Isolation and analysis of virus-specific ribonucleoprotein of tobacco mosaic virus-infected tobacco

VIROLOGY

127,

237-252

isolation

(1933)

and Analysis of Virus-Specific of Tobacco Mosaic Virus-Infected

Y. L. DOROKHOV,*

*Laboratory

Ribonucleoprotein Tobacco

N. M. ALEXANDROVA,? N. A. MIROSHNICHENKO,t AND J. G. ATABEKOV*,’

of Molecular Biology and Bioorganic Chemistry and Department of Virology, University, and tbwtitute of Applied Molecular Biology and Genetics, Academy of Agricultural Sciences, Moscow 1172S’I+, USSR Received

June

29,

1982;

accepted

January

Lomonosov

State

5, 1983

A ribonucleoprotein fraction (vRNP) of a characteristic buoyant density greater than the buoyant density of tobacco mosaic virus (TMV) particles has been isolated from infected tissue by CsaS04 density gradient centrifugation. The vRNP particles appear to be TMV specific because they are synthesized in the presence of aetinomycin D and have RNAs identified as genomic and I-class subgenomic (apparent M, 1.1-1.3 X lo6 and 0.6 0.8 X 106) RNAs by their electrophoretic mobility and hybridization to plasmid-bearing RNA sequences. Polypeptides of apparent molecular weights 17,500 (TMV coat), 31,000, 37,000, and 39,000 were major constituents of vRNP. Of the minor polypeptides, those of apparent molecular weights 70,000, 68,000, 55,000, and 25,000 had electrophoretic mobilities similar to mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. vRNP from common TMV-infected plants, but not from plants infected with a mutant that did not form native coat protein, reacted with immunoglobins against TMV and TMV coat protein. Common TMV and its vRNP differed in the extent of reactivity toward the two immunoglobins, in electron microscopic appearance, and in the higher sensitivity of vRNP to ribonuclease.

Thus, at restrictive temperatures the infection of TMV strains Nil18 or JEavum exists in some “unstable form” when RNA molecules are unprotected or only partially protected from RNase. Possibly such an “unstable form” (presumed to be free viral RNA) may occur in plants infected not only with the coat-protein mutants but also with normal temperature-resistant plant viruses. It can be speculated that the “unstable form” of viral infection is not the free viral RNA but an informosomelike nucleoprotein complex serving for the transport of viral genetic material from originally infected to healthy plant cells. The present work shows that virus-specific ribonucleoprotein particles (vRNP) differing from intact TMV virions in buoyant density are formed at 25 or 33” in‘cells infected with TMV vulgare or ts mutant Nil13 The structure of vRNP was studied, and the sets of constituent polypeptides and RNAs were analyzed.

INTRODUCTION

Eukaryotic messenger RNA (mRNA) occurs in two types of cytoplasmic particles. The bulk of it is usually found in polysomes, and the remainder is associated with specific proteins in a class of lowermolecular-weight particles known as free cytoplasmic messenger ribonucleoprotein (mRNP). The ribonucleoprotein particles of nonribosomal nature were termed informosomes (reviewed by Spirin, 1969, 1972; Preobrazhensky and Spirin, 1978). Among various tobacco mosaic virus (TMV) strains a number of temperaturesensitive (ts) mutants have been discerned that can spread systemically in the infected leaf though forming no functional coat protein at elevated temperatures (3035O) (Jockusch, 1964, 1966a, b; WittmannLiebold et cd, 1965). 1 To whom all correspondence prints should be addressed.

and requests

for re-

237

0042~6822/83 Copyright All rights

$3.00 Q 1983 by Academic Press, Inc. of reproduction in any form reserved

DOROKHOV

238

Some parts of this work have been reported in preliminary communications (Dorokhov et UC, 1980a, b). MATERIALS

AND

METHODS

Viruses. TMV vulgare was kindly supplied by Dr. H. G. Wittman, and the ts mutant Nil18 was donated by Dr. H. Jockusch. Isolation and purification of TMV were carried out as described by Asselin and Zaitlin (1978). Labeling of TMV-infected leaf material. Leaves of Nicotiana tabacum L. cv. Samsun plants were mechanically inoculated with TMV. After 48 hr at 25 or 32-33”, the inoculated leaves were detached and stripped of the lower epidermis with corundum and a brush (Beier and Bruening, 1976). The leaf material was incubated for l-2 hr in a solution containing 100 pg/ml actinomycin D (AMD) (Sigma Chemical Co.), 100 pg/ml cephaloridin (Pliva, Yugoslvaia), and 10 pg/ml rymicidin (Charles Pfizer and Co.). Then either 100 &i/ml of [3H]uridine with a specific activity of 26 Ci/mmol or 50 &i/ml of a 14C-amino acid mixture with a specific activity of 54 &i/ g-atom C (both from the Radiochemical Centre, Amersham) were added and vacuum-infiltrated into the leaves which were then left for 3 or 20 hr at the specified temperature. Isolation and analysis of vRNP in CsCl density gradients. All steps were performed at 4”. The labeled leaf material was ground with corundum by means of a mortar and pestle in the presence of extraction Buffer A (Jackson and Larkins, 1976) containing 0.2 M Tris-CHl, pH 9.0,0.4 M KCl, 0.2 M sucrose, 30 mil4 MgClz, 25 mM ethylene glycol tetraacetic acid (EGTA). The homogenate was filtered through two layers of Miracloth and centrifuged at 10,000 g for 10 min. The supernatant (postmitochondrial fraction) was supplemented with Triton X-100 to 2%, layered onto onefourth of its volume of 30% (w/v) sucrose in Buffer A and, centrifuged at 100,000 g for 270 min. The pellet was resuspended in Buffer B (20 mM triethanolamine pH 7.7, 5 mM MgCIP, 25 mM KCl, 1 mM mer-

ET

AL.

captoethanol) with a Potter-Elvehjem homogenizer and recentrifuged at 10,000 g for 10 min. Neutralized formaldehyde was added to the supernatant to 4% and the mixture was left for 24 hr at 4”. Then dry CsCl was dissolved in the mixture at 0.2 g/ml, and samples of 1 ml were applied on top of a gradient composed of the following layers (from bottom to top in a Spinco SW 50.1 tube): 1.25 ml of 53%, 1.25 ml of 45%, and 1.25 ml of 39% (w/w) CsCl, all in Buffer B with 4% formaldehyde. The gradients were centrifuged for 20 hr at 40,000 rpm in a Spinco L2-65B centrifuge, and the contents of the tubes were fractionated for refractometry and radioactivity measurements. In control experiments, healthy or TMVinfected leaf material (0.5 g) was rapidly frozen in liquid nitrogen, mixed with 50 /Ig of 14C-TMV RNA or 14C-TMV, and ground to a fine powder in liquid nitrogen with a mortar and pestle. Then 10 ml of Buffer A was added, and the further procedure was as described above. Analysis of vRNP in Cs$04 density gradients. Pellets of vRNP obtained by differential centrifugation (100,000 g, 270 min) were resuspended in Buffer C (40 mM TrisHCl, pH 8.5, 20 mM KCl, 10 mM MgClz) and homogenized in a Potter-Elvehjem homogenizer. The mixture was centrifuged at 10,000 g for 10 min, and the supernatant was applied on top of a gradient which composed of the following layers (from bottom to top in a Spinco SW 50.1 tube): 1.25 ml of 50%, 1.25 ml of 42%, and 1.25 ml of 36% Cs2S04, all in Buffer C. The gradient was centrifuged for 20 hr at 40,000 rpm in a Spinco L2-65B centrifuge. Assay of vRNP RNA sensitivity to RNase. The vRNP pellet obtained by centrifugation of the homogenate of tobacco leaves infected with TMV and labeled with [3H]uridine was resuspended in 20 mM triethanolamine, pH 7.7,5 mM MgClz, 25 mM KCl, 1 mM mercaptoethanol, homogenized in the Potter-Elvehjem homogenizer, and centrifuged at 10,000 g for 10 min. The supernatant was supplemented with RNases A and Tl to 50 pg/ml and 10 units/ml, respectively, and treated for 40 min at 37”.

VIRUS-SPECIFIC

RIBONUCLEOPROTEIN

Then neutralized formaldehyde was added to 4% and the mixture was incubated for 18 hr at 4”. The fixed material was layered on top of a preformed CsCl gradient and centrifuged as described above. Immunoprecipitation ofvRNP. Antisera to TMV (titer 1:1024) and to TMVCP (titer 1:256) from rabbits were freed of ribonuclease as described by Palacios et al. (1972). Fractions of CsCl or Cs2S04 gradients containing vRNP were dialyzed overnight against 10 m&l phosphate buffer, pH 7.2. The reaction mixture contained 100 ~1 of I~TMV or I~TMVCP in a l/4 dilution. After standing at 37” for 40 min, the mixture was diluted with 350 ~1 of phosphate buffer, and immunoprecipitated and nonimmunoprecipitated radioactivity was quantitated by either of two techniques. (1) The mixture was filtered through a Whatman GF/A filter and washed with phosphate buffer; the material retained on the filter is referred to as “precipitate,” and the filtrate as “supernatant.” (2) The mixture was centrifuged at 7000 g for 10 min, and the pellet was washed with phosphate buffer and recentrifuged. Radioactivity was assayed in the precipitates (after resuspension in phosphate buffer) and in pooled supernatant fractions. Results obtained by the two methods did not differ significantly. Isolation and puti$cation of cellular informosomes. All steps were performed below 4“. Leaves of Nicotiana tabacum L. cultivar Samsun were homogenized by grinding with corundum by means of a mortar and pestle in the presence of extraction Buffer A (Jackson and Larkins, 1976) or Buffer Al which is Buffer A but with 60 mM KCl. The homogenate was filtered through two layers of Miracloth and centrifuged at 10,000 g for 15 min. The supernatant was supplemented with Triton X-100 to 2% and layered onto 8 ml of 35% (w/v) sucrose in Buffer D (40 mM TrisHCI, pH 9.0, 0.2 M KCl, 35 mM MgCIB, 5 mM EGTA) in Spinco SW 50.2 tubes; these were centrifuged at 45,000 rpm for 240 min. The pellets were resuspended in 2 ml of Buffer C with a Potter-Elvehjem homogenizer. After spinning at 10,000 g for 10

IN

TOBACCO

239

min, the supernatant was layered on 3 ml of 35% Cs2S04 (p = 1.38 g/cm3) in Buffer C in a Spinco SW 50.1 tube and centrifuged at 40,000 rpm for 240 min. The contents of the tubes was fractionated by puncturing the bottom, and fractions with a buoyant density of 1.33-1.38 g/cm3 were pooled. The material (1.6 ml) was applied onto 3.4 ml of 35% C&SO4 in Buffer C and centrifuged in a Sorvall TV-865 vertical rotor at 50,000 rpm for 5-6 hr. Tubes were fractionated with a Buchler Autodensiflow system, recording uv absorption with an ISCO UA5 monitor. Fractions with densities of 1.33 to 1.39 g/cm3 were selected by refractometry and dialyzed against Buffer C or 5 mM Tris-HCl, pH 8.5. Dialysis sacks and all solutions were pretreated to abolish RNase activity. The preparation obtained after dialysis was analyzed in the range 210-350 nm in a Pye Unicam SP-8000 spectrophotometer. RNP preparations obtained by dialysis against 5 mM Tris-HCl, pH 8.5, were lyophilized and stored at -60°C. Sedimentation analysis of cellular informosome preparations in a sucrose gradient. The RNP preparation was layered over a gradient formed with 2 ml 50%, 3.5 ml 37.5%, 3.5 ml 25%, and 12 ml 12.5% (w/ v) sucrose in Buffer C, from the bottom to the top in a Spinco SW 41 tube, and centrifuged at 39,000 rpm for 180 min. Tube contents were fractionated as above; sedimentation coefficients were determined from the tables of McEwen (1967). Treatment of cell informosome preparations with RNase and DNase. Informosomes were treated for 40 min at 37” with 5 hg/ml RNase A (Serva, Heidelberg) or with 50 kg/ml DNase (Worthington). Polyacrylamide gel electrophoresis. Slab polyacrylamide gradient (8-20%) gels (14 X 16 cm) were cast as described by Laemmli (1970). Cs2S04 gradient fractions containing TMV vRNP labeled with 14Camino acids were dialyzed overnight against Buffer C, and vRNP was precipitated by adding 50% trichloracetic acid (TCA) to 10% at O-4” in the presence of 25 /Ig cytochrome c as a carrier. After at least 2 hr the material was sedimented at

240

DOROKHOV

12,000 g for 10 min at 4” and washed three times with cold 10% TCA and three times with cold acetone. The dried vRNP material as well as lyophilized cellular informosomes was dissolved in the gel sample dissociation buffer (125 mM Tris-HCl, pH 6.8, 12.3% sodium dodecyl sulfate (SDS), 1% mercaptoethanol, 10% glycerol, 0.01% bromophenol blue), and heated for 1 hr at 60°C and for 3 min in a boiling water bath. The samples were electrophoresed in 1.5mm-thick slab gels in an apparatus described by Studier (1973) for 5-6 hr at 30 mA. Gels were fixed with 10% acetic acid in 10% methanol with 0.1% Coomassie brilliant blue R-250 for 30 min, washed overnight in 7% acetic acid, 5% methanol, vacuum-dried, and autoradiographed. Molecular weights denoted on figures were obtained by comparison with the mobilities of marker proteins-cytochrome c (12,400), TMV coat protein (17,500), barley stripe mosaic virus coat protein (23,000), chymotrypsin (25,000), a-subunit of Escherichia coli RNA polymerase (39,000), ovalbumin (43,000), bovine serum albumin (SS,OOO), phosphorylase b (94,000). RNA electrophoresis in agarose. Fractions of CszS04 gradients containing TMV vRNP were dialyzed for at least 18 hr against 1000 vol of Buffer C. To the dialyzed material an equal volume of watersaturated phenol was added and the mixture was shaken for 20 min. After spinning at 10,000 g for 10 min, the aqueous phase was withdrawn and treated again with phenol. RNA was precipitated by adding 2 vol of ethanol and several drops of 3 M sodium acetate (pH 4.0), stored overnight at -20°, centrifuged, washed with 70% ethanol, and vacuum-dried. RNAs were electrophoresed in 2% agarose tube gels with 6 M urea, 25 mM citric acid, pH 3.8, at 4 mA per tube for 4 hr in the cold. Gels were stained with 10 pg/ml ethidium bromide and examined under uv light to determine the positions of markers (plant cytoplasmic ribosomal RNAs); 18 S (0.7 X 106) and 25 S (1.3 X 106) RNA, as well as TMV genomic RNA (2 X 106). Gels were cut into 2-mm slices and RNA was extracted from each slice with 0.5 ml

ET

AL.

of 0.5% SDS with shaking for 18 hr at 50”. The radioactivity of each sample was measured in 10 ml of dioxane scintillator solution in a Nuclear Chicago Mark III radiospectrometer. Preparative isolaticm of RNA from gels. After electrophoresis agarose gels were sliced into 2-mm-thick disks each of which was placed into a vial containing 0.5 ml of extraction Buffer E (10 mM Na2HP04 pH 7.0,5 mM EDTA). Vials were shaken overnight at 5O, extracts were withdrawn, and gel slices were treated with another 0.4 ml of Buffer E for 5 hr at 5”. The first and second extracts were combined, and aliquots were taken for radioactivity measurements. After this, appropriate fractions were selected, pooled, and treated twice with phenol. The aqueous phase was then mixed with 2 vol of cold ethanol, left overnight at -2O”, and centrifuged. The pellet was dissolved in Buffer B and dialyzed overnight against 1000 vol of Buffer B. The dialyzed material was reprecipitated with ethanol, washed twice with 70% ethanol, and vacuum-dried. The yield of RNA was 70-80s. Hybridization of [‘HjRNA with pBRTMV-I plasmid DNA. The plasmid pBRTMV-1 containing sequences complementary to TMV RNA was provided by Dr. Yu. V. Kozlov and Dr. V. Rupasov (Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow). The plasmid contains a DNA sequence complementary to about 3500 bases from the 3’ end of TMV RNA. The plasmid was cleaved with restriction endonucleases into fragments of about 400 pairs, and hybridization was carried out as described by Dawson and Grantham (1981). Reassembly of SH-labeled RNA of vRNP with TMVcoat protein. The reconstitution procedure has been described previously (Atabekov et a.& 1970). The reconstitution material was treated with pancreatic RNase (10 pg/ml) and RNase T1 (10 U/ml) and samples were taken for determination of the RNase-stable material. Electron microscopy of TMV vRNP was performed according to Lebeurier et al (1977). Electron microscopic serology of

VIRUS-SPECIFIC

vRNP was carried (1977).

RIBONUCLEOPROTEIN

out after Otsuki

et al.

RESULTS

Isolation of vRNPfrm Plant Cells Infected with TMV vulgare Upon TMV infection, besides mature TMV particles (buoyant density in CsCl of 1.32-1.33 g/cm3), nucleoprotein material with a buoyant density of 1.36-1.45 g/cm3 (vRNP) was found after different times of labeling in the presence of AMD with rH]uridine (Figs. lA, B) as well as with the 14C-amino acid mixture (Figs. lD, E). Practically no labeled material of similar buoyant density is observed in control extracts from healthy plants (Figs. lC, F), suggesting that the RNA(s) and protein(s) constituting vRNP are virus specific. Separate experiments showed that only Jh( ‘W-uridine)

IN

TOBACCO

trace amounts of vRNP could be sedimented at 10,000 g, and that the bulk of vRNP is recovered in the 100,000 g pellet (not shown). Density gradient centrifugation demonstrates the vRNP population to be heterogeneous, the major portion of it being usually represented by particles with buoyant densities of 1.36-1.40 g/cm3 (see below). To check the possibility of vRNP formation during the isolation procedure, the following control was run: free 14C-TMV RNA or 14C-TMV in Buffer A and healthy or infected tobacco leaves were mixed, frozen in liquid nitrogen, and thoroughly homogenized in liquid nitrogen; then the powder was thawed and processed (see Materials and Methods). As can be seen in Fig. 2, virtually no vRNP-like material is produced under such conditions.

POh(‘ll-widine)

uninfected, 3h (‘II-uridine)

C

3h( “C-aminoacidr)

__ 20h(wC-aminoacids) 19 -I,30

uninfected, 3h (14C-mhoacids)

F

lo

20

241

10 20 Fraction number

FIG. 1. CsCl gradient diagrams of vRNP from plants infected with TMV vz&czre. (A, B) vRNP from leaves infected for 48 hours and labeled with [‘Hluridine for 3 hr (A) or 20 hr (B), as seen in a CsCl gradient; (D, E) the same, but labeled with %-amino acids; (C, F) healthy leaf tissue labeled for 3 hr with [*H]uridine (C) or “C-amino acids (F) in the presence of AMD. Buoyant densities are indicated above zones.

242

DOROKHOV

ET

AL,

IA

175 174 473

10

20 E ‘;

.l- “J

1,325 h

10

Fraction

46 -195

20

number

FIG. 2. CsCl gradient diagrams of a reconstruction experiment to test for possible artifactual vRNP formation during homogenization and extraction. “C-TMV RNA (50 pg) or “‘C-TMV (50 pg) was added to nonlabeled leaves, either healthy (A, E) or infected with TMV for 3 days (B); the mixture was rapidly frozen in liquid nitrogen, ground to a fine powder and processed as described under Materials and Methods. Distribution of radioactivity of “C-TMV (E) or r4C-TMV RNA (A, B) in a CsCl gradient is presented for such homogenates, free “C-TMV RNA (C), and 14C-TMV (D).

Analysis of RNA Species in vRNP of TMV w&are and Proof of Their Virus-Spe&tic Nature For direct RNA analysis, vRNP was isolated in gradients of CszSOd, which has a lesser deproteinizing effect than CsCl (Greenberg, 1976), thus obviating the formaldehyde fixation of the ribonucleoprotein material. The buoyant density of vRNP of TMV vulgare in CszS04 ranged from 1.34 to 1.45 g/cm3 (Figs. 3A, B), the

respective values for intact TMV and TMV RNA being 1.29 and 1.61-1.63 g/cm3 (Figs. 3C, D). It should be noted that a material with a buoyant density of 1.23 g/cm3 was invariably present in CszS04 gradients but was never found in CsCl gradients. It was shown that these structures represent a membrane-bound complex containing vRNP together with TMV particles (data not shown). Agarose gel electrophoresis of rH]uridine-labeled RNA from vRNP with

VIRUS-SPECIFIC

I* ‘H-uridine

7

RIBONUCLEOPROTEIN

labelling

1

I IC

w,v

IN

243

TOBACCO

1 ,,‘f :- aminoacids

D TMV”C-RNA 4.61

20

h .,

Fraction

number

FIG. 3. C&SO, gradient diagrams of vRNP from plants infected with TMV leaves infected for 48 hr and labeled with rH]uridine (A) or with %amino (C, D) preparations of ‘*C-TMV (C) and [%]RNA from TMV.

densities of 1.34-1.49 g/cm3 in C&SO4 reveals three RNA species designated RNAs 1, 2, and 3 (Fig. 4A). By electrophoretic mobility RNA 1 corresponds to TMV genomic RNA (Mr 2 X 106), and RNAs 2 and 3 coincide with intermediate-size subgenomic TMV RNA, respectively Ii (M, 1.1-1.3 X 106) and Iz (Mr 0.6-0.8 X 106),which have been found in TMV preparations by Beachy and Zaitlin (1977). vRNP appears to contain no small subgenomic RNA termed LMC (low-molecular-weight component) which has a molecular weight of 0.28 X lo6 and codes for the coat protein (Hunter et al., 1976; Siegel et al., 1976). A similar analysis of the 1.29 g/cm3 material corresponding to the mature virus reveals a single RNA species with the mobility of genomic TMV RNA (Fig. 4B). To prove the virus-specific nature of RNA contained in vRNP, RNAs 1, 2, and 3 recovered from agarose gels (see Materials and Methods) were subjected to hybridization analysis with a pBR-TMV-1 plasmid carrying a nucleotide sequence complementary to TMV RNA. As shown

wulgare. vRNP from acids (B) for 18 hr;

in Table 1, all three RNA types can interact with plasmid DNA to form RNase-resistant hybrids. It should be noted that increasing the plasmid DNA concentration in the hybridization mixture did not enhance the percentage of hybridization (data not shown), signifying that DNA concentration was saturating. It was shown by Beachy and Zaitlin (1977) that intermediate RNAs are present in small quantities in the virus preparations. Thus, the ability of being encapsidated can be regarded as one of the specific properties of subgenomic TMV RNAs of the I class. Therefore, we examined the possibility of in vitro reassembly of TMVspecific RNAs 1 (genomic RNA) and 2 and 3 (supposedly, subgenomic I-class RNAs) with TMV coat protein (TMVCP) (Table 2). It can be seen that RNAs 2 and 3 (as well as genomic RNA 1) can be effectively reconstituted. It was shown in a separate experiment that the material reassembled from labeled RNAs 2 and 3 was stable upon CsCl gradient centrifugation and its buoyant density (1.30-1.31 g/cm3) was rather

DOROKHOV

244

lo

TMV RNA 258 188

4A

ET

AL.

no mature virus (no material with a density of 1.29 g/cm3 in Cs2S04 characteristic of TMV), but vRNP with densities of 1.361.45 g/cm3 was observed. Analysis of RNA from vRNP preparations revealed mostly genomic TMV RNA (Fig. 6). This result indicates that the genomic RNA found in vRNP of TMV wulgare (see above) does not originate from the mature virus contaminating vRNP preparations.

‘: s x Q

Sensitivity of RNA in TMV vRNP to RNase

Unlike the RNA of the mature virus, vRNP RNA is almost completely deTMV RNA

B

4I

16l

I c

‘Ii

TABLE HYBRIDIZATION

OF RNA

PLASMID pBR-TMV-1 PLEMENTARY TO TMV

R

1 FROM

CONTAINING RNA

vRNP

WITH

SEQUENCES

THE COM-

Radioactivity (wm) RNA species

10

20 Fraction

30 40 number

50

FIG. 4. Electrophoretic analysis of RNA from TMV vRNP. (A) fractions of C&SO, gradient with densities of 1.34-1.49 g/cm’ containing vRNP of TMV vulgare were dialyzed against Buffer C and treated with phenol (see Materials and Methods); RNA was electrophoresed in 2% agarose gels; (B), the same for material with a density of 1.29 g/cm*. Arrows denote the positions of RNA markers (from left to right); TMV genomic RNA and 25 S and 18 S rRNA. Numbers refer to particular species of TMV RNA (see text) with molecular weights of 2 X lo6 (RNA l), l.l1.3 X lo6 (RNA 2), 0.6-0.8 X lo6 (RNA 3).

close to that of intact TMV (1.325 g/cm3). Electron microscopic analysis of the reassembled material supported the presence of rod-shaped particles similar to the TMV virions (data not shown). Anal&s of RNA in vRNP of the ts Mutant Nil18

Figure 5 shows that at nonpermissive temperature NilWinfected cells contain

Expt

Total

RNaseresistant

Hybridization (%)

RNA

1

1 2

2540 2392

643 897

25 34

RNA

2

1 2

9800 4760

3430 2572

35 54

RNA

3

1 2

3000 3140

520 738

17 24

[*H]rRNA

1 2

3148 4671

64 138

2 3

1 2

5106 5350

1377 1872

27 35

RNA from 14C-TMV

Note. The hybridization mixture contained the tested rH]RNA, and 2 pg of plasmid DNA in a total volume of 0.2 ml of 0.12 M potassium phosphate, pH 6.8. The mixture was heated for 2 min at 100” and incubated for 48 hr at 60’. The reaction was stopped by freezing in liquid nitrogen. After thawing, 20X SSC (0.15 MNaCI, 15 mMsodium citrate, pH 7.0) was added to 3X SSC. Samples were incubated for 1 hr at 37” without additions or with RNases A (10 pg/ ml) and T1 (10 U/ml), and washed with 7% trichloroacetic acid on a Whatman GF/A filter. Each value is a mean of three parallel determinations.

VIRUS-SPECIFIC TABLE

IN VITRO REASSEMBLY WITH

RIBONUCLEOPROTEIN

245

TOBACCO

TMV , RNA

2 OF RNAs

25s

188

OF vRNP

TMVCP

Radioactivity

(cpm) Percentage of RNaseresistant radioactivity

Expt

Total TCA-insoluble radioactivity

After RNase treatment

RNA-l

1 2

8280 4500

6040 1720

56

RNA-2

1 2

9260 8120

3300 4080

40

RNA-3

1 2

5340 9860

2580 3660

43

RNA species

IN

Note. Coating of aH-labeled RNA (concentration less than 1 #g/ml) with the coat protein was carried out at 20’ in 0.1 M phosphate buffer, pH 7.0, at a TMV coat-protein concentration of 20 pg/ml. Prior to mixing, the components of the reconstitution mixture were preincubated at 20” for 20 min. The reconstitution procedure has been described by Atabekov et al. (1970). The reconstituted material was treated with pancreatic RNase (10 &g/ml).

stroyed on treatment with RNase A and T1 (Fig. 7B). RNAs in different vRNP fractions seem to differ in their susceptibility to RNases. Thus, RNA of vRNP with a buoyant density of 1.44 g/cm3 was entirely broken down with RNases A and T1 whereas small amounts of “light” vRNP

f L x

IO 20 30 Fraction number

FIG. 5. CsaSOI gradient diagram of vRNP plants infected for 48 hr with TMV Nil18 at Cesium sulfate gradients were run as described Materials and Methods for nonfixed material leaves labeled for 18 hr with [‘Hjuridine.

from 32°C. under from

-

FIG. 6. Electrophoretic analysis of RNA from vRNP of TMV Nil18 Tobacco leaves infected with Nil18 for 48 hr at nonpermissive temperature (33’) were labeled with rHjuridine, and vRNP was isolated and fractionated in G&O, as described under Materials and Methods. Fractions with densities of 1.33-1.45 g/ cm3 containing vRNP of Nil18 (see Fig. 5) were dialyzed against Buffer C and treated with phenol. RNA was electrophoresed in 2% agarose gels. Arrows denote RNA markers (from left to right): TMV genomic RNA and 26 S and 18 S rRNA.

(1.36-1.3’7 g/cm3) were still observed upon RNase treatment. Consequently, RNA in vRNP is partly or completely accessible to RNase.

Polypeptide

Fraction numbor

40

Analysis of TMVwulgare

vRNP

To study the polypeptide composition of vRNP, TMV-infected tobacco leaves were labeled with 14C-amino acids, homogenized, and fractionated in Cs2S04. Samples with densities of 1.33-1.45 g/cm3 were subjected to SDS-slab polyacrylamide gel electrophoresis (see Materials and Methods). An autoradiogram presented in Fig. 8 shows that vRNPs comprise a set of six polypeptides having molecular weights of 17,500 (TMV coat protein), 31,000, 37,000, 39,000, 55,000, and 76,000. Staining of gels with Coomassie reveals, besides these polypeptides, small amounts of another three peptides with molecular weights of 25,000, 68,OOO,*and70,000 (data

246

DOROKHOV

IQ

ET

AL.

lb

20 Fraction

io

Y

number

FIG. 7. Sensitivity of vRNP RNA to RNases. Tobacco leaves were infected with TMV vulgare for 48 hr, and labeled with rH]uridine for 2 hr at 32’. Isolated vRNP was applied on top of preformed CsCl gradients (A) before and (B) after treatment with RNase A (50 pg/ml) and RNase Tr (10 units/ml).

not shown). Until recently TMV virions were believed to contain only the coat protein. However, Asselin and Zaitlin (1978) have detected, in addition to the coat protein, minor amounts of a polypeptide of host origin with a molecular weight of 26,500. Our experiments with TMV prep-

A

0

-952”id w-69 --46 -30 -143 FIG. 8. Slab polyacrylamide gel electrophoresis of polypeptides of vRNP of TMV vubare. Fractions with densities of 1.33-1.45 g/cm3 (in CszSO1) were electrophoresed in polyacrylamide gradient gels as outlined under Materials and Methods and autoradiographed. (A) Polypeptide pattern of vRNP of TMV ~&are (B) ‘“C-Methylated marker proteins (Amersham, England): lysozyme (14,300), carbonic anhydrase @WOO), ovalbumin (46,000), bovine serum albumin (SS,OOO), phosphorylase 6 (92,500).

arations purified according to Asselin and Zaitlin (1978) also show the presence of a protein with a molecular weight of 25,000 in TMV vulgare. Lh$rences between TMV Immunoprecipitation

and vRNP

in

Immunoprecipitation with IgTMV (immunoglobin against TMV) was used to differentiate between vRNP and mature virus. Table 3 shows that vRNP of TMV vu& gare only partially reacted with IgTMV, unlike mature TMV. Only 6144% of the radioactivity present in vRNP with buoyant densities of 1.36-1.38 g/cm3 was precipitated with IgTMV, while precipitation of the mature virus was virtually complete. As shown in Fig. 5, vRNP was found upon infection of tobacco leaves with the TMV ts mutant Nil18 which is unable to form mature virus particles at a norrpermissive temperature (32-33”) owing to a defect in the coat-protein gene entailing denaturation of the protein (Jockusch, 1966a, b). It could be expected that vRNP produced at nonpermissive temperatures (when the coat protein is denatured) would contain no coat protein and would not interact with IgTMV. As can be seen in Table 3, vRNP of Nil18 formed at a nonpermissive temperature was not precipitated with IgTMV. The insignificant radioactivity found in the precipitate was due to nonspecific reaction

VIRUS-SPECIFIC

RIBONUCLEOPROTEIN TABLE IMMUN~PRE~IPITATION

IN

3 OF VRNP~ Radioactivity

Antibody IgTMV

~gTM”CP

Antigen

Expt

Total

(cpm)

Unreacted material (% of total)

Precipitate

Supernatant

5,680 68,329

2,680 16,586

39 38

1,950 4,500

91 93

vRNP of TMV vulgare*

1 2

89,240 94,640

vRNP of TMV Ni118”

1 2

2,150 4,949

‘%-TMVd

1 2

65,500 28,790

64,842 27,357

110 510

0.1 2

vRNP of TMV vulgare*

1 2

1,940 1,630

1,908 1,527

70 72

4 4

‘%-TMVd

1 2

8,210 8,663

8,193 8,604

63 50

1 1

202 320

’ Each value is a mean of three determinations. * 1.36-1.38 g/cm3 CsCl fraction, rH$ridine label. ’ Formed at nonpermissive temperature, 33’; rH]uridine label. d The TMV preparation isolated from TMV-infected plants totally

as evidenced by control experiments with heterologous antigens (vRNP of Nil18 and TMV) and antibodies (to potato virus X, data not shown). The antigenic properties of TMV and TMVCP are known to differ somewhat (Milton and van Regenmortel, 1979). The observed differences between TMV and vRNP may be due to the fact that some antigenic determinants of TMVCP are not recognized by IgTMV in vRNP. This suggestion is supported by the data on vRNP immunoprecipitation with IgTMVCP (Ig against TMVCP). Table 3 shows that IgTMVCP precipitates up to 96% of vRNP, being thus markedly more efficient than IgTMV. The data of Table 3 give grounds for concluding that vRNP of TMV vulgare contains the coat protein, but its antigenic properties (and consequently, manner of packing) in vRNP are different from those in the mature virus.

Analysis

247

TOBACCO

of Host Cell Informosms

To elucidate the nature of polypeptides found in vRNP, a comparative study was

labeled

in a %O,

atmosphere.

undertaken on some properties and the polypeptide composition of the informosomes of healthy cells. Cell informosomes were isolated from noninfected tobacco leaves (see Materials and Methods) using buffer solutions of low (Al) and high (A) ionic strength. When Buffer Al was used, two peaks of uv absorption were seen corresponding to densities of 1.33 and 1.39 g/cm3 (Fig. 9A); with high-ionic-strength Buffer A, only one band of 1.38 g/cm3 was obtained (Fig. 9B). Spectrophotometric analysis of informosome preparations dialyzed against 5 mM Tris-HCl, pH 8.5, shows A2JAzS0 within 1.5-1.7 and A2,JAB5 of 1.25-1.56. Sedimentation analysis of cell informosomes in a sucrose density gradient (Fig. 9C) revealed a broad peak with an average sedimentation coefficient around 82 S. Treatment of informosomes with low concentrations (5 pg/ml) of RNase A resulted in hyperchromicity (about 26%). Sedimentation distribution of informosomes after exposure to RNase indicates their breakdown to smaller fragments (Fig. 9D). Informosomes are unaffected by DNase. Electrophoresis of cell informosome

248

DOROKHOV

ET

072

AL.

D

I

0,’

I/\--hCFZ--ZO ,

v t Relative

t

9 gredient

: 0.2

0,4

0,6

0,8

I,0

depth

FIG. 9. Cytoplasmic informosome-like RNP from healthy tobacco leaves. Distribution in C&30, gradients of cell informosomes isolated in Buffers Al (A) and A (B) (see Materials and Methods). (C, D), Fractionation of cell informosomes in sucrose density gradients (C) before and (D) after RNase treatment.

protein reveals polypeptides of 70,000, 68,000,55,000,43,000,25,000, and 14,000 and several other minor ones (Fig. 10).

Electron

Microscopy

of vRNP

For these studies it was necessary to differentiate between vRNP and cell informosomes which occur (Fig. 9) in the same fractions of C&SO4 gradients. This difficulty is overcome by fractionating the material not fixed with formaldehyde in CsCl gradients. Cell informosomes proved to be completely destroyed under such conditions whereas vRNP was resistant (at least

partially) to CsCl and could be found in fractions with densities of 1.36-1.40 g/cm3. Figures llA-C are electron micrographs of a vRNP nrenaration obtained from these fractions. A variety of filamentous structures can be seen, which are heterogeneous in both length and thickness. To confirm that the filamentous structures observed are indeed vRNP, immune electron microscopy was carried out with IgTMVCP. As shown above (Fig. 8), TMV vRNP contains the coat protein. Hence, the virus-specific nature of the filaments could be directly proved by an immune electron microscopic test. Figures llD-F depict the

VIRUS-SPECIFIC

RIBONUCLEOPROTEIN

IN

TOBACCO

249

DISCUSSION

FIG. 10. SDS-Slab polyacrylamide gel electrophoresis of polypeptides from cytoplasmic informosomes obtained from a Cs2S0, gradient (see Fig. 9). The gels were stained with Coomassie brilliant blue; informosomes had densities of 1.33 and 1.38 g/cm3.

electron microscopic images of the filamentous structures after interaction with IgTMVCP.The filaments are surrounded by a characteristic “fluff” composed of Ig molecules. The specificity of the reaction is evidenced by the control (Fig. 11G) in which vRNP was reacted with heterologous (bovine) Ig.

It is possible to isolate from TMV-infected tobacco cells a material with a buoyant density of 1.36-1.45 g/cm3 which is labeled in viva with [3H]uridine or 14Camino acids in the presence of AMD. Analysis of the set of RNAs in vRNP testifies to the presence of TMV-specific RNAs-genomic TMV RNA and two types of shorter RNA close in size to the known subgenomic TMV RNAs I1 and IZ. The TMV-specific nature of vRNP RNAs is evidenced, first, by their ability to hybridize with plasmid DNA containing nucleotide sequences complementary to TMV genomic RNA (Table 1) and, second, by their ability to be specifically recognized by the TMV coat protein in vitro and to form RNase-resistant nucleoprotein complexes (Table 2). The presence of TMV-specific mRNA in vRNP suggests the possible involvement of the latter in the synthesis of virus-specific proteins. Extracts from eukaryotic cells are known to contain so-called RNA-binding proteins capable of binding RNA molecules of various origin in homogenates (Baltimore and Huang, 1970). It was therefore necessary to ascertain whether in our hands vRNP had preexisted in TMV-infected cells or was formed upon disruption and homogenization of TMV-infected leaves owing to the interaction of TMV RNA with RNAbinding proteins of host cells. In other words, vRNP formation had to be proved to be an in vivo process rather than an artifact of the experimental procedure. Figures 2A and B show that no vRNP is formed when TMV RNA and healthy or infected tobacco leaves are frozen in liquid nitrogen, mixed, homogenized, and processed according to the protocol designed for vRNP isolation. It should be noted that the extraction solution used was of high ionic strength, high buffering capacity, and high pH, and contained EGTA which efficiently chelates bivalent metal ions, particularly Ca”, providing on the whole conditions for isolating various RNP particles (Jackson and Larkins, 1976). However, it can be supposed that vRNP arises from

250

DOROKHOV

ET

AL.

VIRUS-SPECIFIC

RIBONUCLEOPROTEIN

IN

TOBACCO

251

stable under these experimental conditions (Fig. 2E). Thus, vRNP can be supposed to represent a new type of intracellular TMV-specific nucleoprotein. Note added in proqj When this paper had been completed we found that (1) the polypeptides of M, 3I,OOO, 37,000 and 39,060 revealed in vRNP have the overlapping amino acid sequences unrelated to that of TMV coat protein; (2) upon vRNP translation in a cell-free system (reticulocyte lyzates) the TMV-specific polypeptides of M, 130,000 and 30,000 are produced.

REFERENCES

Frt;. 11. Electron micrographs of vRNP of TMV vulgare isolated in a C&l gradient. (A-C) Tobacco leaves infected with TMV for 48 hr were homogenized, and the nonfixed material was fractionated. Gradient fractions of 1.36-1.40 g/cm3 were dialyzed and processed for electron microscopy as described by Lebeurier et al. (1977). (D-F) Immune electron microscopy of TMV vulgar-e vRNP with IgTMVCP. (G) Control, vRNP with bovine Ig.

virion degradation since the alkaline buffer A used may cause partial uncoating of some particles in the TMV population. We included a suitable control using 14C-TMV and found that TMV virions remained fully

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