Highly infectious nucleic acid from crude and purified preparations of cucumber mosaic virus (Y strain)

22, 131-141

VIROLOGY

Highly

(1964)

Infectious

Preparations

Nucleic

Acid

of Cucumber

T. 0. DIENER,

from

Crude

Mosaic

Virus

I-I. A. SCOTT,

AND

September

Purified

(Y Strain)

J. M. KAPER’

Plant Virology Laboratory, Crops Research Lh’vision, Agricultural States Department of Agriculture, Beltwille, Maryland, and The George Washington University, Washington, Accepted

and

Research Department D. C.

Service,

of

United Botany,

23, 1963

When tobacco leaves infected with the Y strain of cucumber mosaic virus (CM\:) were extracted with pyrophosphate buffer in the presence of phenol, the resulting preparations were nearly as infectious as or more infectious than preparations made with phosphate buffer. The infectious entities responsible for the high infectivity of the phenol-treated preparations were not destroyed in phosphate buffer extracts and were not ribonuclease sensitive before phenol treatment. Citrate-chloroform extracts of CMV-infected leaves were more infectious than phosphate buffer extracts, but phenol treatment of citrate-chloroform extracts led to reduced infectivity. The infectious entities in phosphate buffer extracts sedimented more readily than those in citrate-chloroform extracts. These results indicate that most or all of the infectivity of phenol-treated tissue extracts originates from complete virus particles. Control experiments with healthy leaves to which purified CMV was added before extraction confirmed this conclusion. Nucleic acid prepared from purified CMV retained approximately half of the infectivity of an equal weight of nucleic acid in CMV particles if the virus was suspended in a medium of relatively high ionic strength during treatment with phenol. Nucleic acid preparations had u!traviolet spectra typical of nucleic acid and were ribonuclease sensitive, but analytical ultracentrifugation disclosed two major and two minor components. The faster moving major component moved at about a 10% faster rate than the slower moving major component. Only the faster moving major cornponent appeared to be infectious. The minor components were not infectious. 1NTROI)UCTION

Nucleic acid (NA) prepared from purified plant viruses generally retains only a small fraction of the infectivity of the parent virus (Gierer and Schramm, 1956; FraenkelConrat et al., 1957; Bawden and Kleczkow. ski, 1959; Kaper and Steere, 1959a,b). Bawden and Pirie (1959) showed that, with tobacco mosaic virus (TMV) and its NA, the relative infectivity of the two types of inocula is not fixed, but depends on the plant speciesused for bioassay, on the physiological state of the test plants, and on the concentration at which the two inocula are 1 Supported

by

IISI’HS

grant

No.

AI-O-k::22-O2. 131

compared. Under the most favorable conditions, however, TMV-NA preparations never retain more than 10% of the infectivity per unit phosphorus of the parent virus (Bawden and Pirie, 1959). Exbracts prepared in the presence of phenol i’rom virus-infected tissuesare generally also much less infectious than are aqueous extracts of comparable tissues (Diener and Weaver, 1959; Schlegel, l96Oa,b). In contrast, Schlegel (1960a,b) found that extracts prepared in the presence of phenol from tissues infected by cucumber mosaic virus (CMV), tobacco necrosis virus, or pea enation virus were nearly as infectious as or more infectious than aqueous extracts.

132

IIIENER,

SCOTT,

With CMV, the infectivity of phenol extracts was 0.5-6.6 times that of buffer extracts, but NA extracted with phenol from partially purified CMV had only 0.5 to 0.7 % of the infectivity of the partially purified virus (Schlegel 1960b). Kassanis (1960) treated clarified sap from tobacco leaves infected by a tobacco necrosis virus with phenol and found that the phenolextracted preparations were nearly as infectious as the clarified sap when the two inocula were compared in dilute phosphate buffer. Unlike CMV, purified tobacco necrosis virus yielded XA preparations with relative infect.ivities as high as those of preparations from clarified sap. Another case where extraction of infected tissue with phenol led to highly infectious preparations in comparison with aqueous extracts concerns tobacco rattle virus. Sanger and Brandenburg (1961) discovered that phenolic extracts from leaves of plants systemically infected with the so-called winter-type, or “poorly multiplying” form, of tobacco rattle virus were many times as infectious as aqueous extracts from comparable leaves. Cadman (1962) confirmed these findings and concluded from his experiments that the infectivity of the “poorly multiplying” forms of tobacco rattle virus was associated with XA located in a subcellular component. Babos and Kassanis (1962) described unstable variants of tobacco necrosis virus whose behavior resembles that of “poorly multiplying” isolates of tobacco rattle virus. The authors concluded that the unstable variant,s occur in the plant largely as nucleic acid, which is destroyed by leaf ribonuclease when the leaves are ext,ra.cted wit’h aqueous buffers but is preserved when the leaves are extracted with phenol. In the case of CMV, there could be two explanations for the high infectivity of the tissue extracts obtained with phenol. The additional infectivity could originate from infectious entities distinct from CMV particles, entities whose infectivity would be destroyed in buffer homogenates but would be retained in phenolic extracts. Alternatively, the additional infectivity could be derived from CMV particles. The infectivity of the buffer homogenates might not be a

ANI)

KAPER

full measure of the total virus present, as already proposed by Schlegel (1960b). Conceivably in buffer homogenates, a major portion of the virus might remain attached to cell components or might be present in aggregated form. Attachment or aggregation of the virus could reduce infectivity of the buffer homogenates, and the increased infectivity of phenolic extracts could then be understood since phenol would presumably release infectious n’A from all virus particles. Progress on this problem was clearly hampered by the unavailability of reliable methods for the purification of the virus. Recently a method for the purification of the Y strain of CMV was developed in our laboratory (Scott, 1963), and the present paper describes experiments undertaken in an effort to determine the causes of t,he high infectivity of extracts from CMV-infected tissues prepared in the presence of phenol. MATERIALS

AN11

METHOI)S

The Y strain of CMV was maintained in Kentucky 35 tobacco (Nicotiuna tabacum L.). CMV-infected tobacco leaves (var. Samsun) served as source for the preparation of tissue extracts and for the purification of the virus by the method described by Scott (1963). Infectivity assays were made in the greenhouse on primary leaves of cowpea (Vigna sinensis (Torner) Savi, var. Blackeye) plants kept in the dark for 24 hours before inoculation. All dilutions for bioassay were made with 1% K,HPOa in water, and all infectivity comparisons were made on opposite half-leaves. Liquefied phenol (Fisher* Certified Reagent) was saturated with water at 4” and was used without redistillation. In all cases, one volume of water-saturated phenol was added to the solution to be treated with phenol. The mixture was manually shaken for 2 minutes at 4”. The aqueous phase was separated from the phenol phase by centrifugation at 10,000 g for 5 minutes. The aqueous phase was pipetted off and, after appropriate dilution, was often directly used 2 Mention of specific equipment, trade products, or a commercial company does not constit,ute its rndarscmrnt. hy the TJnitcd States (hwnmcnt over similar products or companies not named.

NUCLEIC

ACID

FROM

CUCUMBER

for infectivity assays. Alternately, the aqueous phase was freed of phenol either by repeated extraction with ether or by precipitating the NA with 2 volumes of ethanol and resuspending it in cold buffer. For density-gradient centrifugation, sucrose gradients were made with an automat.ic device for the preparation of linear gradients. The gradients were centrifuged in an SW 25.1 rotor at 24,000 rpm for Is-16 hours (Spinco model L Centrifuge). The centrifuged density-gradient columns were fractionated with an ISCO density-gradient fractionator and flow densitometer. Ultraviolet (IJV) absorption spectra were determined in a Cary model 14 recording spectrophotometer. Analytical ultracentrifugation was performed in a Spinco model E centrifuge. All operations were performed at O-4”. RESULTS

Experiments

with Tissue Extracts

Phosphate vs. pyrophosphate-phenol extracts. Samples of C&W-infected tobacco leaves were extracted with phosphate buffer and others with pyrophosphate buffer and phenol according to the methods of Schlegel (1960b). The resulting preparations were equally diluted, and their infectivities were compared. As shown in Table 1, the pyrophosphate-phenol-extracted preparations produced 0.4-3.9 times as many lesions as the phosphate-extracted preparations. Ribonuclease sensitikty oJ i?zJectious icarTABLE RELATIVE

hpt.

INFECTIVITIES

no.

1

4

2

1

3 4 5

5 7 10 of lesions (19OOk)).

Dilution in l’>h KnHPO4 I:50 1:lOO 1: 50 1:lOO 1:lOO 1:lOO 1:zoo per

half-leaf

tides in phosphate extracts. Aliquants of phosphate-extracted preparations were incubated with ribonuclease. Other aliquants of phosphate-extracted preparations were treated with phenol, and still others were first incubated with ribonuclease and then treated with phenol. Bioassays were made with all preparations and also with pyrophosphate-phenol-extracted tissue samples (Table 2). It is evident that the infectivities of samples that were first extracted with phosphate buffer and then treated with phenol were as high as those of samples extracted solely with pyropliosphate and phenol. Incubation of phosphate-extracted preparations with ribonuclease did not cause a reduction in the infectivity released by treatment with phenol. Phosphate vs. citrate-chloroJom extracts. Samples of C&IV-infected tissue were extracted with phosphate buffer (as above) and others with citrate-chloroform (Scott, 1963). The phosphate extracts were expressed through cheesecloth. The citratechloroform extracts were centrifuged, the resulting aqueous phases were combined with the pulp, and the mixtures were expressed through cheesecloth. To equalize the medium in which the treatment with phenol was perforrned, one volume of a phosphate extract from healthy tobacco leaves was added to the citrate-chloroform extract from infected tissue and conversely one volume of the aqueous phase and pulp of a citrate-chloroform extract frorn healthy I

Tissue extracted with phosphate C.9

inoculated

LE,YVES

Tissue extracted with pyrophosphate-phenol (B)

93 12 118 74 16 249 150 on 9-12

133

1’IRUS

OF EXTR.WTS MADE FROM CMV-INFECTED TOBACCO OR WITH PYROPHOSPH.\TE AND PHENOLQ

Age of infection (days)

a Average numkjer according to Schlegal

MOSAIC

239 84 158 2(iO IO 111 157 primary

conpea

\YITH

PHOSPHATE

Ratio

B :A

2.6 2 0 :: .!I 3.5 0.0 0.1 1.0 leaves.

Extracts

rnade

134

DIENER,

SCOTT,

AND

KAPER

TABLE

R.EI..LTIVE IVITH

INFECTIVITIES

OF EXTRACTS

AND

1i~uB.4TIoN

WITHoI!T

no.

FROM

-

-

T Expt.

2 CMV-INFECTED Tosacco LEAVES WITH PHOPSH.ZTE WITH P.4NcREhTIc RIBONUCLEASE AND WITH AND WITHOIIT TRE.YTMENT WITH PHENOLS M.WE

Tissue Dilution in 1% K~HPOI

Not incubated Not treatec Nith pheno

phosphate*

Incubated

I

Tissue extracted RNas ec I with pyrophosphate-phenol Treated Nith phen 101

with

IXot treatec Treated Nith phenl 01 1Nith pheno

G

7

8

9

1:50 1:lOO I:50 1:lOO 1:25 1:25 1:50 1:50 1:lOO 1:50” 1:lOO” 1:200 I:200

4

4

5

10

n * c d

with

-

Age of infection (days)

__.-~

extracted

29 12 155 -

123 30 178 51

3G

127 -

162 137 53 169 -

52 230 212 51 105 234

-

93

-

29

207 -

-

-

Average number of lesions per half-leaf on N-10 primary cowpea Extracts made according to Schlegel (196Ob). Incubated with lo-* fig of pancreatic ribonulcease per milliliter Tissue estract expressed through cheesecloth before dilution.

tobacco leaves was added to the phosphate extract from infected tissue. The resulting mixtures were treated with phenol, and the aqueous phases were tested for infectivity. As shown in Table 3, citrate-chloroform extracts were much more infectious than phosphate extracts. Phenol-treated phosphate extracts retained all or most of their infectivity, but treatment of the aqueous phases of citrate-chloroform ektracts with phenol led to consistent reductions of infectivity as compared with that of the preparations not treated with phenol. In spite of this reduction, phenol-treated citrate-chloroform extracts always had higher levels of infectivity than phenol-treated phosphate extracts. Low speed CentriJugation of tissue extracts. Samples of CMV-infected tobacco leaves were extracted with phosphate as before, and the extracts were expressed through cheesecloth. Other samples of leaves were extracted wit,h citrate-chloroform and were centrifuged to separate the aqueous phases from the chloroform phases. The resulting

-

-

1% 62

133

-

-

- I

186

leaves. for

1 hour

:tt, 4”.

preparations were centrifuged for 15 minutes at 5000 g. Aliquants of the supernatants and of the resuspended pellets were tested for infectivity, and the remainders of the supernatants were centrifuged for 15 mitiutes at 10,000 g. Sampling of the supernatants and resuspended pellets was repeated, and the remainders of the supernatants were centrifuged for 15 minutes at 15,000 g. The resulting supernatants and resuspended pellets were also tested for infectivity (Table 4). It is ekdent that most of the infectious entities of the phosphate extract sedimented at 5000 g and virtually all at 10,000 g, whereas the infectious entities of the citratechloroform extract remained in the supernatant even after centrifugation at 15,000 g. Phenol treatment of mixtures oj” healthy leaces and purified CM V. Samples of CMVinfected tobacco leaves were extracted with citrate-chloroform, the emulsions were separated by centrifugation, the resulting aqueous phases were combined with the pulps, and the mixtures were expressed through cheesecloth. Similar extractions

NUCLEIC

ACID

FROM

CUCUMBER TABLE

REIATIVE

INFECTIVITIES AND WITH

OF EXTRACTS MADE CITRATE-CHLOROFORM

MOSAIC

135

VIRUS

3

FROM CMV-INFECTED TOBAWO LEAVES WITH FOLLOWED BY TREATMENT WITH PHENOLS

PHOWHATE

Tissue extracted with Expt. no.

10

Age of infection (days)

Phosphate” Not treated with phenol

4

72

~_ Treated with phenol

Citrate-chloroformc __ Not treated with Treated with phenol phenol

73

11

7

29

155

273 353

67 127 27

313 150 93

15

88

4

74

a Average number of lesions per half-leaf on 4-9 primary cowpea leaves. All preparations were diluted 1:50 with 1% KzHPOI prior to inoculation. ) Ten grams of CMV-infected leaves were ground in a mortar with 20 ml of 1% KsHPO( solution. The suspension was expressed through cheesecloth, and 1 volume of a citrate-chloroform extract of healthy leaves (prepared as described under footnote c) was added. c Ten grams of CMV-infected leaves were ground in a mortar with 20 ml of citrate buffer and 20 ml of chloroform. The emulsion was centrifuged for 15 minutes at 5400 g. The resulting pulp was suspended in the aqueous phase, expressed through cheesecloth, and 1 volume of a phosphate extract of healthy leaves (prepared as described under footnote b) was added.

were made with healthy tobacco leaves to which purified CMV had been added before extraction. Aliquants of the preparations from diseased and healths leaves were treated with phenol, and the-resulting aqueous phases and the original preparations were tested for infectivity. As shown in Table 5, phenol treatment of the aqueous phases of citrate-chloroform extracts led in all cases to decreased infectivitv of the preparations. Phenol-treated preparat.ions from infected leaves sometimes retained a higher percentage of the infectivity of the original extract than did phenol-treated preparations from healthy leaves to which CMV had teen added before extraction, but this trend was not consistent. Experiments with Purified Virus Phenol treatment of virus. Equal amounts of purified CMV were suspended in 0.05 M borate buffer, pH 8.5, and in 0.5 M phosphate buffer, pH 6.5. Aliquants of these suspensionswere diluted with 1% K2HPOI . Other aliquants were treated with phenol, and the aqueous phases were then diluted with 1% K,HPOa . Equal dilutions of the

TABLE

4

RELATIVE INFECTIVITIES OF CENTRIPUGED PHOSPHATE AND CITRATE-CHLOROFORM EXTRACTS FROM CMV-INFECTED TOBACCO LEAVES”

-

Phosphate extracth Centrifugation

Supernatant

Citrate-chloroform extract”

Resuspended pellet

.___ 5,000 9

48

10,000 g

I

15,000 g

2

~____ 124

GS

0 108

G

0 119

0 L a Average number of lesions per 7-13 primary cowpea leaves. b Extract made from 10 grams fected leaves according to Schlegel c Ten grams of CMV-infected ground with 40 ml of citrate buffer chloroform.

0 half-leaf

on

of CMV-in(1960b). leaves were and 40 ml of

original suspensions and of the phenoltreated preparations were compared for infectivity (Table 6). The infectivity of the

1X6

DIENER,

SCOTT,

AND

TABLE I~ELATIVE AND

INFECTIVITIES.OF

FROM

HEALTHY

5

CITRATE-CHI.OROFORM TOBACCO

LEAVES

AND

EXTRACTS

TO WHICH

THOSE

KAPER

FROM

CMV-INFECTED

CMV

PURIFIED

OF PHENOL-TREATED

WAS

Dilution in 1% K*HPO,

no.

Infected

extract

tissue

Not treated with phenol

BEFORE

LEAVES

EXTRACTION

EXTRACTS”

Citrate-chloroform Espt.

TOBACCO

ADDED

of

Healthy

tissue

+ CMV

Treated with phenol

Not treated with phenol

Treated with phenol

12”

1:25 1:50

140

20

50 20

19 3

13*

1:25 1:50 1:lOO

88 63 19

A4 55 1

71 16

40 11

u Average number of lesions per half-leaf on 4-9 primary cowpea leaves. * In experiment 12, 10 g of leaves were extracted with 20 ml of citrate-thioglycolate buffer and 20 ml of chloroform. In experiment 13, 10 g of leaves were ext,racted with 40 ml of citrat,e-thioglycolate buffer and with 40 ml of chloroform. TABLE REL,ZTIVE

INFECTIVITIES AND

6

SUSPENDED

OF PHENOL-TREATED

Buffer Composition

OF CMV

of buffer Dilution

IN

BORATE

Phenol-treated

suspension (A) ______ Infectivity”

OR PHOSPH.\TE

OF CMV

SCSPENSIONS

suspension

(B)

Dilution

Infectivity

u

Ratio

Boric acid-NaOH, 0.05 M, pH 8.5

1:250 1:500 1: 1000

160 68 25

2:5 1:5 1:lO

67 176 78

Phosphate, pH 6.5

I:250 1:500 1:lOOO 2:5 I:5 1:lO

3 0.1 0 139 76 38

2:5 1:5 1:lO 2:5 1:5 1:lO

208 198 283 115 192 188

n Average

0.5 M,

number

of lesions

per half-leaf

on 8-11

phenol-treated borate suspension was much less than that of the virus suspension, but the infect.ivity of the phenol-treated phosphate suspension was from 0.69 to almost 20 times as great as that of the virus suspension. Samples of purified CMV were suspended in various media, and the suspensions were treated with phenol. Aliquants of the original virus preparations were diluted and their infectivities were compared with those of equally diluted phenol-treated preparations (Table 7). When virus suspensions in 0.005 iM borate or 0.08 M KC1 were treated with phenol, the resulting preparations had very

with

primary

conpea

B : i\

0.004 0.026 0.03 0.69 19.8 0 .83 2.53 4.95

leaves.

low or no activity; hut when virus suspensions in suspending media with higher salt concentration were treated with phenol, the resulting preparations retained IS-56 % of the infectivity of the virus suspensions: Specific

infectivities

of CM V and

CM V-

NA. Preparations of CMV and of CMV-NA (prepared by treating CMV suspended in 0.4 M KsHP04 with phenol) were diluted to equal NA concentration (based on their respective UV spectra and on an NA content of CMV of 20%) (unpublished work) and inoculated on opposite half-leaves of cowpea. Table 8 shows that at the highest concen-

NUCLEIC

ACID

FROM

CUCUMBER TABLE

RELATIVE

OF CMV-NA

INFECTIVITIES

CMV

Suspending

Molar

medium

cont.

IN

137

VIRUS

7

QBTAINED V.~RIOLJS

Dilution

MOSAIC

BY PHENOL MEDIAN

in l’:‘b

KeHPOa

TREATMENT

CMV suspension (A)

OF PURIFIED

Phenoltreated CMV suspension @J)

Ratio

B :A

0.005 0.4

1: 5000 1: 10000

72 48

0.2 12

0.003 0.25

KrHP04

0.04 0.08 0.4 0.4

1: 2000 1: 2000 1: 2000 1: 4000

194 172 148 61

34 84 70 34

0.18 0.49 0.47 0.56

KC1

0.08 0.8

1: 5000 1: 10000

201* 63*

0 16

0 0.25

0.4

1: 1000

119

29

0.24

0.4 0.4

1:20 1:40

220 248

75 129

0.34 0.52

Borate-NaOH,

pH

Tris-HCl, Citrate,

pH 8 pH

0.5

(1 Average number 6 Virus suspended

9

of lesions per half-leaf on 6-12 in 0.005 ii/r borate, pH 9.0.

tration tested, CMV-NA produced nearly half as many lesions as a virus preparation containing an equal quantity of NA. At lower concentrations, the NA preparations produced progressively fewer lesions than virus preparations of equal NA concentration. Alteinatively, preparations of CMV were diluted to give approximately equal numbers of lesions as suitably diluted preparations of CMV-NA on opposite half-leaves of cowpea. As shown in Table 8, approximately equal numbers. of lesions resulted if the virus suspensions contained 60% of the nucleic acid present in the nucleic acid inoculum. Comparison of the infectivities of CMV and tobacco ringspot virus. The question arose why CMV-NA prepared under optimal conditions was half as infectious as CMV suspensionsof equal NA concentration. Conceivably, at a given virus concentration, CMV could be less infectious on cowpeas than other viruses, yet the NA’s prepared from these viruses might be equally infectious, resulting in a high relative infectivity of NA to virus with CMV but not with other viruses. Table 9 shows, however, that the number of lesions produced by CMV was comparable to the number pro-

primary

cowpea

leaves.

duced by tobacco ringspot virus when both viruses were tested at equal concentrations on the same host. Properties of CM V-NA Ribonuclease sensitivity. Suspensions of purified CMV and of CMV-NA were diluted and incubated with pancreatic ribonuclease. Incubat,ion for 1 hour at 0” with 1OW pg ribonuclease per milliliter was sufficient to eliminate all infectivity of CMV-NA but had no significant effect on the infectivity of virus preparations. Ultraviolet spectrum. CMV-NA preparations were freed from phenol by repeated extractions with ether. The ultraviolet spectra of these preparations were typical of those of nucleic acids (maximum at 258 rnp, minimum at 230 rnp, maximum :minimum ratio from 2.2 to 2.6). Protein content. The nucleic acid preparations contained approximately 1% of protein as determined by the method of Lowry et al. (1951), with ovalbumin as a standard. Type of nucleic acid. Preparations of CMVNA gave positive reactions with orcinol and no reactions with diphenylamine, indicating that CMV-NA is a ribonucleic acid.

138

DIENER, TABLE

RELATIVE

8 OF CMV

INFECTIVITIES

Concentration hdml)

SCOTT,

Infecti

AND

-

CMV-NA Per cent nfectivity

vit Y” -

CMV-NA

CMV

CMV-NA

CMV

1.G 0.8 0.4 0.2 0.1 2 2 2 2 2 1 1 1 1 1 0.5 0.5 0.5 0.5 0.5

8 4 2 1 0.5 10 8 6 6 4 5 4 3 3 2 2.5 2.0 1.5 1.5 1.0

95 39 13 11 6 137 115 170 236 250 107 119 110 129 153 47 61 49 179 142

197 212 105 72 61 231 162 193 186 148 175 188 147 120 122 121 93 122 104 63

48 18 12 15 10 59 57 53 76 68 61 51 45 65 50 39 52 24 103 90

= Average number of lesions 9-16 primary cowpea leaves. b No. of lesions (NA) No. of lesions (CMV) ’ Concentration Concentration

INFECTIVITY RINGSPOT

Virus cont. (i&ml) 2 1 0.5 0.25

half-leaf

of NA (CMV) of NA (NA)

TABLE RELATIVE

per

on

x

loq

AND

KAPER

pattern. Two major and two minor components can be discerned. To determine their sedimentation constants, four dilutions of a typical nucleic acid solution were centrifuged. The apparent sedimentation coefficients were computed and subsequently corrected to 20” and water basis. The reciprocals of the sedimentation coefficients thus obtained for the two major and the trailing minor component were plotted against the total NA concentration. The 1/~!5’~0,~versus concentration relationship was linear. Extrapolation to zero concentration yielded 21.0 S, 18.6 S, and 11.9 S, respectively. Density-gradient centrifugation. Preparations of CMV-NA were subjected to densitygradient centrifugation in sucrose gradients and then fractionated. The resulting fractions, together with unfractionated CMVNA solutions of equal NA concentration, were tested for infectivity. Figure 2 shows a typical recorder trace and infectivity data. The NA preparation was separated into at least three components (A, B, C). The three components had UV spectra typical of N&s. Component (A) was evidently noninfectious; component (C) was infectious. The specific infectivity of the fractions comprising component (II) was consistently much lower than that of the fractions comprising component (C), suggesting that these fract,ions were contaminated with component (C) and that component (B) was ncninfectious.

9 OF CMV VIRUS*

CMV

AND

Tobacco ringspot virus

120 69 20 11

* Average number of local lesions on 5-10 primary cowpea leaves.

TOBACW

92 70 31 32 per

half -leaf

Analytical ultracentrifugation. Preparations of CMV in0.8 M KC1 were treated with phenol, and the NA was concentrated by precipitation with ethanol. The NA was resuspended in 0.02 M phosphate buffer, pH 7, and subjected to analytical ultracentrifugatinn. Figure 1 shows a typical sedimentation

DISCUSSION

Extraction of CMV-infected tissue with pyrophosphate-phenol often led to more infectious preparations than extract,ion with phosphate (Table 1). Thus, Schlegel’s results could be confirmed, but the differences in infectivity between the two inocula were not as large as those found by Schlegel, and in many cases the pyrophosphate-phenol extracts had lower infectivities than the corresponding phosphate extracts. Phosphate extracts subsequent’ly treated with phenol were as infectious as pyrophosphate-phenol extracts (Table 2)) indicating that the high infectivity of these extracts could not be due to the presence in the tissue of incomplete (or partially degraded) ribonuclease-sensitive virus particles, to free RNA, or to entities similar to the unstable variants

NUCLEIC

AC111

FROM

CI:CURIBER

MOSAIC

VIRUS

139

FIG. 1. Sedimentation pattern of CM\.-NA in 0.02 M phosphate buffer, pH 7.0. NA concentration: 1.5 mg/ml. The phot)ograph was taken 28 minutes after a speed of 59,780 rpm was reached. Angle of the schlieren diaphragm, 30 degrees; sedimentation is from right to left.

of tobacco necrosis virus described by Babos and Kassanis (1962). These conclusions are strengthened by the fact that highly infectious preparations resulted even if the phosphate extracts were incubated with ribonuclease before phenol treatment (Table 2). The infectivity of phenol-treated preparations could have originated from complete virus particles or from entities similar to the so called “nonmultiplying” forms of tobacco rattle virus as described by Cadman (1962) or from both types of entities. In the latter case, the infectious entities would have been protected from attack by ribonuclease by being located within some subcellular particles. Extraction of tissue with phosphate buffer would not release the infectious entities from the subcellular particles, thus ex-

plaining both the resistance to attack by ribonuclease and the low infectivity of the extracts. Treatment with phenol would release the particles and render them both ribonuclease sensitive and highly infectious. Our .data do not exclude the possibility that an infectious entity of this type exists in CMV-infected tissues; but, in the light of our results, such an assumption is unnecessary. Extraction of CMV-infected tissues with phosphate led to pr,eparations with much lower infectivity than did extraction of tissues with citrate-chloroform (Table 3). Thus, the high infectivity of phenol-treated preparations, as compared with that of phosphate extracts, could simply have been due to incomplete extraction of virus from t.he tissue with phosphat’e buffer, whereas extrac-

140

DIENER,

5 FRACTION

NO

10

15

SCOTT,

-

FIG. 2. Absorbance (broken line) at 253.6 mp of a centrifuged density gradient. column containing CMV-NA and relative infectivity (unbroken line) of consecutive l-ml fractions taken therefrom. The CMV-NA was prepared by phenol treatment of CMV suspended in 0.8 M KCl, followed by three extractions with ether and one precipitation with ethanol. The NA was dissolved in 0.02 M phosphate buffer, pH 7.0, and 0.48 mg of the NA was floated on a linear density gradient prepared from 0.02 Jl phosphate buffer, pH 7.0, containing 8-23y0 sucrose. The column was centrifuged for 16 hours at 24,000 rpm. Absorbance at 260 rnp was determined of all fractions to be assayed for infectivity, and dilutions containing 2 pg/ml of NA were inoculated on half-leaves of primary cowpea leaves. Opposite half-leaves were inoculated with noncentrifuged CMV-NA (2Mg/ ml) stored overnight at 4”.

tion with pyrophosphate-phenol led to a much more complete release of virus (as nucleic acid) from the tissue. On the other hand, it is known that CMV suspended in phosphate aggregates (Scott, 1963); and possibly this aggregation was responsible for the relatively low level of infectivity in the phosphate extracts. Centrifugation of phosphate extracts showed that the virus could readily be sedimented at 5000 g, a result indicating aggregation or lack of release from tissue fragments, whereas in citrate-chloroform extracts,

the virus

remained

suspended

even at 15,000 g (Table 4). The infectivities of phenol-treated citratechloroform extracts from CMV-infected leaves were not consistently higher than those of equally treated preparations from healthy leaves to which purified CMV had been added before extraction (Table 5). Thus, the hi&h infectivity of the phenoltreated preparations from diseased leaves could have originated solely from virus particles.

AND KAPER

With purified CMV, phenol-treated preparations that had only 0.004 times as much infectivity or almost 20 times as much infectivity as the parent virus suspensioncould be produced (Table 6). This presumably depended on whether the suspending medium did or did not lead to aggregation of the virus (Scott, 1963). The high relative infectivity of NA to virus could thus readily be explained without the necessity of assuming infectious entities other than complete virus particles. Treatment of citrate-chloroform extracts from CMV-infected tissues with phenol, however, consistently led to preparat,ions that retained a considerable fract,ion of the infectivity of the virus suspensions (Table 3). Here, neither aggregation nor lack of release of virus could have been responsible for the apparent high level of infectivity after treatment with phenol. When purified suspensionsof CMV were treated with phenol, the infectivities of the resulting iYA solutions depended on the ionic strength of the medium in which the virus had been suspended during treatment with phenol (Table 7). Phenol treatment of CJIV suspended in buffers of low ionic strength jed to NA preparations with low levels of infectivity, but treatment of CMV suspended in buffers of higher ionic strength led to NA preparations with high levels of infectivity. That the ionic strength, not the molar concentration of the suspending medium, must exceed a certain minimal value for the production of highly infectious NA preparations is indicated by the ineffectiveness for this purpose of 0.08 M KC1 and the effectiveness of 0.04 M K2HP04 . Since CMV produced as many lesions on cowpea as did equal amounts of tobacco ringspot virus (Table 9)) it is unlikely that the high CMV-X-A to CMV infectivity is an illusion causedby poor lesion-producing ability of CMV. Furthermore, countable lesions were regularly produced when cowpea leaves were inoculated with CMV-T\‘A at a concentration of 0.1-2 pg/ml; whereas with NA from tobacco ringspot virus, a concentration of 10-50 rg/ml was required on the same plant species (Kaper and Steere, 1959a). It thus appeared that the high ratio of infectivity of NA to virus was due to a high

NUCLEIC

ACID

FROM

CUCUMBER

ievel of infectivity retained by CLIV-KA, not to poorly infectious virus. No unusual properties of CMV-NA could be detected with respect to its sensitivity toward ribonuclease or its UV-absorption spectrum. Unusual properties were discovered, however, when CMV-NA was subjected to analytical ultracentrifugation. Two high-molecular components whose sedimentation constants differed by about 10% could consistently be found (Fig. 1). Ko two-component system similar to the one reporbed here has been described with KA prepared from plant viruses. The sedimentation behavior of CMV-NA is, however, reminiscent of that of the Dn’A from bacteriophage +X174 as described by Sinsheimer (1959) and by Fiers and Sinsheimer (1962). These authors demonstrated the presence of two discrete components in preparations of this DNA: a minor component, which comprised about 20-40% of the total, and a major component, which usually comprised 50-70% of the total and which moved at about a 10Y~ faster rate than the minor component. By treatment with pancreatic deoxyribonuclease and by thermal denaturation, the authors showed that the minor component is the first degradation product of the major component, formed by a single scission of the major component but without significant decrease in molecular weight. The authors concluded from their experiments that the major component is a covalently linked ring structure and the minor component is the corresponding openchain degradation product and that only the ring-structured component- is infectious. With RNA preparations from CMV, preliminary evidence indicates that only the faster moving component in a sucrose gradient is infectious and that incubation of CMV-KA with low concentrations of ribonuclease leads to partial loss of the faster moving component before the overall concentration of the slower moving component diminishes. Further work is needed to correlate the analytical ultracentrifugation and density-gradient patterns in order to determine the configurat’ion of the faster moving component and ‘to investigate whether this particular configuration is responsible for the high level of infectivity of ChlV-NA preparations.

MOSAIC

VIRUS

111

REFERENCES

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