaic virus (BMV) R N A was kindly provided by Dr A. O. Jackson. Plants. Seeds of cowpea [Vigna unguiculata L. (Walpers) cv. Blackeye] were planted in vermiculite moistened with Long Ashton culture solution (Hewitt, 1952 ) and watered daily with the same solution. Plants were maintained in a growth room with a daily cycle of 14 h light (28 °C) and to h dark (22 °C). Illumination was supplied by a mixture of 65 W white fluorescent tubes and 6o W incandescent bulbs to give a total intensity of approx.. l I ooo lux at leaf height. Isolation of protoplasts. Protoplasts were isolated essentially as described by Beier & Bruening 0975, I976) from 9-day-old plants, except that the enzyme solution used was l.o°,~, cellulase Onozuka R-lo, o . I % Macerozyme R-to (Kinki Yakult Manufacturing Co. Ltd., Japan) and o.l ~)~obovine serum albumin, and neither the leaves nor the enzyme solution was sterilized. After incubation of the leaf pieces (approx. I g per IO ml) at 3° °C for 2 h, the flask was swirled to release the protoplasts, the suspension filtered through muslin to remove undigested material and the protoplasts washed three times with sterile o'45 M-mannitol by centrifuging at J o o g for 2 min. This procedure yielded 8 x io 6 to 12 x io ~ protoplasts per gram of leaf tissue, 8o to 9 o % of which were intact as assessed by phase contrast microscopy. Inoculation ofprotoplasts. Protoplasts were pelleted by centrifugation at lOO g for 2 min and resuspended (Io6/ml) in the inoculum of sterile 0.025 M-potassium phosphate buffer, pH 5"6, o'45 M-mannitol, 2/zg/ml poly-L-ornithine (Sigma, Type I-C, approx, mol. wt. I22ooo) and 2/~g/ml CMV (unless otherwise stated) which had been pre-incubated at 25 °C for 5 min just before use. After incubation at 25 °C for 15 min with occasional shaking to keep the protoplasts in suspension, the protoplasts were pelleted by centrifugation and washed twice with sterile o'45 M-mannitol. Mock infection was carried out in the same way but in the absence of CMV. Incubation ofprotoplasts. Protoplasts were incubated at 25 °C and at a concentration of about 8 × 1C/ml in a sterile medium of o'45 M-mannitol, 50 mM-MES (N-morpholinoethane sulphonic acid), pH 5"4, 1o mM-CaCl2, I.O mM-MgSO4, 1.o mM-KNOa, o'2 mMK H2PO4, l 'o/~M-K I, O'Ol/~M-CuSO4, 250 #g/ml carbenicillin, lO # g / m l gentamycin and 25 units/ml nystatin. Illumination at 2000 lux was provided by a cool white fluorescent tube. The percentage of infected protoplasts was determined by fluorescent antibody staining essentially as described by Otsuki & Takebe (]969, I973) using rabbit antiserum to formaldehyde-fixed Q-CMV (Francki & HabiIi, I972). Extraction of virusfi'om protoplasts. All procedures were carried out at o to 4 °C. Stored ( - 8o °C) pelleted protoplasts (4"8 × IO6 to 8"4 × Io ~) were thawed in 1.5 ml of 0"5 M-sodium citrate, pH 6"5, 5 mM-EDTA, o'~ % thioglycollic acid. The homogenate obtained with a Potter-Elvehjem homogenizer was centrifuged at IOOOOg for lO rain and the supernatant shaken vigorously with ['5 ml of chloroform. The emulsion was centrifuged at I o o o g for lo rain and the clear aqueous phase dialysed overnight against 500 vol. of 5 mM-sodium borate, o'5 mM-EDTA, pH 9"o. The dialysed solution was then used for local lesion infectivity assays on Vigna unguiculata (Peden & Symons, 1973) and for sucrose gradient centrifugation to determine virus yield as follows. Analysis ofprotoplast extracts on sucrose gradients. The dialysed extract (o'5 or 1.o ml) was layered on to an 11 ml 5 to 3o ~o (w/v) sucrose gradient in 5 mM-sodium borate, 0"5 mMEDTA, pH 9'o, and centrifuged at 4oooo rev/min and 4 °C for 90 min in a Beckman SW4I rotor. Gradients were analysed using an Isco Model 640 density gradient fractionator on the

C M V replication in protoplasts

725

o.i absorbance scale and virus was quantified by comparing the areas of the peaks on the absorbance trace corresponding to CMV with the areas of peaks containing added known quantities of virus. Virion numbers were calculated using a particle weight for CMV of 5"5 × lO6 (Van Regenmortel et al. I972). When uninoculated protoplasts were extracted as described above in the presence of a known quantity of CMV and the extract analysed on a sucrose gradient, 55 % of the initial quantity of virus was recovered. All data in Results were corrected for this recovery. Radioactive labelling of protoplast proteins in vivo and their analysis by SDS-polyacrylamide gel electrophoresis. Infected and mock infected protoplasts in incubation medium were labelled with either 3H-leucine (5o Ci/mmol) or 14C-protein hydrolysate at about 2o #Ci/ml, either continuously or for 2-h pulses at various times after infection (p.i.). Samples of o'3 ml were centrifuged and the protoplast pellet stored at - 8o °C. When required, pellets were resuspended in 8o/~1 of loading buffer (Laemmli, 197o) and lO to 3o #1 samples were analysed by electrophoresis on discontinuous polyacrylamide slab gels (Laemmli, 197o; Schwinghamer & Symons, I977). Marker proteins were labelled with aH-borohydride (Kumarasamy & Symons, 1979a). Radioactive proteins were detected by fluorography using pre-flashed film (Bonner & Laskey, 1974) and fluorograms scanned on a Joyce-Loebl densitometer. Isolation of single-stranded (ss) RNA from protoplasts. Protoplast samples were thawed in 2.o ml of o-o5 M-NaC1, o.oI M-tris-HCl, p H 8"5, o.oI M-EDTA, 1% (w/v) SDS, and the aqueous phase extracted twice with an equal vol. of water-saturated phenol and then three times with an equal vol. of ether. The solution was made o'25 M in NaCI and the nucleic acids precipitated with 2"5 vol. ethanol overnight at - 1 5 °C. The precipitate was collected by centrifugation, dried and dissolved in o.2 ml o f o . I mM-EDTA, p H 7, when total nucleic acid was required, or in o'5 ml of 15:85 (v/v) ethanol:STE (o.1 M-NaCI, o'o5 M-tris-HC1, pH 6"85, I mM-EDTA) for further fractionation on CF-11 cellulose. The ssRNA, isolated by chromatography on CF-I 1 cellulose, essentially as described by Franklin (1966) and Jackson et al. (I97I), was precipitated with ethanol as described above and dissolved in o.z ml of o-I mM-EDTA, p H 7. Preparation of 82P-eDNA probes. CMV R N A s t to 4 were purified by the method of Symons (1978) and a~P-cDNA to each R N A was prepared by the method of Taylor et al. (I976), essentially as described by Gould & Symons (1977). For the preparation of 3~P-cDNA to sequences unique to R N A 3 (eDNA 3U), the e D N A fragments in e D N A 3 which were complementary to R N A 4 sequences were removed as follows. To 3o to 6o ng of zZP-cDNA 3 (I.5 × Ion to 6 × Io 6 ct/min) was added 12 #g each o f C M V RNAs I and 2 and I #g o f C M V R N A 4 in a total volume of 25o #1 of hybridization buffer (o.18 M-NaCI, o.oi M-tris-HCl, pH 7"o, I.omM-EDTA, o ' o 5 % SDS). After overlaying with paraffin oil, the solution was heated at lOO °C for 3 rain and then hybridization was allowed to occur for 3o rain at 6o °C; thus, R N A s I, 2 and 4 were hybridized to Rot values (initial R N A concentration in tool nucleotide residues/1 × time of hybridization in s) of 2"5 × lO -1, 2"5 × lO -1 and 2.o × lO -3 mol s/I, respectively. After cooling to o °C, 23/zg BMV R N A 4 [purified from BMV R N A (Symons, 1978) and for use as a marker] was added and 2oo/zl of the reaction mix loaded on to an I 1.6 ml, Io to 4 o % (v/v) sucrose gradient in o.1 M-NaCI, lO mM-tris-HC1, p H 7"4, I mM-EDTA. The gradient was centrifuged at 38ooo rev/min in a Beckman SW4I rotor for 16 h at 4 °C and then fractionated with an Isco model 640 density gradient fractionator into o'5 ml fractions. Samples of 5 or IO/zl from each fraction were counted, appropriate fractions pooled (Fig. 4), made o'3 M in N a O H and incubated at 37 °C for 2 h to degrade RNA. After neutralization with acetic acid and the addition of IOO # g / m l of carrier RNA, the 3~P-cDNA was ethanol precipitated, dried and dissolved in hybridization buffer.

726

T. J. G O N D A

AND

R. H. S Y M O N S

Hybridization procedures and analysis of hybridization kinetics. The hybridization of a2P-cDNA to RNA in hybridization buffer and the nuclease $1 assay for the extent of hybrid formation were essentially as described by Gould & Symons (I977)- R0t~ values were determined using the computer program of Pearson et al. (I977). To determine the proportion of particular CMV R N A sequences in the total RNA, the P~t~ for the pure R N A species (Gould & Symons, I977) was divided by the measured R0tk. Since cDNA 3U measured sequences unique to RNA 3, which comprise 59 % of RNA 3, the standard Rot~ for these sequences was taken at 59 % of that for RNA 3. RESULTS

Optimal conditions for infection of co,Tea protoplasts with Q-CM V The conditions given in Methods for the inoculation of cowpea protoplasts with Q-CMV resulted in 40 to 80 % of protoplasts becoming infected, as detected by fluorescent antibody staining, whilst the proportion of viable protoplasts at 4o to 48 h p.i. varied from 5o to 70 % (results not shown). Infection was completely dependent on poly-L-ornithine and was reduced by increasing the virus concentration in the inoculum above 2 #g/ml. These conditions are very similar to those reported by Alblas & Bol (I977) for the infection of cowpea protoplasts with alfalfa mosaic virus (AMV) but are very different to those of Koike et al. 0977) for the infection of Vigna sesquepedalis protoplasts (called cowpea by the authors) with CMV. The latter authors found that infection was not completely dependent on the presence of poly-L-ornithine and used a Io: t ratio by weight of virus to poly-L-ornithine. We suspect that these differences in infection conditions are due to the different strains of CMV used.

Quantification and time course of virus multiplication Three methods were used to assess virus production in protoplasts harvested at o, 16, 32 and 48 h p.i. : fluorescent antibody staining, local lesion assay of extracts on cowpea and sucrose gradient sedimentation analysis of extracts. Results of three such experiments are shown in Table I. Virus production could be detected at 16 h p.i. by both fluorescent antibody staining and infectivity assay. (It is important to note that the CMV antibody only reacted against intact virions and not against isolated coat protein in immunodiffusion tests; our unpublished data.) As measured by these techniques, the level of virus increased appreciably between o and 32 h p.i. but very little, if at all, between 32 and 48 h p.i. It is unlikely that the increase in proportion of protoplasts infected with time was due to secondary infection, since there was no poly-L-ornithine in the incubation medium. Protoplast extracts were analysed by sedimentation on a sucrose gradient (Fig. I) in order to estimate the average number of virions/cell (Table I). Although an absorbance peak due to virus was detected in extracts from protoplasts 32 and 48 h p.i. no peak was detectable in the I6 h sample (Fig. I). However, it was estimated that the smallest quantity of virus detectable by this technique was about io 5 virions/cell; hence, although infectious virus was present at I6 h p.i. (Table I), the level of virus must have been below IO5 virions/cell. Overall, therefore, the three methods employed in Table r provided reasonably consistent results in showing an increase in virus production from o to 32 h p.i. with either further or no increase for the next I6 h.

C M V replication in protoplasts Table

727

Time course of virus synthesis in cowpea protoplasts infected with Q-CMV as determined by three different methods

I.

Time p.i. (h)

Intact protoplasts infected* Mean number of Virions/cell:~ (%) lesions/hal f leaft ( x ~0 -5) r ~ ~, ~ ~c ~-Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. I

2

3

1

2

3

t

2

3

0

0

0

0

0

0

0

0

0

0

16

34

16

25

84

33

5

o

o

o

32 48

57 69

68 73

53 51

248 233

50 52

178 222

5"I 4"6

2'5 5"0

3"6 5"5

* Determined by fluorescent antibody staining. ~" Produced by dialysed protoplast extracts prepared and assayed as described in Methods. Calculated from sucrose gradient analyses (Fig. t) of dialysed protoplast extracts as described in Methods. (a)

(b)

48 h

Sedimentation Fig. I. Sucrose gradient analysis of protoplast extracts. (a) Standards of 3 and 1o ttg of purified Q-CMV in the absence of protop[ast extract. The same peak areas were obtained when virus was mixed with uninfected protoplast extract prior to centrifugation. (b) Extracts of infected protoplasts collected at o, ~6, 32 and 48 h p.i. Each gradient was loaded with extract from the equivalent of ~'3 to 1.8 × to e infected protoplasts, as determined at 48 h p.i. Further details are given in Methods.

Time course of coat protein synthesis P r o t e i n s s y n t h e s i z e d in i n f e c t e d a n d m o c k - i n f e c t e d p r o t o p l a s t s w e r e l a b e l l e d w i t h r a d i o a c t i v e a m i n o a c i d s a n d a n a l y s e d b y S D S - p o l y a c r y l a m i d e s l a b gel e l e c t r o p h o r e s i s f o l l o w e d b y f l u o r o g r a p h y . B o t h c o n t i n u o u s a n d p u l s e - l a b e l l i n g w e r e u s e d in a n a t t e m p t t o i d e n t i f y t h e synthesis of virus-stimulated proteins.

T. J. GONDA AND R. H. SYMONS

728 Mol. wt. x 1~ a

S

M

I

M

I

M

I

M

I

M

I

M

I

M

S

I

Mol, wt. X 1 0 -3

115

115

68

68 53

53

36

36

24.5

24.5

I

I -- 5--

I 1

10--

I ~

15 1

I ~20~

I ~

30~

I ~40

1

I ~50~

Time p.i. (h) Fig. 2. Fluorogram of a 15 o/ polyacrylamide-SDS gel of proteins labelled with aH-leucine (added at i h p.i.) in mock-infected (M) or infected (I) protoplasts. Protoplasts (78 O/,oinfected) were collected at the indicated times p. i. Further details are given in Methods. Mol. wt. standards (S) were: pyruvate carboxylase ([ [ 5 ooo); bovine serum albumin (68 ooo); glutamate dehydrogenase (53 ooo); glyceraldehyde-3-phosphate dehydrogenase (36oo0): and CMV coat protein (z4 500). Table 2. Time course of labelling of CMV coat protein in infected eowpea protoplasts Continuous labelling

Time p.i. (h)

Pulse labelling

Radioactivity in Radioactivity in coat protein as coat protein as % total Beginning of % total radio2-h radioactivity* pulse (h p.i.) activity?

5 to I5 2o 30 40 50

o o 1"5 3'4 4'1 6.] 6"5

8 16 24 32 42

o'75 7"75 7"4 4"9 4"3

* Calculated from densitometer scan of fluorogram of Fig, 2. ? Calculated from densitometer scar, of fluorogram of Fig. 3.

Continuous labelling of protoplasts Proteins synthesized in protoplasts labelled c o n t i n u o u s l y from I to 50 h p.i. were analysed o n a I 5 % polyacrylamide slab gel (Fig. z). Only one protein was detectable in C M V infected protoplasts which was n o t present in mock-infected protoplasts; this co-migrated with the coat protein m a r k e r (Mr 14 5oo) a n d was shown to be coat protein b y t w o - d i m e n sional gel electrophoresis (A. R. Gould, T. J. G o n d a a n d R. H. Symons, u n p u b l i s h e d data). The same pattern as that of Fig. z was obtained when the same samples were electrophoresed o n a I2 % gell which gave better resolution of high mol. wt. proteins. F u r t h e r analysis of proteins from the protoplasts labelled up to 3o or 43 h p.i. on 7"5 % or Io % gels also failed to reveal virus-induced proteins other t h a n coat protein (not shown).

C M V replication in protoplasts Mol. wt.

S

M

I

M

I

M

I

M

I

M

729 I

X I 0 -3

S

Mol. wt X 10 -3

115

115

68

68

53

53

36

36

24-5

24.5

1_8 _1_ ,6 _1_ 24 _i_ 32_1_ 42 _1 Time p.i. (h) Fig. 3. Fluorogram of a 32 % polyacrylamide-SDS gel of proteins synthesized in mock-infected (M) or infected (I) protoplasts and pulse-labelled with I6/~Ci/ml of 14C-protein hydrolysate for 2-h periods beginning at the indicated times p.i. At 46 h p.i., 96 % of the protoplasts were infected. Mol. wt. standards (S) were as in Fig. 2; other details are given in Methods.

The coat protein was clearly detectable at I5 h but not IO h p.i. (Fig. 2). Densitometer scanning of the fluorogram of Fig. 2 showed that the proportion of total radioactivity incorporated into coat protein increased rapidly between t5 and 40 h p.i. with little increase between 40 and 50 h p.i. (Table 2). The percentage of total radioactivity which was incorporated into coat protein is not necessarily proportional to the amount of coat protein synthesized, as the rate of incorporation of SH-leucine was not constant over the labelling period (data not given). However, an increase in the proportion of label in coat protein after any time period clearly demonstrates synthesis of coat protein.

Pulse labelling of protoplasts Rottier et al. (I978) reported that pulse labelling of cowpea protoplasts was essential for the detection of cowpea mosaic virus-specific polypeptides. Therefore, infected and mockinfected protoplasts were pulse labelled for 2-h periods at various times until 44 h p.i. and the labelled proteins were analysed on a I2 % polyacrylamide-SDS gel (Fig. 3). The fluorogram shows that coat protein was still the only virus-induced protein detectable, although it could now be detected in the 8 to Io h p.i. interval; coat protein was not detected at IO h p.i. when continuous labelling was used (Fig. 2). Densitometer scanning of the fluorogram of Fig. 3 showed that coat protein synthesis, as a proportion of total protein synthesis, was maximal during the I6 to I8 h and 24 to 26 h p.i. pulses (Table 2). Synthesis of virus RNAs The synthesis of CMV RNAs in tobacco protoplasts has been followed by the incorporation of SH-uracil (Takanami et aL I977); a combination of actinomycin D treatment and u.v. light irradiation was needed to suppress host RNA synthesis. A more direct approach

730

T. J. G O N D A

.,__ A ___~~__ B - - ~ ..~--C - - ~

AND

R. H. S Y M O N S

100 t

? O x

._= "~ 60

I

Z4°f

.2

20

0 2 4 6 8 1012 14 16 18 20 22 24 Fraction number Sedimentation

-4

J

i

3

a

-2

--

I1

0

log Rot (tool.s/l)

i

Fig. 4

Fig. 5

Fig. 4. Distribution of radioactivity after sucrose gradient centrifugation o f the hybridization reaction mixture of32P-cDNA 3 to CIV[V R N A s I, 2 a n d 4, as described in Methods. T h e vertical arrows indicate the positions o f B M V R N A 4 (left) a n d C M V R N A s T a n d 2 (right), determined by their absorbance at 254 n m during fractionation o f the gradient. Fractions A, B a n d C were pooled as shown. Fig. 5. Hybridization kinetics of 3~P-cDNA recovered f r o m peak A o f Fig. 4 to C M V R N A s 3 (11) a n d 4 (A). Hybridization procedures were as described in M e t h o d s .

has been used here which does not rely on the suppression of host RNA synthesis but which employs quantification of the four CMV RNAs by hybridization analysis with specific complementary 82P-DNA (cDNA) probes. The data provide quantitative estimates of total RNA present, something which cannot readily be achieved by in vivo labelling techniques.

Preparation of a2P-cDNA probes Since RNA 4 is completely contained at the 3'-end of RNA 3 (Gould & Symons, I977), it was necessary for the present work to prepare a cDNA probe for sequences unique to RNA 3 (eDNA 3U). An additional problem in the preparation of this probe was the presence in RNA 3 of breakdown products of the same size derived from RNAs I and 2 (Gould & Symons, I977). The method developed (see Methods) was to hybridize 32P-eDNA to total RNA 3 with excess RNAs I, 2 and 4 and to separate the hybridized cDNA from the unhybridized cDNA 3U by sucrose gradient centrifugation. This was feasible because cDNA3 had an average tool. wt. of approx, r.z to I'5× t@ (Gould & Symons, I977) whereas RNAs 1, z and 4 have mol. vet. of ].35, 1.I6 and o'35 × Io 6, respectively (Peden & Symons, I973). Fig. 4 shows that, after hybridization to RNAs ], 2 and 4, the majority of a2P-cDNA to RNA 3 (peak B) sedimented a little more slowly than brome mosaic virus RNA 4 (mol. wt. o.28 × to6; Lane & Kaesberg, T97t). When z2P-cDNA from the slowest sedimenting peak A was hybridized to RNAs 3 and 4, the kinetics shown in Fig. 5 were obtained. The Rotk for the hybridization of RNA 3 was 4"2 x ]o -3 mol. s/l, a value essentially the same as that obtained by Gould & Symons 0977) for the hybridization of RNA 3 to eDNA 3 and the result expected for eDNA 3U. Hybridization of peak A to RNA 4 (Fig. 5) showed very little hybridization at the R0t~ of RNA 4 (2.1× io -3 mol. s/l; Gould & Symons, I977) which indicates no more than about 5 % contamination of cDNA 3U by cDNA 4 sequences. This


80/jj

100

0

5

lO

15

60 ~ 40

73 t

; o o/*-1100

60 40

2o

2o

0 1oo 20 80 ~ =- 60 {t3 40 20 0 . . . . . . I, - -2-1 0 1 2-3-2-1

/

•

"

~

O0 0 80 60 40 20

0 0 l 2-3-2-1 0 1 2-3-2-1 0 1 2 log Rot (tool.s/l) Fig. 6. Hybridization kinetics of a2p-cDNA 2 (prepared against CMV RNA z) to RNA extracted from infected protoplasts at the indicated times in hours p.i. All procedures were as described in Methods. T a b l e 3. Amounts of the four virus R N A sequence classes in CMV-infected protoplasts* R N A sequence class RNA )

RNA 2

R N A 3U

RNA 4

r

Time Amp.i. punt (h) (#g) o 5 lo I5 20 30 40 50

pmol

Ams.d. IVlol- ount

(To) ar%

pmol

(/zg)

0.o57 o.o42 3'7 13 o.o61 0'073 o'054 7"0 I5 oqo 0'065 0"048 )o 9 oqo 0'33 0"24 I2 )5 o'32 0-85 0"63 )8 [3 r-o o'77 o'57 )6 )3 ' l ' l 1.8i 1"3 9"8 15 2'2 H6 0.86 )6 7 3 .6

Ams.d. Mol- ount

(To) ar % o'05z r3 o.086 6.2 0.086 4'5 0.28 9'4 0-86 ]6 0'95 14 I' 9 26 3 '1 21

Ampmol s.d. IV[ol- ount

s.d. Mol-

(°o) ar% (#g)

pmol (%) ar%

(p,g)

I6 23 I7 I8 )7

0"o59 o'I2 0"059 o'I2 o.io o.zo 0 " 2 9 o'58 0"76 1"5

21

1"0

2"0

21 25

I' 5 3"0

2'9 6"0

IZ 9"9 ~3 24 27

37 32 39 37 30 25 44 34 32 2t 49

0"o37 o'037 o'o63 o'16 0"7 0'36 I'O o.8)

o.11 o'~I o.I8 o'46 2.0 Po 2"9 2. 3

6'4 8"7 9"6 33 32

34 3o 35 3o 40

28

22

33 46

32 )9

* Each sample contained I × to e infected protoplasts.

curve had a R0t~ of 4"3 × lO-3 mol. s/1 which is about xo times the R0t~ of R N A 3 ; this indicates that the R N A 4 used here contained about Io % of sequences derived from R N A 3U. Similar experiments on the hybridization of c D N A 3U to RNAs ~ and 2 showed that c D N A 3U contained less than 5 % of sequences hybridizing to R N A s I and 2 (data not given). Overall, therefore, these results show that peak A c D N A hybridized to at least 9o % to sequences unique to R N A 3 and was c D N A 3U. Material from peaks B and C (Fig. 4) hybridized extensively to all four virus R N A species with peak C showing some enrichment for c D N A to RNAs I and 2 (data not given).

Time course of virus RNA synthesis T h e a p p r o a c h u s e d was to e x a m i n e the h y b r i d i z a t i o n kinetics o f specific c D N A p r o b e s to s s R N A e x t r a c t e d at v a r i o u s t i m e s p.i. f r o m C M V - i n f e c t e d p r o t o p l a s t s . Since, u n d e r R N A excess c o n d i t i o n s , t h e rate o f h y b r i d i z a t i o n is directly p r o p o r t i o n a l t o the c o n c e n t r a t i o n o f h y b r i d i z i n g R N A s e q u e n c e s (Birnstiel et al. I 9 7 2 ; Y o u n g & Paul, T973) , t h e p r o p o r t i o n o f e a c h virus R N A s e q u e n c e in t h e t o t a l R N A e x t r a c t e d c o u l d be d e t e r m i n e d by c o m p a r i s o n o f t h e o b s e r v e d rate w i t h t h a t o b t a i n e d o n h y b r i d i z a t i o n to t h e p u r i f i e d virus R N A species to t h e c D N A p r o b e ( G o u l d & S y m o n s , I977, I978). A n y virus d s R N A p r e s e n t w o u l d c o n t a i n + s t r a n d s e q u e n c e s a n d t h e r e f o r e it was r e m o v e d by C F - I i cellulose c h r o m a t o g r a p h y p r i o r t o t h e h y b r i d i z a t i o n r e a c t i o n s (see M e t h o d s ) . Fig. 6 s h o w s t h e kinetics o f h y b r i d i z a t i o n o f s 2 p - c D N A 2 to s s R N A f r o m infected p r o t o -

732

T . J . GONDA AND R. H. SYMONS

plasts sampled at various times p.i. ; corresponding curves for the other three cDNAs are not given. The R0t~ values plus standard deviations for all curves were determined using the computer programme of Pearson et al. (I977) and the data used to calculate the amounts of the four virus R N A sequence classes (Table 3). In general, the data are internally consistent, although some discrepancies are apparent. For example, there was a decrease in the amount of R N A 4 from 20 to 30 h p.i. while the doubling of the amount of RNA 3U from 4o to 5o h p.i. was not accompanied by a similar change in the other RNAs. Such variation is considered to be due to a combination of experimental errors, only some of which were reflected in the standard deviations. In spite of these variations, the following features of the data of Table 3 are significant. The R N A from the virus particles taken up by or adsorbed to the protoplasts during the inoculation procedure could be detected as given by the data at o h p.i. For three of the four RNAs, there was a significant increase above this level by IO h p.i. while all species showed at least a threefold increase by 15 h p.i. Little or no increase in the level of any of the virus RNAs was detected between 2o and 30 h p.i. Except for the 50 h sample, the level of R N A 3U sequences was approximately equimolar with R N A 4 sequences; the important implications of this are considered below. The relative molar amounts of the four sequence classes remained fairly constant for all time samples; certainly there was no large selective increase in the level of any one species.

Proportion of the RNA species in virion RNA The observation that the molar ratio of R N A 3U to R N A 4 sequences was approximately I : I in the o h p.i. sample was unexpected since R N A 4 sequences are present in both R N A 3 and R N A 4 in virions. When the total virion R N A was fractionated by electrophoresis on a polyacrylamide tube gel (Peden & Symons, I973), the molar ratio of full-length R N A 3 to full-length R N A 4 was I:2.2. However, hybridization of this total virion R N A to c D N A 3U and to e D N A 4 gave a molar ratio of I :o'93 (data not shown), in good agreement with the o h p.i. result of I :o'92 (Table 3). From these data, it must be concluded that there were R N A 3U sequences in the virion R N A other than those in intact RNA3.

Estimates of initial and final numbers of virions per infected cell Since all the virus R N A in the o time sample of Table 3 must have been derived from virus in the inoculum, it was calculated that 2.8 × i O virions/infected protoplast were taken up or adsorbed. This value is roughly io-fold higher than that reported for several other virus-protoplast systems (Motoyoshi et aL I973; Hibi et al. T975; K u b o et aL r976; Shaw, T978). I f all of the virus R N A in the infected protoplasts at 4o to 5o h p.i. was encapsidated, it was calculated that there should have been 2 to 3 × los virions per infected cell. This is four to six times higher than the values found in three different experiments shown in Table I and indicates that a considerable proportion of virus R N A synthesized in protoplasts was not encapsidated. Comparison of time courses of virus, RNA and coat protein synthesis From the data in Tables I to 3 and Fig. 2 and 3, the accumulations of virus, virus R N A and coat protein, as well as the rate of coat protein synthesis, as a function of time after infection were calculated as percentages of their maximum values and plotted in Fig. 7. For these calculations, the final level of R N A was assumed to be that at 4o h p.i., the proportion of total radioactivity incorporated into protein found in coat protein was taken as a measure of the amount of coat protein, the results of the three experiments in Table T on the numbers of virions/cell were averaged and at I6 h p.i. virion numbers/cell were taken as 20% of


733

100 80

E

60

E

2G 0

I

0

l0

2;

30

I

d0

50

Time p.i. (11) Fig. 7. Comparison of the time courses o f total virus R N A (O), virus (C)) and coat protein U3) synthesis in CMV-infected protoplasts. Also shown is the rate o f coat protein synthesis (A) as determined by pulse-labelling. The level of total virus R N A was determined by summing the amounts of each sequence class in Table 3. For further details, see text.

the maximum value on the basis of the lesions/half leaf (Table I) and of considerations above on the sensitivity of the estimation of virion numbers/cell. In view of the origin of the data for Fig. 7, the overall pattern must be considered approximate. Fig. 7 indicates that virus RNA and coat protein synthesis and the assembly of virions occurred co-ordinately while the rate of coat protein synthesis reached a relatively early peak at about 15 h p.i. DISCUSSION

The results presented here have allowed the first comparative analysis of the time course of synthesis of C1MV coat protein (both total and rate) of the four CMV RNAs and of intact virions in CMV-infected protoplasts. The only other system in which similar data are available is for TMV-infected tobacco protoplasts (Takebe et al. t975). In contrast to the coordinate synthesis of virus, RNA and coat protein found with CMV, there was a lag between TMV RNA synthesis on the one hand and TMV and coat protein synthesis on the other in tobacco protoplasts. Further, the peak rate of CMV coat protein synthesis at about T5 h p.i. is to be compared with an increasing rate of TMV coat protein synthesis with time p.i. (Siegel et al. I978). This difference between CMV and TMV may be due in part to different assembly mechanisms for rod-shaped and spherical viruses. The only virus-induced protein detected here by one-dimensional polyacrylamide slab gel electrophoresis was CMV coat protein. It appears, therefore, that other virus-induced proteins are synthesized at much lower levels than that of coat protein and hence are obscured by protoplast proteins on the fluorograms. Further studies aimed at detecting other virus-induced proteins by the more sensitive technique of two-dimensional gel electrophoresis are in progress. Single-strand CMV RNA synthesis was measured here by hybridization analysis with specific ~2P-cDNA probes in RNA excess. Virus RNA present as replicative form or replicative intermediate was removed before hybridization analysis ; synthesis of these will be followed at a later date as will the detection and quantification of any single-strand minus RNA sequences. It is important to note that the hybridization method used here determined total virus RNA sequences and did not distinguish between intact virion RNAs and lower mol. wt. fragments of them. Techniques for the hybridization analysis of RNAs on the basis of size are now available (Alwine et al. I977; Stark & Williams, ~979).

734

T.J.

GONDA

AND

Ro H. S Y M O N S

The usual procedure of incubating infected protoplasts in the presence of radioactive precursors, usually a2P-phosphate or aH-uridine, followed by gel electrophoresis in order to examine virus RNA synthesis has a number of disadvantages over the use of labelled cDNA probes as described here. One of these is that the virus RNAs may be close in size to cellspecific RNA species: thus, Takanami et al. 0977) needed to use a combination of u.v. irradiation and actinomyin D treatment to suppress completely ribosomal RNA synthesis in tobacco protoplasts infected with CMV as CMV RNAs t and 2 are very close in size to 25S ribosomal RNA. Further, the use of u.v.-irradiation in particular has disadvantages as it probably interferes with virus replication. Sakai et al. 0977) found that u.v.-irradiation of tobacco protoplasts altered the kinetics of cowpea chlorotic mottle virus (CCMV) synthesis while, in the present work, u.v.-irradiation severely inhibited CMV infection of cowpea protoplasts (data not given). In addition, interpretation of time course studies relies on the assumption that the specific radioactivities of the virus RNAs remain constant; Bancroft et al. (I975) found differences in the specific activities of the CCMV 3~P-RNA species synthesized in tobacco protoplasts. The use of hybridization analysis with cDNA probes circumvents these problems and allows the actual amount of virus RNA to be determined. From the observation that, at most times after infection, the molar ratio of RNA 3U to RNA 4 sequences was close to l : I (Table 3), it follows that all the RNA 4 sequences detected could be accounted for by those present as RNA 3- This conclusion is inconsistent with any model for the replication of RNA 4 which involves transcription from a negative RNA 4 strand or partial transcription from a negative RNA 3 strand but is consistent with RNA 4 being produced by the specific nucleolytic cleavage of RNA 3. Hence, RNA 3U sequences detected by hybridization would be a mixture of those present in intact RNA 3 and those cleaved off during the production of RNA 4. Since the results of Takanami et al. 0977) did not show any peak corresponding to full length RNA 3U in RNA isolated from tobacco protoplasts infected with CMV and labelled with ZH-uracil, it is possible that R N A 3U after cleavage is randomly degraded but to sizes still large enough to be detected by hybridization. It is of considerable interest that Takanami et al. (I977) found only trace amounts of dsRNA corresponding to CMV RNA 4 while Bancroft et al. (I975) obtained no evidence for a dsRNA form of RNA 4 of either BMV or CCMV in tobacco protoplasts; the latter data contrast with the reported isolation of the double-strand replicative form of BMV RNA 4 by Bastin & Kaesberg (1976). The authors thank Dr R. I. B. Francki for the use of glasshouse facilities, Dr W. R. Pearson for providing a copy of his computer program and Dr J. C. Wallace for his assistance in the use of it. This work was supported by the Australian Research Grants Committee. REFERENCES ALBLAS, F. & BOL, J. F. 0 9 7 7 ) . Factors influencing the infection o f cowpea mesophyll p r o t o p l a s t s by alfalfa mosaic virus. Journal of General Virology 36, ]75-185. ALWINE, J. C., KEMP, D. J. & STARK, G. R. (]977)- M e t h o d for detection o f specific R N A s in agarose gels by transfer to diazobenzyloxymethyl-paper a n d hybridization with D N A probes. Proceedings of the National Academy of Sciences of the United States of America 74, 535o-5354 • BANCROFT, J. B., MOTOYOSHI, F., WATTS, J. W. & DAWSON, J. R. O. (]975). C o w p e a chlorotic mottle a n d b r o m e mosaic viruses in tobacco protoplasts. In Modification of the Information Content of Plant Cells, pp. 133I6o. Edited by R. M a r k h a m , D. R. Davies, D. A. H o p w o o d a n d R. W. Horne. A m s t e r d a m : N o r t h Holland. BASTIN, M. & KAESBERG, P. (I976). A possible replicative f o r m o f b r o m e mosaic virus R N A 4. Virology 7a, 536-539. 8EIEg, H. & 8RUENtNG, G. (I975). T h e use o f an abrasive in the isolation o f cowpea leaf protoplasts w h i c h s u p p o r t the multiplication o f cowpea mosaic virus. Virology 64, 272-276.


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