Potato spindle tuber “virus”

VIROLOGT

&,

411-428

(197:)

Potato IV. A Replicating,

Spindle

Tuber

Low

”

Molecular

T. 0. DIEKER Plard

T7irologu

Laboratory, Plant U. S. Department

Science Research Division, of ilgriculture, Bebtsville, Accepted

April

Research

Agriculhral

Marulancl

Service,

20705

6, 1971

Earlier results had indicated that the spindle tuber disease of potato is incited by free RNA, and that neither conventional virions liar proteins that could be construed as viral coat proteins are synthesized in infected plants. By a combination of density-gradient centrifugation and polyacrylamide gel electrophoresis, using internal marker RNAs, it is now shown that the infectious RKA occurs in t,he form of several species with molecular weights ranging from 2.5 X 1W to 1.1 X 1Q5 daltons. So evidence for the presence in uninoculated plants of a latent helper virris was found. Thus, potato spindle tuber “virus” RP\‘A, which is too small to contain the genetic information necessary for self-replication, must rely for its replicaLion mainly on biosynthetic systems already operative in the uninoculated plant. Several possible mechanisms are discussed. The term “viroid” is proposed to designate potato spindle Tuber “virus” RKA and other RNAs with similar properties. INTRODUCTIOK

The spindle tuber disease of potat’o is characterized by symptoms and natural means of transmission that are basically similar to those of many virus diseasesof plants (Diener and Raymer, 1971). Phosphate buffer extracts from spindle tuber-diseased tissues, however, are radically different from similar extracts prepared from tissues infe&ed wit(h known plant viruses in that: (a) t&hepresence of virions cannot readily be demo&rat’ed (Diener and Raymer, 1969) ; and (b) such extracts contain very slowly sedimenting, nuclease-sensitive, infect,ious matserial(Diener a,ndRaymer, 1969). Several lines of evidence indicate that neither conventional virions (Diener, 1971), nor proteins that could be conskued as viral coat proteins (Zaitlin and Hariharasubramanian, 1970), are synt,hesized in pot#ato spindle tuber-diseased tissues, The infectious moiety is sensitive to treatment with ribonuclea’se, but’ not. \ith deoxyribonuclease (Diener a,nd Raymer, 1967, 1969). Because of its slow rate of sedimenta411

tion, its sensitivity to RNase,’ its insensitivity to treatment, with phenol, chloroform, n-butanol, or et&no& and because of its chromatographic properties, the infectious moiety is believed t’o be free RNA (Diener and Raymer, 1967, 1969). Attempts have been made t’o separate PSTV-RNA from host R?JA (Diener and Raymer, 1969). Although considerabie enrichment of the infectious RNA was achieved (as judged by comparing the infectivit tion end point with the quantit,y of present), analysis by density-gradient eentrifugation or by chromatographic procedures revealed that even in the most highly purified preparations, essentially ail the nucleic acid detectable by UV absorption was host RX-4 and not, PSTV-RSA (Diener and Raymer, 1969). PreparAons w&h in1 Abbreviations used: DNase, deoxyribona-~ clease; PSTV, potato spindle tuber “virus”; RNase, ribonuclease; rRKA, ribosomal RKA; S-TRW, satellite-like component of TRW ; TlMY, tobacco mosaic virus; t.Rh’A, transfer R.K;P; TRSV, tobacco ringspot virus.

412

DIENER

fectivity dilution end points as high as 1O-6 or lo-’ did not contain UV-absorbing components that could be correlated with infectivity distribution (Diener and Raymer, 1969). Evidently, PSTV-RNA has an unusually high specific infectivity and occurs in the infected tissue in exceedingly small amounts. Thus, infectivity remains, so far, the only parameter useful for investigating the properties of PSTV-RNA. The slow sedimentation rate of the infectious RNA indicates that the molecule is either very small or has a structure that lowers its rate of sedimentation. A combination of density-gradient centrifugation and gel electrophoresis is useful to distinguish differences in structure from differences in weights of RNAs (Loening, 1967). With either method, infectivity is the only necessary parameter, provided that, appropriate marker nucleic acids are analyzed concurrently. The present report describes the results of such a combined analysis of PSTV-RNA. MATERIALS

AND

METHODS

Reagents. To prepare phenol solutions nearly saturated with water at 4”, 1 volume of water was added to 5 volumes of 90 % phenol (liquid phenol, Fisher2 certified reagent). DNase (electrophoretically purified, free of RNase) was obtained from Worthington Biochemical Corporation. RNase-free sucrose (Schwarz BioResearch, Inc.) was used to prepare sucrose density gradients. Hydroxyapatite (Bio-Gel HT) was obtained from Bio-Rad Laboratories. Pathogens. PSTV was grown in tomato plants (Lycopersicon esculentum, cv. Rutgers), as previously described (Raymer and Diener, 1969). Leaves were harvested 3-4 weeks after inoculation, and were stored frozen until used. RXA from the satellite-like component of tobacco ringspot virus (Schneider, 1969) was a gift of Dr. I. R. Schneider. Bioassays. PSTV was assayed on Rutgers tomato plants (Raymer and O’Brien, 1962). Infectivity of samples was evaluated by use z Ment,ion of specific equipment, trade products, a commercial company does not constitute its endorsement by the United States Government over similar products or companies not named. or

of a modification of a previously described infectivity index (Raymer and Diener, 1969), which is based on the dilution of inoculum, the time required for symptom expression on tomat,o plants, and the number of plants infected at each dilution. The index was computed by adding together the number of plants infected for each dilution over the entire recording period; by multiplying each of these figures with the appropriate dilution factor (not the logarithm of the dilution factor, as previously used) ; and by adding together these products for all the dilutions tested. Infectivity profiles of fractionated density-gradient tubes and of sliced polyacrylamide gels were plotted by averaging the infectivity index determined for the fract’ion or gel slice in question with the average infectivity index of the two neighboring fractions or gel slices. This transformation served to smoothen fluctuating values inherent in a bioassay based on systemic symptom expression. Nucleic acid extraction and purification. One hundred grams of PSTV-infected, frozen leaves were triturated in a blender with 200 ml of 0.1 M Tris-HCI buffer, pH 7.5, containing 10 g of Wyoming bentonite. The resulting slurry was pressed through four layers of cheesecloth. The pulp was scraped off the cheesecloth and was again triturated with 200 ml of buffer and 10 g of bentonite. The liquid that passed through the cheesecloth was pooled, and was centrifuged for 10 min at 3000 rpm (Servall RC-2 refrigerated centrifuge, GSA rotor). The resulting supernatant solui;ion was discarded. The pellets were combined with the pulp scraped off the cheesecloth; and the mixture was triturat’ed with 200 ml of 1 M &HP04, 100 ml of chloroform, and 100 ml of n-butanol. A l-gallon Waring Blendor was used with a l-quart stainless steel container. The mixture was blended for 1 min at low speed and then for 1 min at medium speed. The slurry was pressed through cheesecloth, and the pulp in the cheesecloth was discarded. The liquid that passed through the cheesecloth was centrifuged for 10 min at 5000 rpm (Servall RC-2 refrigerated centrifuge, GSA rotor). The aqueous phase, as well as the material at the interface was retained. The chloro-

form-butanol phase was discarded. Watersaturakd phenol, 200 ml, was added to the retained malerial; t’he mixture wa,s mechanically stirred for 20 min at, 4’, and was then cenkifuged at 4’ for 10 min at 5000 rpm (Servall SS-3 centrifuge). The aqueous (lower) phase, as well a,s mat,erial collect’ed at. the interface were retained. The phenol phase was discarded. Tke retained materkl was once more treated with phenol, stirred, and centrifuged as above. This time, only the aqrrieotis (lower) phase was ret#ained. Two volumes of cold 95 % ethanol were then added to t’he retained solut!ion, and the mixture was stored overnight at, -20”. Next morning the liquid in the flask was immediately decanted into centrifuge bottles, leaving most of the precipitate behind; and it was then centrifuged for 10 min at 5000 rpm (Servall RC-2 centrifuge, GSA rotor). The pellets were suspended in 20 ml of 0.5 91 KZHPO~ ; and the mixture was combined kth the precipit’ate (left behind in the flask, and suspended in 20 ml of 0.5 M &HPOJ. Twj volumes of cold 96 % ethanol were t,hen added to t’he preparation. The mixture was stored for 1 hr at! O”, and was t’hen centrifuged for 10 min at G500 rpm (Servall SS-3 centrifuge). The nucleic acid precipitate [which accumulated as a skin at the interface between t,he phosphat)e-rich (lower) phase and the ethanolic phase] was freed as completely as possible of liquid by careful decant,ation, and was then suspended in 7 ml of I M NaCl, 0.01 I!1 Tris.HCl buffer, pH 7.8. Preparations at this stage of purifkat(ion had UV spectra typical of nucleic acid wit’11 maximum (260 nm) to minimum (230 nm) rat#ios of 2.2-2.4, but light scattering was far in excess of t(hat, due to the nucleic acid present. To remove the components responsible for the light’ scat#tering (presumably polysaccharides), five such preparations (from a tot(al of 500 g of leaves) were combined and furt,her purified by a modification of the Kirby procedure (Ralph and Bellamy, 1964). One volume of 2.5 M phosphate buffer, pH 8, and 1 volume of 2met’hoxyet’hanol were added to the nucleic acid preparation; the emulsion was vigorously shaken for 2 min for 5 min at 4”, and was then cerkifuged

at 5000 IJ. The ciear supernat’ant layer ~a:: carefully withdrawn, avoiding interface m:~terial; it was mixed with an equal volume oi 0.2 M sodium aeet’ate; and the nucleic acid was precipitated by addition of 0.5 voiumr:s of 1% ce~~yltrime;hy:ammonium bromick (CTA). The suspension was stored for 5 min at O”, and was then centrifuged for Z min at 5000 i/. The purified CTA-nucleic acid was washed three times with cold 70 ‘;i. etjhanol containing 0.1 M sodium acetate Ii] convert, CTA-nucleic acid back to K:i nucleic acid and t’o remove CTA-aeetatc. The nucleic acid was recovered by cent’rifugntion, and the pellet was washed witti 95 ‘Y ethanol. The nucleic acid watt finally dissolved in 5-7 ml of 0.02 M phosphate buffer, pH 7, The resulting nucleic acid preparation-: wre devoid of light, scattering. Each step of the purification procedure Y-:L;, evalua,ted by bioassay. Essentially 11) infectious material was lost in any of the iraetiow that were discarded, and the final prepaw tions consistently had infect)ivity dilutions end poink of 1F or IO-‘. Analysis bs density-gradient centrifugation and po!yacrylamide gel electrophoresis showed that, preparat)ions at this stage contained DKA, tRNA, and 5 S KYA, as well as smu!I amounk of 17 S ribosoma! RKA. Further purifieakion of PSTV-RKA v,w. achieved by incubating nucleic acid preparations for 1 hr at 25” with 10 pg/ml of DKase (in buffer conkning lo-? M MgC&), followed by gel filtration in columns of Sephadex G-100 (Watson and Ralph, 1967). YLIcleic acid preparations treated v&h DSase and dialyzed against, 0.02 II glycine-NaQ9 buffer containing 3 X 1OW IW MgC12 I p 9.0 (GM buffer) were added (in 2..M.cml portions) to Sephadex G-100 co!umns (1 cm diameter X 200 cm). Nucleic acid was eluted wit,11 G&3 buffer, and K-ml fractions were collected. Bioassays showed that most infectivity -vi-as contained in the excluded RKL4 fract)ion. DNA fragment’s and tRNA were completely separat,ed from PSTV-RNA, but a port’ion of 5 S RNA eluted simultaneously wit’h PSTV-Rr\;A. The fractions cont,aining PSTV-RD;A lvere cornbined, and were reconcentrat!ed by &KI

414

D IENER

matography on hydroxyapatite columns (1 cm diameter X 1 cm) (Bernardi, 1969). Nucleic acids were eluted with 0.15 M KzHPOJ, in GM buffer, and were dialyzed against GM buffer. The preparations (3-5 ml) usually contained 0.1-0.5 mg/ml of RNA (mostly 5 S RNA), and had infectivity dilution end points of low5 to 10-7. For gel electrophoresis, the preparations were further concentrated by ethanol precipitation and resuspension in electrophoresis buffer (seebelow). Density-gradient centrifugation. To determine the sedimentation properties of PSTVRNA, preparations were analyzed in linearlog sucrose density gradients (Brakke and Van Pelt, 1970). Gradients were prepared by successively layering 5 ml each of sucrose solutions (in 0.02 M phosphate buffer, pH 7.0) of decreasing concentrations into SW 25.1 rotor tubes. Sucrose concentrations from the bottom of the tubes to the top were: 0.78 M, 0.60 M, 0.47 M, 0.37 M, 0.29 M, and 0.228 M. Before use, the gradients were stored at 4” for 24-48 hr. One milliliter of an appropriately diluted RNA preparation (in 0.02 M phosphate buffer, pH 7.0) was then floated on each of the gradients, and the tubes were centrifuged at ea. 4” for 24-26 hr at 24,000 rpm (Spinco Model L Centrifuge, SW 25.1 rotor). The gradients were then fractionated in an ISCO Density Gradient Fractionator and UV Analyzer (Instrumentation Specialties Co., Lincoln, Nebraska) . Polyacrylamide gel electrophoresis. Polyacrylamide gels, cross-linked with methylene bisacrylamide, were prepared by the method of Loening (1967) by using materials and procedures provided by Bio-Rad Laboratories (Richmond, California). For gels in which the final acrylamide concentration was 3 % (w/v), the bisacrylamide concentration used was 5 % of that of the acrylamide. For gel concentrations of 5, 7.5, or 10 % (w/v), the bisacrylamide concentration was 2.5 % of that of the acrylamide. Gels were prepared in Plexiglas tubes (0.4 cm internal diameter). After polymerization, the gels were transferred to 1 liter of 0.04 j@-Tris, 0.01 M sodium acetate, 0.001 M sodium-EDTA buffer, adjusted to pH

7.2 with glacial acetic acid. The gels were then stored at room temperature for 2-4 days. Eleetrophoresis was performed in Plexiglas tubes (0.4 cm internal diameter). Gels were supported by dialysis membranes stretched across the lower end. They were prerun at 4” for 1 hr at 5 mA per tube in a Shandon disc electrophoresis apparatus with a constant current power supply. The electrophoresis buffer was composed of 0.04 M Tris, 0.02 M sodium acetate, 0.001 M sodium EDTA, and 0.2 % (w/v) sodium dodecyl sulfate, adjusted to pH 7.2 with glacial acetic acid. Samplesof nucleic acid in electrophoresis buffer containing 5 % sucrose (RNasefree) and bromophenol blue (added as a visible marker) were added to the top of the gels. Usually, lo-20 ~1 of nucleic acid solution were applied to each gel. Electrophoresis was carried out at 4” at 5 mA per tube (constant current) until the dye had reached the desired position in the gels. After the run, the gels were placed into rectangular quartz cuvettes and were scanned at 260 nm in a Gilford Model 2400 Recording Spectrophotometer equipped with a linear transport system. For bioassay, the gels were sliced into disks (2 mm thick) with an assembly of spaced razor blades. Gels of 10 % and 7.5 % polyacrylamide concentration were sliced immediately after UV scanning; whereas gels of lower polyacrylamide concentration were frozen and partially thawed prior to slicing. Each slice was suspendedin 0.5 ml of 0.02 M phosphate buffer, pH 7.0. The gel was thoroughly dispersed with a Q-tip, and the mixture was applied to plants after appropriate dilution in 0.02 M phosphate buffer, pH 7.0. Isolation of hypothetical helper virus. One hundred grams of fresh (not frozen) leaves from uninoculated Rutgers tomato plants were ground in a blender with 200 ml of 0.05 M KxHP04 (method l), or 0.02 34 KzHPO( containing 0.1 M ascorbic acid, adjusted to pH 8 (method 2), or with 100 ml of 0.05 M I&HPO, and 100 ml of chloroform (method 3). All operations were performed at 4”. The homogenates were expressed through four layers of cheesecloth, and the expressed suspensionwas cent,rifuged for 15

x

REPLICATIKG

LOW

MOLECULAR

min at 5000 rpm (Servall SS-3 Centrifuge). The supernat’ant solutions (the aqueous phase in method 3) were then centrifuged for 2 hr at 40,000 rpm (No. 40 rotor, Spinco Model L centrifuge). The result’ing pellets were suspended in 0.02 M phosphat#e buffer, pB 7, aad the suspensions were centrifuged for I5 min at 5000 rpm. The resulting supernatant, soMions lvere used.

WEIGHT

RNA

413

in previous experiments (Diener and mer, 1969)) PSTV-RNA sedimented slowly. Peak infectivity was associated the fraction that corresponds to an

Es)-very with s~C,,~.

RESULTS

Sedinzentation Pv-operties of PSTV-RNA To accurately determine sedimentation coefficients of RKAs by density-gradient centrifugation, two conditions must be fulfilled: (1) RNAs with accurately known sedimentation coefficients (determined by analytical ultracentrifugation in t,he same solvent as that used for densit#y-gradient centrifugation) must be used as internal markers. At least one of these marker RKAs should have a sedimentation coeEcient reasonably close to that of the unknown RNA. (2) The rates of sedimentation of RiYAs in the gradient used must be proportional to their sedimentation coefficients. The RNA of the satellite-like component of tobacco ringspot virus (S-TRSV) (Schneider, 1969) appeared most useful as a marker RNA. In addit’ion, the two RKAs of TRSV were used as markers because szo,w values of these RNAs, determined in 0.02 M phosphate buffer, pH 7, were available (Diener and Schneider, 1966). Figure 1 b shows that the rates of sedimentation of S-TRSV-RNA and of TRSV-RKA 4: and II in the linear-log gradients used are proport’ional to their ~~0,~values. In addition, the sedimenf’ation rates of tRNA, 5 S RNA, and Mbosomal Rn’As prepared from tomato leaves are compatible with the sedimentation coefficients dehermined for tRNA, 5 S R,NA, and ribosomal RNAs, respectively, isolated from different sources (Table 1, I?ig. lb). Thus: these RSAs could be used as secondary marker RN,4s. Their positions are indicated in Fig. la by arrows. Centrifugation of a mixture composed of PSTV-RNA, 5 S RNA, S-TRSV-RIYA, and t’he two TRSV-RKAs gave the result’s shown in Fig. la. Bioassays of consecutive 0.5ml fractions from the gradient revealed that, as

b

20 I ,.i

0

I

I 10

iAL IO

JO

ml

FIG. 1. (a) UV absorption profile (-----I and infectivity distribution (O---O) of centrifuged sucrose density gradient containing PSTV-R_UA (P and f’), S-TRSV-RKA (S), and the two TRSV-RNAs (TR I and II). Position in gradient of other marker RNAs is indicated by asrows: tRNA (T), rRNAs (R I and II), and 5 S RKA (6)Linear-log gradient, centrifugation for 26 Br at 24,000 rpm, SW 25.1 rotor, Spinco Model L centrifuge. Centrifugation is from left to right. Azji = absorbance at 254 nm; I@“. = Infect,ivity index. (b) Relation bet,ween rate of sedimentation in of marker RN-As. gradient and SZ, %,.values

416

DIENER TABLE PROPERTIES

1

OF MARKER

RNAs Molecular

Sedimentation RNA

species

C?O,W

tRNA” 5 S RNAb S-TRSV-RNA rRNA Ic

4.2 4.5 7.3 17

rRNA

23

IIc

TRSV-RNA TRSV-RNA TMV-RNA

I II

24 32 31

Solvent 0.1 M NaCl 0.15 M NaCl 0.02 M phosphate 0.025 M TRIS 0.025 M NaCl 0.025 M TRIS 0.025 M NaCl 0.02 M phosphate 0.02 M phosphate 0.02 M phosphate

Ref.

Calculated from SZ~,,” (daltons)”

1 1 2 3

2.59

x 3 x 8.7 x 5.6 X

3

1.08

4 4 5

1.2 2.2 2.1

weight

From literature (daltons)

104 104 104 lo5

2.6 3.6

X lo4 X 104

1 1

5.6

X 105

1

x

106

1.1

x

10”

1

x x x

106 106 106

2.1

x

106

5

a By the formula of Gierer (1958). b Sedimentation data and molecular weights refer to Escherichia coli RNA. c Sedimentation data refer to tobacco pith cell RNA; molecular weights to E. coli RNA. References: 1. Bishon et al. (1967): 2, Schneider. unpublished; 3, Ralph and Bellamy (1964); and Schneider (1966) ; 5, Gierer (1958):

value of 5.5. A minor peak of infectivity was found at s = 7.0. Since all markers used are single-stranded RNAs, we may tentatively calculate their molecular weights from their ~~0,~ values (determined in 0.02 M phosphate buffer, pH 7) by Gierer’s (1958) formula. Table 1 shows that. these calculated values are in reasonable agreement with those determined by physical parameters. The molecular weights of S-TRSV-RNA, and of the two TRSVRNAs, have not been independently determined; thus, the values calculated may be in error. According to Gierer’s formula, the major fraction of PSTV-RNA would have a molecular weight of 4.7 X lo4 daltons; whereas the minor fraction would have a molecular weight of ea. 7.5 X lo4 daltons. These values, however, are even more questionable than are those of the TRSV-RNAs, inasmuch as it, is not known whether PSTVRNA is single-, double-, or multistranded, or whether its secondary structure is comparable to that of the marker RNAs. Electrophoretic

Mobility

of PSTV-RNA

The relative electrophoretic mobilities of RNAs in polyacrylamide gels are generally inversely proportional to the logarithms of their molecular weights (Bishop et al., 1967). The effects of different’ secondary structures

Ref.

4, Diener

on electrophoretic mobility of RNAs are the opposite of those on their rate of sedimentation (Loening, 1967). Thus, unfolding of an RNA molecule should lower its electrophoretic mobility so that it will move with molecules of higher molecular weight in polyacrylamide gels, whereas it will move with molecules of lower molecular weight during density-gradient centrifugation. A combination of density-gradient centrifugation and gel electrophoresis can therefore be used to distinguish differences in structure from differences in molecular weight of RNAs (Loening, 1967). We now apply this rationale first to the marker RNAs used and then to PSTV-RNA. Electrophoretic mobility of PSTV-RNA in 3 % polyacrylamide gels. Figure 2a shows the results of an electrophoretic analysis of a mixture composed of PSTV-RNA, 5 S RNA (present in the PSTV-RNA preparation), S-TRSV-RNA, and the two TRSV-RNAs in a 3 % polyacrylamide gel. The relative electrophoretic mobilities of other marker RNAs were established in additional gels, and are indicated in Fig. 2a by arrows. The molecular weights of TMV-RNA and of tRNA are well established, and are thus used in Fig. 2b to construct the line that relates relative electrophoretic mobility with molecular weight. For all other marker

A REPLICATING

LOW

MOLECULAR

WEIGHT

RNA

-+ a

Ri 50

0/

]

c

2

i 70

20

30 FRACTION

Fro. scrylamide aration the two arrows: Infectivity marker

40

NO.

2. Electrophoret,ic mobilities of marker RlYAs and of PSTV-RSA (P and P’) in a 3$1 polygel. (a) UV a,bsorption (---) and infectivity (O--O) profiles. A highly purified RX-4 prepfrom PST];-infected tissue, containing mainly 5 8 KKA (.5), to which S-TRSV-RKA (8) and TRSV-RNAs (TR I and 11) were added, was used. Position of other markers is indicated by tKXA (T): rRNAs (JZ I and II), and TMV-RNA (TM). d zig = absorbance at. 260 am; Ii/j. = index. (b) Relation between electrophoretic mobihty and log molecular weight (MW) of RNAs.

RKAs, t,he tentaGve molecular weights calculated from their s?~,%~values were used. Figure ‘Lb shows that t(he electrophoretic mobilities of all marker RNAs are in reasona,ble agreement, with t)he calculated molecular weights. Consequently, we conclude that the calculat,ed molecular weights of t,he marker RNAs cannot be grossly in error. Rioassays of individual gel slices revealed

the infect,ivity distribut8ion illustrat,ed in Fig. 2a. Two major and several minor infectivity peaks are apparent. Highest inieetivity was in fraction Ko. 16, which indicates a relative electrophoretie mobility of PSTVRNA corresponding to a molecular weight’ of 5 X lo4 daltons. The second major in&z-~ t,ivity peak (fraction No. 20) corresponds to a molecular weight of ea. 9.4 X 10” dniions.

418

DIENER

Minor peaks of infectivity are apparent at positions corresponding to molecular weights of 2.5 X 104, 6 X 105, and 1.2 X lo6 daltons. The presence of more than one infect,ious species of PSTV-RNA is indicated both by sedimentation analysis and by gel electrophoresis. The molecular weight of the major infectious component, as calculated from its sedimentation coefficient (4.7 X lo4 daltons), is in reasonable agreement with that deduced from its relative electrophoretic mobility (5 X lo4 daltons). No close agreement exists, however, between the calculated molecular weight of the minor densitygradient component (7.5 X lo4 daltons) and that of the second major electrophoretic component (9.4 X lo4 daltons). To achieve better separation of the infec-

tious RNAs, preparations of PSTV-RNA were subjected to electrophoresis in gels of higher polyacrylamide concentration; i.e., smaller pore size. Electrophoretic mobility of PST V-RNA in 5 and 7.5% polyacrylamide gels. Electrophoresis of PSTV-RNA in 5 % polyacrylamide gels (not illustrated), using tRNA, 5 S RNA, S-TRSV-RNA, and rRNA I as internal markers, resulted in excellent inverse proportionality between the electrophoretic mobilities of the marker RNAs and the logarithms of their molecular weights. Bioassays again indicated the presence of more than one infectious species. Peak infectivity was found in the fraction that corresponds to a molecular weight of 1.06 X 105 daltons, and secondary infectivity peaks INF.

AldO

MW XW’

I

I

10

I

I

I

I

I

I

I

I 20

I

I 30

I

I 40

FRACTION

NO.

FIG. 3. Electrophoretic mobilities of marker RNAs and of PSTV-RNA (P and P’) in a 7.5% polyacrylamide gel. (a) UV aljsorption (--) and infectivity (O--O) profiles. A partially purfied RNA preparation from PSTV-infected tissue, containing DNA fragments (D), tRNA (T) and 5 S RNA (J), to which S-TRSV-RNA (8) was added, was used (preparation was treated with DNase, but was not filtered through Sephadex). A260 = absorbance at 260 nm; 1f. = Infectivity index. (b) Relation between electrophoretic mobility and log molecular weight (MW) of marker RNAs.

A REPLICATING

LOW

MOLECULAR

appeared in fractions corresponding t’o moleccular weights of 4.8 X 10” and 2.7 X lo4 daltons. Figure 3a illustrat~es the results of an electroph.or&c analysis of PSTV-RNA in a 7.5 “i;, polyacrylanude gel. Of the marker RNAs, only t,R,NA, 5 S RNA, and S-TRSVRNA enter a gel of this eoncent’ration. As shown in Fig. 3b, inverse proportionality again existed between the electrophoretic mobilities of the marker RSAs and the logarithms of their molecular weights. Rioassays revealed that peak infectivity was present in the fraction that corresponds t)o a molecular weight of 1.08 X lo5 dalhens (I’ig. 3b). Significant, infect’ivity was also found in fraet’ions corresponding to molecular weights of ea. 5 X lo4 and 7.5 X 104 da,ltons. Evidently, PSTV-RNA is able to enter a 7.5 c/o polyacrylamide gel. It is known that

WEIGHT

PXA

419

“9 S messenger RKA” (with a presumed molecular weight of ca. 1.4 X 105 daltons) just enters 7.5 % polgacrylamide gels (Loening, 1967)) vihereas all higher molecular weight RSAs are excluded from such gels. Thus, PSTli-RKA must have a, molecular weight. of less than 1.4 X lo5 daltons. Three other preparations of PS~FV-RS~?L were analyzed, t,ogether wit811internal markei RNAs, in 7.5 5% gels. Results were simiiar, a,nd molecular weight’s of PSTV-RKA de duced from the fractions with peak infectivity were: 1.06 X lo;, 1.10 X IO”, and 1.15 X lo5 daltons. In each case; minor infec tivity peaks were apparent at positmions carresponding to ca. 5 X 10” and 7.5 X 10J dalt’ons . Electrophoretic mobiiity 0s PST?‘-I&VA ia IO % polyacrylamicle gels. Figure 4 shows the results of an electrophoretic analysis in a 10 % polyacrylamide gel. Two major and aeverni

b

FIG, 4. Eiect,roDhoretic mobilities of marker RNAs and of PSTV-PISS (P) in a 10% polyacrylamide gel. (a) UV absorption (-) and infectivity (O--O) profiles. A partially pilrified RKA preparation from PSTV-infected tissue, containing tRNA (T) and 5 S RNA (5), to which S-TRSV-RFA (8) was added, was used (predaration was treated with DNase, but was not filt,ered through Sephadex). z1-260 = absorbance at 260 nm; Ins. = Infectivity index. (b) Relation between electrophoret,ic mobility and log molecular weight (MW) of marker R?JAs.

420

DIENER

minor infectivity peaks are apparent. The major peaks correspond to molecular weights of 4.5 X lo* and 7.6 X lo4 daltons; the minor peaks to molecular weights of 3 X lo*, 6 X 104, and 9.6 X lo* daltons. Question of Spurious Penetration of PSTVRNA into Polyacrylamide Gels

solution immediately above the gel surface, and that the infectivity measured in the gels is only a small fraction of RNA that somehow managed to enter the gel in spite of its high molecular weight. To investigate this possibility, a small volume of buffer (usually 0.5 ml) was regularly withdrawn after each run from immediately above the gel surface. Bioassays showed that these solutions were always devoid of infectivity, even when they originated from the space above 10 % polyacrylamide gels. Finally, if most of PSTV-RNA were high molecular weight RNA, the small amounts of infectivity in the high molecular weight region of 3 % gels (Fig. 2a) could not be explained. We must conclude, therefore, that all the PSTV-RNA is able to penetrate into the gels, and that it moves through the gels in typica bands.

Is it possible that the molecular weights indicated by polyacrylamide gel electrophoresis are in error? Could PSTV-RNA be a molecule similar in size to self-replicating viral RNAs; and are the infectivity bands in the gels due simply to spurious penetration of this high molecular weight RNA into the gels? This question is particularly pertinent since biological activity was the only measurement available, and since, therefore, the quantities of RNA involved are not known. To answer this question, it is necessary to compare the infectivity distribution across Question of Paw&lisper&y of PSTV-RNA a “band” of PSTV-RNA with that across a band of genuine low molecular weight RNA. Gel electrophoresis indicated the existence The question needs to be asked whether the of more than one infectious speciesof PSTVdrop of infectivity found on either side of a RNA. The question arose whether the varposition in the gel coinciding with peak ious infectivity bands represent RNA moleinfectivity is or is not typical of that found cules of different molecular weights, or with a genuine RNA band. whether at least some of the bands are Comparisons of the infectivity distribuartifacts of electrophoresis. To investigate tions across the band of S-TRSV-RNA this question, the following experiments were (Schneider, unpublished), and across that of performed. PSTV-RNA (analyzed as a mixture in one (i) PSTV-RNA, together with S-TRSV7.5 % polyacrylamide gel) indicated t,hat the RNA as a marker, was subjected to deninfectivity dilution end points of either RNA sity-gradient centrifugation. After fractionadropped to the same levels (from Ho0 to tion, the fractions from 0 to 7.0 ml (fraction >$o and from ?$‘o to undiluted) at approxiI) and those from 7.0 to 15.0 ml (fraction mately the same distances on either side of II) were pooled (seeFig. l)- Each sample was the infectivity peaks. dialyzed to remove sucrose, and was reconIf we assume PSTV-RNA to be a high centrated by hydroxyapatite chromatogmolecular weight RNA, and its penetration raphy, followed by ethanol precipitation of into the gels to be due to nonspecific causes the RNA and resuspensionin electrophoresis such as wall effects, we would expect highest buffer. The two sampleswere then analyzed levels of infectivity in fractions closest to the by electrophoresis in 10 % polyacrylamide origin and diminishing amounts in fractions gels. further and further removed from the origin. Bioassays of the gel slices revealed two This is clearly not the case. On the contrary, prominent infectivity peaks with either prepfractions close to the origin were regularly aration, one corresponding to a molecular devoid of infectivity or contained only weight of ca. 5 X lo4 daltons, the other to negligible amounts (Figs. 3 and 4). one of ea. 7.5 X lo4 daltons. In fract’ion I, It is conceivable, however, that most of however, most infectivity was in the 5 X lo4 the high molecular weight RNA is completely dalton region; whereas in fraction II, most excluded from the gel and still present in the infectivity was in the 7.5 X lo4 dalton region.

Thus, iow sedinient8ation rat,e was correlated nit)h high electraphoretic mobilit#y (and viceversa’), indicating that in either system, RNA molecules of different molecular weight,s xere separated from one another. (ii) Portions of one preparation of PSTVRNA were added t’o 6 gels of 7.5 %, and to 6 gels of 10 % polyacrylamide. S-TRSV-RNA was added t,o each gel as an internal marker. Aft,er electrophoresis, a, ‘J-cm port’ion of each 7.5% gel, from the S-TRSV-RP\TA peak toward the origin, was removed. A similar portion was removed from each 10% gel, but, in t’his case, from the peak of S-TRSVRNA t,oward the bott#om of t’he gel (away from the origin). The removed port’ions mere ground in tissue grinders with 0.02 dd phosphnt’e buffer, pH 7.0. MostJ of t’he gel was removed by low-speed CentrifugaGon. Portions of the supernatants were subject’ed to density-gradient centrifugation. The centrifuged gradients xere fractionated, and consecutive 0.5-ml fractions were bioassayed. As shox-n in Fig. 3a, infectious RNA

eluted from 10 % gels sediment.ed slower tIh:tn RKA eluted from 7.5 % gels. Thus, a eorreiat,ion between electrophoretic mobi!ity and rate of sedimentation was again evident. The infectivit,y peak of the former HKL4 was located at EL 4.2 8 (corresponding to a molecular weight of ca. 2.3 X IO* da,ltoxw); whereas t)hat, of the latter RXA was located at ca. 5.7 S (corresponding tcJ ea. ,? X 10” daltons) . These molecular lx-eight, values do not coincide with t,hose deduced from the electrophoretic mobilities of the respective RNAs. Thus, although all -the RIV:$ elcted from 7.5 % gels had electrophoretic mobiliiles that’ of S-TRSV-IQ\:4, smaller than most, of t.he RX12 sedimented slo~e-er than S-TRW-RNA. Also, in spite of the narrow selection of RNAs with respect to t’heir eiectrophor&e mobilities, RKA molecules sedimenting with rates corresponding to molecular weights ranging from 2.5 X 10” to 1 X 105 daltons were again evident in either sample. The remaining eluates from the 7.3 ‘~5 INS?

7

I

!

I

1

FIG. 5. Rate of sedimentation (a) and electrophoretic mobility (b) in 7.5% polyacrylamide PSTV-RKA previously eluted from 7.570 gels (O--O) and from 1054 gels (@--S-TRSV-RIYA added as an int,ernal marker; Inf. = Infectivity index.

gels

of

422

D IENER

and 10 % gels were reconcemrated by ethanol precipitation of the RNA and resuspension in electrophoresis buffer. Each sample was analyzed by electrophoresis in 7.5 % gels, followed by bioassay of consecutive slices cut from the gels. Figure 5b shows that most of the RNA eluted from the 10% and 7.5% gels had an electrophoretic mobility in 7.5% gels corresponding to a molecular weight of 1.1 X lo5 daltons. Question of a Helper Virus The low molecular weight of PSTV-RNA suggests that its replication may depend on a latent helper virus that is present in Rutgers tomato plants. The following experiments were performed to demonstrate the presence of such an entity. Are all Rutgers tomato plants susceptible to low molecular weight PSTV-RNA? In our tests, tomato plants inoculated with highly infectious preparations of PSTV-RNA occasionally did not develop symptoms of the disease. Are these plants nonsusceptible to PSTV-RNA because they do not contain the necessary helper virus? To test this possibility, nine plants which had been inoculated with low molecular weight PSTV-RNA (eluted from 10 % polyacrylamide gels) and which did not show symptoms 25 days after inoculation (whereas all other plants inoculated at the same time with the same preparation showed symptoms) were reinoculated. To preclude the possibility that helper virus (or its nucleic acid) was introduced concurrently, the nine plants were reinoculated with PSTV-RNA eluted from that region of 10 % polyacrylamide gels that corresponds to an RNA molecular weight of 4 X IO* to 8.7 X lo4 daltons. Eight of the nine plants developed typical symptoms of PSTV infection 15-21 days after reinoculation. Twenty-four days after reinoculation, transfers to small tomato plants were made from each of the nine plants. All these plants developed typical symptoms of PSTV infection. Reinoculation with low molecular weight PSTV-RNA was thus successful with all nine plants. Evidently, if a helper virus is required for PSTV-RNA replication, this virus must be

TABLE

2

EFFECT OB “VIRUS” FRACTIONS FROM UNINOCULATED TOMATO LEAVES ON THE INFECTIVITY OF Low MOLECULAR WEIGHT PSTV-RNA Infectivity Isolation methoda

1 2 3

PSTV-RNA %irus”

+

indexb PSTV-RNA buffer

156 102 105

a See Materials and Methods. 6 Assayed undiluted and diluted 10-3, and 10-4.

+

175 95 108

l&l,

lW,

present in each individual Rutgers tomato plant; i.e., transmission of this virus through the seed must be obligatory. E$ect of “virus” fractions on infectivity of P&TV-RNA. To determine whether addition of “virus” fractions from tomato leaves to inoculum enhances the infectivity of low molecular weight PSTV-RNA, such fractions were isolated from uninoculated tomato leaves by three methods (see Materials and Methods). Tenfold dilutions of low molecular weight PSTV-RNA (eluted from 10 % polyacrylamide gels, corresponding to RNA molecular weights of 4 X lo4 to 8.7 X lo4 daltons) were mixed with equal volumes of undiluted “virus” fractions or with 0.02 M phosphate buffer, pH 7, and were bioassayed. Table 2 shows that no detectable enhancement of PSTV-RNA infectivity occurred when “virus” fractions prepared by any of the three methods used were added to low molecular weight PSTV-RNA. DISCUSSION

The spindle tuber disease of potato has been known for almost 50 years (Martin, 1922). The disease is of considerable economic importance, and has, therefore, been rather thoroughly studied. None of these studies suggest that potato spindle tuber differs phenomenologically from similar diseases incited by viruses. Thus, symptoms, although distinctive, are not radically different from symptoms of plants infected with certain viruses. The disease is readily spread to healthy plants by contact of

A REPLICATIKG

LOW

MOLECULAFl

WEIGHT

RNA

42::

healthy with diseased foliage or with mathe higher molecular weight species a:*e chinery (Ma~nzer and Merriam, 1961). Eviapproximate multiples of the smallest, indently, the disease is both infectious and fectious emity (2.5 X 10* daltons) ; namely, contagious. A11 experience indicates that the 5 x lo4 7.5 x lo*, and 1 x 10” da~iow 21 A&, inciting agent is able to multiply in otherwise The demonstration of a low molec&,r healthy plant’s; i.e., that its multiplication weight RKA that, replicates in a variety does not depend on the presence of a known of host species apparently uninfected by helper virus. another agent’ entails a num In view of these similarities wit,h known important, implications for virology-, moiecvirus diseases of plants, our finding (Diener ular biology, and genet,ics. and Raymer, 1969) that the disease is inAbove all, one wonders by what me&a,. cited by free RNA is surprising. Even more nisms an RNA of Uris size is replicated in an surprising is the apparent low molecular otherwise uninfected host. The minimal weight of t,he infectious RNA, which seems genetic information necessary to induce in a ineompahible wilth present concepts of selfsusceptible host all the synthetic machinery replicating viral RKA molecules. In light of for virus replication is believed t,o require a the results presented, however, the conclunucleic acid strand with a molecular weight sion that PSTV-RNA’ is of low molecular of about 1 X 10G daitons or more. PSTVweight8 appears inescapable. RKA, not. requiring the genetic informa&n ot,h densit’y-gradient centrifugation and to code for a coat protein, presumably could gel electrophoresis indicate the existence of be self-replicating with less genetic informs-~ more than one infect’ious species. Our ex- tion. However, the molecular weight of t’he periments suggest’ that the paucidisperse smallest infectious entity is sufhcient only to behavior of PSTV-RNA is not an artifact of code for approximately 2.5 amino acids; i.e., the analyCca1 met’hods used, but is due to it’ is not suflicient to code for even one timall the presence of RNA molecules with different prot’ein, and certainly not for a specific RIVAmolecular weights. Different preparat’ions dependent’ RIG4 polymeras of PSTV-RNA contain different amounts Self-replication of PSTV if the genetic information of the several infectious species. The species protein were contained on two or more with higher molecular weight’s may thus represent aggregates of a minimal infectious separate I’JA strands of similar lengt unit. As was shown earlier (Diener, 1971), but different. nueleotide sequence. Such a scheme, although theoretically possible, has crude extracts prepared from PSTV-infected tissue contain infectious agents t’hat sedino precedent and would appear to necessitate complex recognit’ion mechanisms to ensure ment at higher rates than the ones in highly proper sequential readout of each cist’ror; purified RNA preparations examined here. fragment Furthermore, infee+&y diMon. It is possible, therefore, that in situ PSTVcurves of PSTVRNA (Raymer and Diener, RNA occurs in large, loosely bound aggre1969) are typical of single-hit events and do gates that gradually break up during extracnot indicate t,he necessity of two or more tion and purification. On the other hand, part’icles at each infect’ion site. certain manipulat8ions, such as ethanol precipitation of the R?;A, may result, in reagWe are thus forced t’o the conclusion t.hat most of the biosynthetic syst)emsneceqsary gregation of smaller molecules. Disaggregafor PSTV-RiYA replication must be pretion and reaggregat’ion may also occur during gel electrophoresis, and may. explain some of formed in susceptible hosts, and cannot’ be induced by genet#ic information contained the het,erogeneous infectlvlty dist,ribution on the RKA strand. Most plausiblet at and t,he observation that, all of PSTV-RiYA first glance, is t’he assumption that PSI3 penetrates int,o 10 % polyacrylamide gels. R,KA is similar t,o a sat,ellit,e RNA that reThe hypothesis, that the several infectious quires a helper virus for its own replication. species of PSTV- KA represent a minimal infectious unit and its dimers, trimers, and The relationship between t#his presumed helper virus and PSTV-RYA would be someletramers, is also suggested by the fact that

424

D IENEP,

what similar to that of adenovirus and the healthy and PSTV-infected tomato tissue DNA from adenovirus-associated virus (Hogfailed to disclose the presence of virion-like gan et al., 1968) or of latent avian leukosis particles in cells of either healthy or infected virus and the RNA of Rous sarcoma virus tissue (R. H. Lawson, personal communica(Vogt, 1967). Basic differences between these tion). Sap from uninfected tomato leaves is systems and the PSTV-RNA-(hypothetical) only weakly antigenic (Bagnall, 1967). helper virus system are, however, evident. Furthermore, double-stranded RNA could be First, PSTV-RNA is not encapsulated by a demonstrated in extracts prepared from coat protein. Second, PSTV-RNA occurs TMV-infected, but not in extracts from in the infected tissue in such small amounts healthy tobacco leaves (Ralph et al., 1965). that its detection by other than biological None of these observations rule out the means has so far proved impossible. possibility that a latent helper virus might As shown, efforts to demonstrate the be universally present in uninfected tomato presence of a helper virus gave negative re- and tobacco tissue; they do, however, indisults. They revealed, however, that, if such cate that such a virus, if it exists, must be a virus exists, it must be vertically transpresent in very small amounts. If so, it mitted through the seed to every single appears reasonable to suppose that the tomato plant. Although seed transmission of amount of helper virus available at infeccertain plant viruses is a well-known tion sites limits PSTV-RNA replication, phenomenon, high rates of transmission are and that addition of helper virus t,o the inoculum should enhance PSTV-RNA inrare and mainly restricted to nematodeborne viruses. Of about 52 viruses known to fectivity. No enhancement could, however, be seed-transmitted, seed transmission be demonstrated when “virus” fractions, reaches 100% only with five viruses (all prepared by several methods from uninnematode-borne) and with these only under fected tomato leaves, were added to PSTVcertain conditions and in certain hosts (BenRNA prior to inoculation. nett, 1969). With solanaceous plants, such Absence of a universally present latent high rates are unknown. Thus, no virus is virus cannot conclusively be demonstrated; known to be transmitted through the seed of but if all tobacco and tomato plants conpotato (except potato spindle tuber “virus”) ; tain such a virus, we might, with equal and only two viruses are known to be transjustification, question the self-replication of mitted through the seed of tomato [aside such viral RNAs as t(hose of tobacco mosaic from tomato bunchy-top “virus,” which is virus, tobacco ringspot virus, and tobacco identical with PSTV (Diener and Raymer, necrosis virus. In view of these considera1971)]. With these two viruses, both of tions, it appears most unlikely that a conwhich are nematode-borne, percentage transventional helper virus is involved, and mission ranges from 1.8 to 19% (Bennett, mechanisms of PSTV-RNA replication that 1969). obviate the need of a helper virus need to Since PSTV-RNA is able to replicate in a be considered. number of solanaceous plant species other Such schemes fall into two categories. than potato and tomato (Q’Brien and RayOne assumes that PSTV-RNA is synthemer, 1964), hypothetical helper viruses sized by the normal RNA synthesizing would have to be present also in these machinery of the cell; i.e., that it is transpecies. Among these are Samsun and scribed from a DNA template; the other Burley tobacco, varieties that for many assumes that PSTV-RNA is self-replicating; years have been extensively used in plant i.e., that its synthesis is DNA independent. virological studies and have been repeatedly In the former case, we must assume that examined by electron microscopy of thin one or more DNA segments coding for sections. No reports of viruslike particles in PSTV-RNA are present in the genome of sections from uninfected tobacco plants are all plant species in which PSTV-RNA is known to the author. Similarly, electron able to replicate. If so, this genetic informamicroscopy of thin sections prepared from tion must be completely repressed in unin-

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LOW

MOLECULAR

oculated organisms and PSTV-RNA, when introduced into a susceptible host, must act as a trigger to derepress this DNA segment. Such a scheme invites the question of whv a completely repressed DYA segment that contains genetic information detrimental to the organism should be mainta,ined during evolution. Also, one would expect that “spontaneous” derepression of the DNA segment should occasionalIy occur. Largescale commercial product,ion of tomato plants without reported outbreaks of the disease incited by PSTV-RNA bears witness to the fact that this is not t.he case. X!ternatively, a novel DKA might be produced in the infected plant, with PSTVRN-4 serving as a t,emplate, as has recent’ly been demonstrated to occur in the case of several oncogenie RNA viruses (Baltimore, 1970; Temin and Mizut#ani, 1970). In view of the low molecular weight of PSTV-RNJ4, and in view of the absence of virions (which might contain requisite enzymes), this assumption appears untenable. A novel DKA might, however, be produced as a consequence of infection with PSTV-IINA4 if RXA to DNA information transfer systems operate in normal cells (Scoinick et al., 1971). The second alternative (i.e., that PSTVRNA, in spite of its low molecular weight, is a self-replicating RNA4) contradicts apparently conclusive evidence that all RNA synthesis in uninfected cells occurs by transcription from DKA templates (Allfrey and Minsky, 1962). Recent observations, however, may indicate t,hat RiYA-dependent RNA re+cation occurs to a limited ext.ent in most, if not. a,ll, cells. In new of our present knowIedge of cellular and viral RXA synthesis, demonstrarion of double-stranded RXA in cells represents presumpt,ive evidence of RNAdirected RSA synthesis. Small but significant amounts of double-st,randed RNA have recently been found in a variety of apparent,ly uninfected tissues and cells (Monmgnier, 1968a; Duesberg and Colby, 1969; Stern and Friedman, 1970; Stellar and Stellar, 1970). Significantly, synthesis of these double-stranded RNAs is lesssensitive

WEIGIiT

RN.4

,t*-,-;

to treatment with actir,omycin D thaw ii; that of cellular RXA (Montagnier, 2968a..; Stern and Friedman, 1970)_ Although different interpretations are passible, it has been suggested t,hat ‘Lsome self-replicating RNAs may have become part of cell genetic information” (liontagnier, 1968b). If such self-replica&g RNAs occur in most, if not, all, seemingly healthy cells, systems for DNA-independent RP\‘A replication must operate in t!hesecells,. If so, PSTV-RXZ4 may be repii&ed b,y these systems, and no need for a con::entional helper virus exists. In this view? self-replicating R%‘A species occ’ur in most, if not all, organisms. They have long remained undetected because (b’) they constitute only a minute fraction of the total RKA complement of a cell; (2) they replicate more slowly thaa either eellular or viral RKA (and are, therefore, diEi-. cult to detect by radioactive labeling); and (3) their biological effects, if any, were LIEknown. They have come to light (a) because their replicative forms are the onIy doublestranded R,KA species present in host,s non. infected by viruses, and (b) because one of these RNAs (PSTV-RN-4) is pa,t~hogeaic i, cert’ain organisms. Such RXAs have characteristics of bot,h cellular and viral RI%&. They resemble cellular RKAs because they may become part of cellular genetic information, and because they do not code for coat proteins (but may or may not’ code for specific replicases). Unlike viral RXAs, these IWAs need not exceed a eerta,in minimal size. They resemble viral RXL4s because .&eg are self-replicating, and because at leas: some of them are pathogenic in rertaiu organisms.” Known properties of PSTV-R-U-4 illust&e t’hese characteristics. PSTt’-RKA (in contra& t)o all known potato \-irusesj is transmitted through both the seed and pollen of infected plant,s (Hunter et al., 1969; Singh, 1970); i.e., it may become part of cellular genetic information. Its small size precludes the possibility that PSTJT-RK;A 3 At least one other planb pathogen ties similar to those of PSTV-RKA described (Semancik and Weathers,

with

properbaii bee!: 1908j.

426

DIENER

codes for a specific replicase or for a coat protein. The RNA evidently replicates and causes viruslike diseases in potato and tomato. In several other hosts, however, PSTV-RNA replicates and reaches infectivity titers similar to those in potato or tomato, yet doesnot incite discernible symptoms (O’Brien and Raymer, 1964). In these hosts, PSTV-RNA may thus be analogous to the RNAs that have been detected by the presence of double-stranded RNAs (presumably their replicative forms) in a number of uninfected animal tissues where their presence similarly does not cause discernible damage to the host. Irrespective of its mode of replication, we may consider PSTV-RNA as a primitive viral RNA that has not achieved the genetic sophistication to induce in susceptible hosts novel biosynthetic machinery for its own replication, but relies instead mostly on mechanisms already operative in its hosts. Alternatively, PSTV-RNA may have originated from a conventional viral RNA by loss of these functions. Other similar RNAs may be more advanced (or less degenerate) than PSTVRNA, and may code for specific enzymes to ensure their own replication. Thus, doublestranded RNAs from certain uninfected animal cells have been estimated to have molecular weights of from 4.5 X lo5 to 2 X lo6 daltons (Montagnier, 1968a). Evidently, molecules of this size could code for one or more proteins. Unlike conventional viral nucleic acids, however, none of these RNAs code for specific coat proteins that are capable of self-assembly into capsids. These entities, therefore, exist only in the vegetative phase; the dormant phase so characteristic of viruses (i.e., the virion) is absent. I propose the term “viroid” for such entities. Altenburg (1946) introduced this term to designate hypothetical symbionts, akin to viruses, that were supposed to occur universally within the cells of higher organisms and to give rise, by mutation, to viruses. The viroid theory has not been widely accepted, presumably because experimental verification of its principal tenets did not materialize. If, however, the “viroid” is redefined operationally and in

modern terms to encompass nucleic acid species with the properties discussed here, the term serves a useful function. To distinguish pathological conditions incited by viroids from those incited by viruses, the term“ viroid disease” is proposed. It appears unlikely that PSTV-RNA is the only pathogen of its type or that such pathogens should be restricted to higher plants. The ease with which PSTV-RNA is mechanically transmitted may be connected with its propensity to aggregate or with its peculiar structure (Diener, 1970) and may not be typical of other representatives. Contrary to PSTV-RNA, which is readily transmitted horizontally without the protection afforded by a capsid, other pathogens of this type may be transmitted horizontally only with difficulty, or not at all, and may depend mainly, or entirely, on vertical transmission for their maintenance. This may explain why other similar pathogenic agents have not so far been discovered. Such RNAs may represent the “missing link” between viruses and genes (Temin, 1970) and may be agents of extrachromosomal inheritance. Such RNAs may also be involved in cell transformation and oncogenesis, and may represent the postulated “oncogenes” (Huebner and Todaro, 1969). Finally, in view of the extremely small genetic message possible on PSTV-RNA and the seriousness and type of disease produced, it is tempting to speculate that PSTV-RNA does not function as a messenger RNA, but is an abnormal regulatory RNA. This is also suggested by the observation that the RNA occurs in the nucleus, but not in the cytoplasm, of infected cells (Diener, 1971). Its low molecular weight, its association with chromatin (Diener, 1971)) and its slow rate of synthesis are properties which PSTV-RNA has in common with a recently discovered group of low molecular weight nuclear RNAs (Prestayko et al., 1970). ACKNOWLEDGMENTS I wish to thank Dr. gifts of S-TRSV-RNA his excellent technical

I. R. Schneider and Mr. D. assistance.

for generous R. Smith for

A REPLICATING

LOW

MC ILECULAR

REFEREP\‘CES

ALLFREY: 8. G., and MIRSKY, A. E. (1962).

Evidence for the complete DNA-dependence of R14’A synthesis in isolated thymus nuclei. Proc. Nat. Acad. Sci. U.S. 48, 1590-1596. ALTEXWXG, E. (1946). The “viroid” theory in relation to plasmagenes, viruses, cancer, and plastids. Amer. Natur. 80, 559-567. BAGNALL, R. H. (1967). Serology of the potato spindle tuber virus. Phytopathology 57, 533-534. B~LTIMOEE, D. (1970). RNA-dependent DNA poiymerase in virions of RiYA tumour viruses. flatwe (London) 226, 1209-1211. BENXETT, C. W. (1969). Seed transmission of plant viruses. A&an. vi’irus Res. 14, 221-261. BERNXEDI, G. (1969). Chromatography of nucleic acids on hydroxyapatite. III. Chromatography of X.NA and polyribonucleotides. &o&m. Biophys. Acta 174> 449-457. BISSOP, D. W. L., CL~YBEOOK, J. R., and SPIEGELMAN, S. (1967). Electrophoret,ic separation of vira1 nucleic acids on poIyacryIamide gels. J. Mol. Riol. 26, 373-387. BRUXE, M. K., and VAX PELT, N. (1970). Linearlog sucrose gradient’s for estimating sedimentation coefficients of plant viruses and nucleic acids. Anal. Biochem. 38, 56-64. DIENER, T. 0. (1970). IsoIation of exonucleaseresist’ant ribonucleic acid from healthy and potato spindle tuber virus-infected tomato Ieaves. Phytopathology 60, 1014 (Abstract). DIESER, T. 0. (1971). Potato spindle tuber virus: A plant virus with properties of a free nucleic acid. III. Subceilular location of PSTV-RNA and the question of whether virions exist in extracts or in s&L. Virology 43,75-89. DIENER, T. O., and RAYMER, W. B. (1967). Potato spindle tuber virus: A plant virus with properties of a free nucleic acid. Science 158, 378-381. DXESEX? T. O., and RAWER, W. B. (1969). Potat,o spindle tuber virus: A plant virus with properties of a free nucleic acid. II. Characterization and partial purification. Yirology 37, 351-366. DIENER,T.O., ~~~RAYMER, W.B. (1971).Potato spindle tuber virus. 1n “Descriptions of Plant Viruses,” Commonwealth Mycological Institute and Association of Applied Biologists (Ferry Lane, Kew, Surrey, England), in press. DIENER, T. O., and SCHKEIDER, I. R. (1966). The two components of tobacco ringspot virus nucleic acid: Origin and properties. ViroEogy 29, 100-105. DPESBERG, P. H., and COLBY, C. (1969). On the biosynthesis and structure of double-stranded RXA in vaccinia virus-infected cells. Proc. Nat. Acad. Sci. U. S. 64, 396-403. GIERER, A. (1958). Gr6sse und Struktur der

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Ribosenucieinsiiure dea Tabakmosaikvirus. IS. Naturforsch. B 13, 477-484. HOGGAN, ;ii. D., SHITKIN, A. J., &~rtx.ow~ r\‘. R., Koczo~, F., and ROSE, J. A. (lQ68). Helper-dependent, infectious deoxyribonucleic acid from adenovirus-associated virus. J. ‘ii‘roi. 2, 850-851. HUEBNER, R. .I., and Ton.4~0, 6. J. (1969). Oncogenes of RP\‘A tumor viruses as determinants of cancer. Proc. Xal. Acad. Sci C. s’. 64, 1087-1094. HUNTER, D. YE., ~RLING, Ii. M., and BEMZ; W. L. (1969). Seed transmission of pot&o spindle tuber virus. $naer. Potato J. 46, Xi-2500. LOESING, U, E. (1967). The fractionation of highmolecular-weight ribonucleic acid by poly.. electrophoresis. Biochem. J. acrylamide-gel 102, 251-257. M.~WZER, F. E., and MERRIAM, D. (1961). Field transmission of the potato spindle timber virus and virus X by cultivating and hilling equipment. Amer. Potato J. 38, 346-352. MARTIN, W. PI. (1922). “Spindle tuber,” a new potato trouble. Hints to Potato Groujer-s, New Jersey State Potato Ass. 3, 8. MONTBGNER, L. (1968a). Pr&ence d’un ncide ribonuclBique en double chaine dans des eellules animales. C. R. Acacl. Sci. Ser. D 267, 1417-1420. MONT~GNIER, L. (1968b). The replication of viral RNA. Symp. Sot. Gen. Microbial. 118, 125-148. O’BRIEN, M. J., and RHYMER, TV. 73. (19G4). Symptomless hosts of the potato spindle tuber virus. Phytopathology 54, 1045-1047. PRESTSYKO, A. W., TOSATO, M., and BTJS~E, B. (1970). Low molecular weight RNA associated with 28 9 nucleolar RNA. J. Mol. Biol. 47, 505-515. RALPH, R. K., and BEIUJVIY, A. R. (1964). Isobtion and purification of undeg. ribonue!eic acid. Biochim. Biophys. Acta. X7, 9-16. RALPH, R. H., MATTNEWS, R. E. F., P&TKS, A. I., and M~NDEL, 33. 6. (1965). Isolation and properties of double-stranded viral RNA from virus-infected plants. J. Mol. Biol. 11, 202-212. RAYMER, W. B., and O’BRIEX, M. 3. (1962). Tmnsmission of potato spindle tuber virus to tomato. ilmer. Potato J. 39, 401-408. RAY&IER, W. B., and DIENER, T. 0. (1969), Poktto spindle tuber virus: A plant virus with propert,ies of a free nucleic acid. I. Assay, extraction, and concent,ration. ViroEogy 31, 343-350. SCHNEIDER, I. R. (1969). Satellite-like particle of tobacco ringspot virus that resembles tobacco ringspot virus. Science 166, 1627-1629.

ScOi,XICK,

E. M.,

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