VIROLOGY 39, 82-96 (1966)

Reconstitution of Turnip Yellow Mosaic Virus RNA with

TMV Protein Subunits

R. E. F. MATTHEWS

Microbiology Department, University of A.uckland, Auckland, New Zealand

Accepted lvlay 12, 1966

When TYMV RNA is incubated with TMV protein subunits in 0.25 M phosphatebuffer at pH values near 7.0 at 30° for several hours, some of the RNA is coated withprotein to form rods that appear very similar to TMV except fOI' length distribution.Most of the rods are shorter than the length expected for a full TYMV RNA comple­ment. Yields of reconstituted material vary widely (1-30%) and are much lower thanobtained with TMV RNA near pH 7.0. The reconstitution reaction (as judged bychemical criteria) has a second pH optimum near pH 4.8 for both TYMV RNA andTMV RNA. At this pH, the reaction is much more rapid and higher yields of coatedRNA are obtained. However, as judged by infectivity of TMV, very few completerods are formed at this pH.

TYMV RNA coated with TMV protein at pH 6.7 or pH '1.8 and isolated by highspeed sedimentation may retain some infectivity for Chinese cabbage, but all, oralmost all, this infectivity is lost on treatment with ribonuclease. The infectivity inthe reconstituted preparations probably resides in particles consisting of an intactTYMV RNA complement partially coated with TMV protein.

INTRODUCTION

Fraenkel-Conrat and Williams (1955)showed that infectious tobacco mosaic virus(TMV) rods could be reconstituted fromT1VIV RNA and the viral protein subunits.Hart and Smith (1956) obtained evidencesuggesting that rods could be formed usingTMV protein and yeast RNA or varioussynthetic polyribonucleotides. Fraenkel­Conrat and Singer (1964) obtained recon­stitution with certain artificial polymers, butnot with RNA from wheat germ or ascitescells. Holoubek (1962) found that the RNAfrom various strains of TJ'vIV could be re­constituted into infectious rods with proteinsubunits from a type strain.

In his early work Fraenkel-Conrat usedthe term reconstitution in a strict sense, forthe in vitro formation of infectious TlVlVrods. For convenience the term will be usedhere for the formation of rods of TMV pro­tein containing RNA, without necessarilyimplying biological activity. In the workdescribed below it has been found that under

82

appropriate conditions of incubation near pH7.0 or pH 4.8 TMV protein subunits will coatturnip yellow mosaic virus (TYMV) RNAto form rods. A low level of infectivity forChinese cabbage may be retained by suchrods. Most of the TYMV RNA is not com­pletely coated in such preparations since allor nearly all the infectivity is lost on treat­ment with ribonuclease. A preliminary ac­count of some of this work has appeared(Matthews and Hardie 1966).

MATERIALS AND METHODS

Viruses. TMV was cultured in tobacco(Nicotiana tabacum L. val'. White Burley).TYMV was cultured in Chinese cabbageiBraeeica pekinensis (Lour.) Rupr. val'.Wong Bok). TMV was isolated by threecycles of high speed sedimentation from 0.1M phosphate buffer pH 7.2 containing 0.2111ethylenediaminetetraacetic acid (EDTA).TYMV was isolated by the pH 4.8 procedure(Matthews, 1960).

Nucleic acids. RNA was isolated from

RECONSTITUTION OF TYMV RNA WITH TMV PROTEIN 83

purified viruses by a modification of thephenol procedure of Ralph and Bellamy(1964). Virus in 0.01 M phosphate buffer pH7.2 containing 0.02 111 EDTA was extractedtwice with 3 volumes of water-saturatedphenol containing 0.1 % 8-hydroxyquinoline.Phenol was removed with ether, and theRNA was precipitated as the cetyltrimethyl­ammonium salt. This was converted to thesodium salt in 70 % ethanol and taken to adry powder as described by Ralph andBellamy (1964). RNA was stored in a desic­cator over phosphorous pentoxide.

TMV protein. TMV protein subunits wereisolated by the acetic acid method of Fraen­kel-Conrat (1957) from freshly preparedTMV and stored in 0.01 MpH 6.7 phosphatebuffer at 2°.

Radiochemical methods. 32P-Iabeled viruswas prepared as described previously (Mat­thews, 1960). Radioactivity was measured onplanchettes using Millipore-type filters anda Phillips thin-end window ONI tube (PM18515).

Spectrophotometry. Ultraviolet absorptionmeasurements were made with a Zeiss PMQII spectrophotometer using cells of J-cmpath length.

Suctose density gradient fractionation. Sam­ples were layered over a 5-20 % linear su­crose gradient. The sucrose contained 0.14 MNaCI and 0.01 M phosphate buffer pH 7.3.The gradients were then centrifuged for 4hours at 35,000 rpm in a Spinco SW 39 ro­tor at about 4°. Samples were collected by adrip out procedure.

Assay for reconetiuuion. To detect andestimate reconstitution three methods wereused: (a) Sedimentation for 1 hour at 3.5,000rpm at pH 6.7-7.3 followed by ultravioletabsorption measurements on the redissolvedpellets, as used by Fraenkel-Conrat andSinger (1964). After incubations near pH 7.0for several hours at 300 virtually no freeRNA remained sedimentable, However,after short incubations near pH 4.8 somefree UNA may sediment. For this reasonmixtures incubated at pH 4.8 were some­times treated with pancreatic ribonucleaseat pH 6.7 (1-2 J-lgjml, 20 minutes at roomtemperature) before isolation of any recon­stituted rods. (b) Electron microscopy. Insome experiments solutions containing re-

constituted material were mixed with anequal volume of phosphotungstic acid (2.0 %at pH 6.7) and the mixture was placed oncarbon films, dried, and examined. In othersa standard solution of polystyrene latex par­ticles was mixed with the reconstitutedmaterial, and the mixture was sprayed ontogrids. After shadowing with palladium­platinum, appropriate small drops were pho­tographed for estimating size distribution ofany rods present. (c) Resistance to ribonu­clease. RNA coated with TlVIV proteinsubunits is resistant to attack by ribonu­clease. We used the increase in resistance toribonuclease as a rapid method for assay ofreconstitution. Aliquots of the incubationmixture containing 32P-Iabeled RNA werediluted in saline or buffer and incubated atpH 6.6-7.3 with ribonuclease (2 J-lg/ml) forabout 20 minutes at room temperature.Carrier protein (0.5 mg per sample) andtrichloroacetic acid to a final concentrationof 5 % were then added. The sample wasMillipore-filtered and washed with 5 % tri­chloroacetic acid and the radioactivity wasmeasured. Under these conditions about1.4 % of the radioactivity in free TMV RNAand about 0.6 % of that in TYMV RNA re­main on the filter. This material is presuma­bly the ribonuclease-resistant core. No cor­rection was made for this residue in theresults reported. In most experiments thesequantities would not have been significantlyabove background in the samples measured.

Infectivity assays. Infectivity of TMVpreparations was assayed on N. glutinosa,and TYlVlV was assayed on Chinese cabbage.For both assays half-leaf comparisons be­tween treatments were used. For tests on theresistance of infectivity to ribonuclease,samples in 0.01 M phosphate buffer, pH 6.7,were treated with the enzyme at 0.1 J.lg/mlfor 20-30 minutes at room temperature,

RESULTSHeconeiitnuiot: with TYMV RNA near pH 7.3

As a starting point, the conditions thatFraenkel-Conrat and Singer (1959, 1964)had found to be optimal for reconstitutionwith TlVIV RNA were used. Unless other­wise stated 1 mg TMV protein subunits and.50 p.g of RNA was used per milliliter in allexperiments.

84 MATTHEWS

F IG. 2. Effect of molarity of phosphate b ufferon recons t i tu tion at pH 7.3. The mix lure COli t ai uedTMV pro tein subunits at 1 mg /rnl and UP-labeledR)\A at 50 pg/ml; it was incubat ed for 24 ho urs at300

• Reconstit ut ion was estima ted by ribonucleaseresistance . 0--0 = TM V R NA; X -·· --X =T Yi\IV RNA.

0.01Molarity af buffer

1.0

,..;X,X"I

/I

/I

/x__-.

4 0

0.0 01

s 20oC<I>W

cf. 10

50

Effect of teniperoiure. F igure 1 shows re­sults of an experiment to determine thetemperature optimum for reconstit utionwith TYMV R NA at pH 7.3 . All threemethods noted above were used to detectreconsti tuton, Aliquot s of the incubationmixtures were used to determine the pro­portion of RNA tha t had become resistan tto ribonuclease. The bulk of each incubationmixtur e was cent rifuged at 38,000 rpm for 1hour in a Spineo No. 40 rotor . Any pelletedmate rial was redissolved in buffer at pH 7.3and centriguged a second time. Yields ofsediment able nucleoprot ein followed closelythe p attern shown for ribonuclease resistance(Fig . 1). Examination of samples by electronmicroscopy revealed rods (mostly short) onlyin the 24°, 30°, and 40° sam ples. Proteinsubunits incubated at pH 7.3 and 30° with­out R NA produced no rod s. I t was con­clude d that TlVIV protein subuni ts can packaround T Yi\IV R NA to form rods and t hatthe temperature optimum for t he process atpH 7.3 is close t o 30°, as found for T MVRN A by Fraenkel-Conrat and Singer (1959) .

FlO. 1. Temperature optimum for reconst.it u­tion at pH 7.3. Aliquots of u mixture contain ingper millilitre 1 m.g TMV prote in subuni ts and50 I'g of ~2P -labeled TTIvIV RNA in 0.1 M phos­phate buffer were in cubate d a t vari ous tempera­tures for 6 ho urs. P ercentage of reconstitution wasest ima ted by ribo n uclease resis tanc e (X ----- X )and by yield of sedimentable nucleoprotein<e- e) .

3.0...~

~ \· \· ', \· \I \\\\\

~\\\\\

Temperature

M olal'l:ty of buffer. Figure 2 shows themarked dependence of reconsti tution ofT YMV R NA on molarity of the pH 7.3phosphate buffer used, as found by F raenkel­Conrat and Singer (1964) for T l\>fV. In ourtests virtually no reconstitut ion occurred foreit her T YMV or T MV RNA at 0.033 M orbelow.

Time cow'se of reconstitu tion. Figure 3shows t he increase in reconst itut ion ofTY1VIV RNA with time. After () hours at 30°and pH 7.3 in 0.1 111 phosphate buffer some4 o/cl of the RNA had become resistant toribonuclease. Figure 3 illustrates a m ajordifficulty with TYl,IV RKA under t heseconditions. More and more of the R N A notcoate d with Tl\IV protein becomes so furdegraded that it is no longer insoluble in 5 %trichloroacetic acid. This is presumably dueto traces of nuclease present in the ine uba­tion mixture,

E:O'ecl of pH. Figure 4 shows tha t in therange pH 5.0-8.0 the opt imum for recon­sti tution of T YMV R NA is neal' pH 7.0 , asit is for T l\IV.

In va rious experiments with TYMV R NAusing different batches of materials the pH

RECONSTITUTION OF TYMV RNA WITH TMV PROTEIN 85

7.5 8.07.06.56 .0o....~_-A._-A.__L-....

70

80

60

c.2

~ 50;;;c-o(J

1!OJ 400>o

"EOJ

~ 30a.

values TMV protein sub units aggregat e toform rod s of indeterminate length and thatt~le.se rods aggregat e to form visible pre­cipitates, The RNA protected from ri­bonuclease attack at pH 4.5-5.0 but notat pH 6.7 was probably trapped non­specifically in the aggregates of rodsformed at the low er pH, and thus given pro­t ection from rib onuclease at tack. To avoidt his complicat ion in furt her experiments ali­quots of samples incubated in the low pHrange were brought to pH 6.7- 7.0 beforebeing tested for resistance to ribonuclease.

Temperature optimum at pH 4.8. The ex­periment summarised in Fig. 6 shows thatthe temperature optimum for reconsti tutionas judged by resistance of the RNA to ribo­nucl ease, is about 5.5- 50° for both T l\'[VR NA and TYMV R NA. The differenee in

ph

FIG. .1. Effect of pH 0 11 recons ti tut.i ou nearpH 7.0 TMV. T ile mixture contained p roteinsubuni ts a t 1.0 rngyml and 82p -lah eled RNA at50 Jig/mI., it was in cub ated for 23 hours a t. 30° in0.25 M phosphate buffer. Reconatituti ou wasestimated by ribouuolease r esistance. 0 - - 0TMV RNA; X ----- X = TYMV RNA.

6o 2 4Hours of incubohon

FIG. 3. Time course of rec oustitution and RNAbreakdown at pH 7.3. The mix tu re con tained TMVpr otein subunits at 1 mg /ml and siP-labeledTYMV RNA at 50 /Lg/ml it, was incuba ted in 0.1 illphosphate hulf'er at 30°. X-----X = percen tageof RNA rendered r ibnllllel eas e res is tnn t. , 0--0'= percentage RNA rend ered so luble in 5% tr i­chloroacetic acid.

op timum varied somewhat and frequentlywas as low as pH 6.7.

Yields. Under optimum couditions at pH6.7-7.3 (0.2-0.2G JlI phos pha te buff er , at 300

for 0-24 hours) th e yields of reconsti t utedm ateri al have v aried widely, rungi ng fromabou t If} to 50 (1,) for T MV UN A a nd 1 to30 % for T Yi\lV UNA.

R econsntuiion. neal' pH 1,.8.

Unless otherwise stated TlVIV protein sub­unit s were used at 1 mg/ml and RNA at50,ug/ml.

EjJect ojpH. The lower pH values tested inthe experiment of Fig. 4 suggested thatthere might be a second, lower pH optimumat least for TYlVIV. The experiment to testthis showed that the re was a second pHoptimum near 4.5-5.0 for both TlVIV RKAand TYMV RNA (F ig. 5), as judged by in­creased ribonuclease resis tan ce of the R NA.In th is experiment aliquots of the incub ationmixture were dilu ted in sa line and incubatedwith ribonuclease. Subsequent t ests showedthat if the pH of th e samples was broughtto 6.7 before ribonu clease treatment ab outone-third of the apparent ly ribonuclease­resistan t RNA becam e suscep tible t o diges­tion . It is well-known that at these lower pH

86 MATTHEWS

pH 4.8 (Fig. 9) was generally similar to theeffect at pH 7.3 (Fig. 2), However, somereconstitution took place at pH 4.8 at 0.01and 0.03 M whereas none occurred at thesemolarities at pH 7.3.

Yields at pH 4.8. Yields of reconstitutedmaterial in repeated experiments under thesame conditions at pH 4.8 varied quitewidely, as they do at pH values neal' 7.0.However, as shown in Figs. 6-9, the yields ofreconstituted material both for TMV RNAand TYlVIVRNA are much higher at pH 4.8than at pH values near 7.0. In these experi­ments reconstitution was measured by theribonuclease resistance at pH 6.7 of aliquotstaken from the incubation mixtures. Yielclsbased on amount of nucleoprotein materialisolated by two cycles of high speed sedi­mentation at pH 6.7 (1 hour at 38,000 rpm)were always lower. They have ranged invarious experiments from about 15 to 60 % ofthat expected from ribonuclease resistanceassays on aliquots of the incubation mixture.The lower yield was probably due in part atleast to the high proportion of very shortrods formed in incubation mixtures at pH4.8, much of this material being left in thesupernatant fluids. A second centrifugationof the supernatant fluid gave a further yieldof nucleoprotein containing RNA resistantto ribonuclease.

5.54.5 5.0ph

0---'--...."'"'-_.......4.5

80

70

60

".9 50~;;c0u~ 40.,0>0c:.,

30u;;;o,

20

10

FIG. 5. Effect of pH on reconstitution near pH5.0. Conditions as for experiment of Fig. 4 exceptfor pH. 0-0 TMV RNA; X-----X TYMVRNA.

yield between TYMV RNA and TIVIV ismuch less at pH 4.8 and GO° than at pHvalues near 7.0 and 30°.

Ti'llw course at pH 4.8. The experiment ofFig. 7 shows that the rate of increase inresistance to ribonuclease is very much morerapid at pH 4.8 and 60° than at pH 7.0 and30°. The reaction was essentially completefor both TlVIV RNA and TYMV RNA after5 minutes' incubation.

Effect of ratio of protein s'ubunits to RNA atpH 4.8. The experiment of Fig. 8 shows thatat pH 4.8, using an incubation period of 5minutes at 60°, doubling the amount of pro­tein subunits in the mixture over the stand­ard "molar" ratio increased the amount ofRNA protected from ribonuclease attack.Increasing the "molar" ratio of protein toRNA above 2: 1 gave no further increase.

Ellect of phoephatemolariui at pH 4.8. Theeffect of increasing molarity of phosphate inthe incubation medium on reconstitution at

Properties of the Product Produced by Re­constitution. of T111V RNA and TY 111VRNA with 'l'MV Protein Subunits

For examination of various properties,reconstituted material was isolated by highspeed sedimentation from the incubationmixture. For material incubated near pH7,0, reconstituted rods were sedimented at38,000 rpm for 1 hour in a Spinco No. 40rotor. The water-clear pellets were resus­pended in 0.01 JI{ phosphate buffer at pH6.7-7.0 and resedimented under the same con­ditions. The final pellets were taken up in asmall volume of the same buffer. Incubationmixtures at pH 4.8 were first sedimented at30,000 rpm for .5 minutes. Because of thegross aggregation at this pH all reconstitutedmaterial was sedimented under these concli­tions. The pellets were then taken up in 0.01111 phosphate buffer pH 6.7 and the recon­stituted rods were sedimented twice as for

RECONSTITUTION OF TiNIY RAN WITH TMV PROTEIN 87

90

20

80

70

II

II

II

II

X,,I,,,

>('I

II

I/

II

... X10 _---x... ... ...

.,en.e 40c.,~.,a. 30

c: 60o2inl5 50u~

0 ...-""--...----...",--.....- ...---..10 20 30 40 60 70

Temperoture

FIG . 6. Temperature optimum for recons ti tution at pH ·1.8 . The mixture contained TMV protein sub­un its at 1 mg/rnl and 32P-labeled -RNA at 50 pg/ml in 0.25 AI ph osphate. Samples were incubated for 5minutes. Reconstitution was measured by ribonucleas e resistance . 0--0 = TM V RNA ; X -----XTYMV RNA.

material incubated near pH 7.0. This pro ce­dure gave variable but low yields of materialwith a protein-like UV absorption spectrumwhen RNA was omitted from the incubationmixture. In some experiments, the pelletsresusp ended after the first high speed sedi­mentation were treated with ribonuclease(1- 2 I.lg/ml for 20 minutes at room tempera­ture) to remove any RNA th at was notcoated with protein.

UV absorption spectrum. Tl\ IV RNA re­constituted at pH value s neal' 7.0 had a UVabsorption spect rum indist inguishable fromthe parent Tl\fV. TYl\IV RNA recousti­tuted neal' pH 7.0 or at pH 4.8 and isolatedas described abov e had a UV absorptionspectrum with a peak or plateau in the re­gion 2Gii-270 m u, A typical spe ctrum isshown in Fig. 10. The reasons for th e va ri­ability and th e differen ce from reconsti tuted

T 1VIV remain to be determined, but theyprobably include (1) the difference in basecomposition of T lvIV RNA and T YM VRNA, the lat ter having a much highercytidylic acid content; (2) differences in andvariability in the length distribu t ion of theTYiVIV RNA rods, leading to differences int he cont ribut ion of light scattering to theabsorption spectrum ; and (3) variation inthe content of RNA in the TYJ.\IV RNA rodprepar ations.

Size distribution. TY:.\lV RNA is verysimilar in length to T?dV RNA , so com­plete rods formed by TYl\lV RNA and TMVpro tein would be expected to be about thesame length as TMV. Precise data concern­ing th e size dist ribution of T MV R NA andTYl\IV RNA reconstituted under variouscondit ions has not yet been obtained. H ow­ever , a number of preparations, isolated as

c:,g 60::J"=t;c8~

88 MATTHEWS

100.. -.

80 -n ..

,x-- -- --x-----------)t-X; .. , ...

)/(~g, X~ 40 IJ" rrQ; I0. I,,

II

20

I I

5

I

10 20

Time of incubation (minutes)30

FIG. 7. Time course of reconstitution at pH 4.8 ancl60°. Mixture contained TMV protein subunits at1 mg/rnl and 32P-labeled RNA at 50 JLg/ml in 0.25 M phosphate. Reconstitution was measured by ribo­nuclease resistance. 0--0 = 'I'MV RNA; X-----X == TY:1vIV RNA.

described above, have been examined in theelectron microscope using PTA staining orsprayed droplets followed by metal shadow­ing. From these observations the followinggeneral statements can be made: (1) TMVRNA reconstituted near pH 7.0 gives a highproportion of rods about the length of nat­ural TlVIV, as found by Fraenkel-Conrat andSinger. (2) TlVIV-RNA reconstituted at pH4.8, at least in the temperature range 30-60°,consists of numerous rods much shorter thanintact TMV, with very few full-length rods.(3) TYlVIV RNA preparations reconstitutedat pH 6.7-7.0 and 30° and examined by PTAstaining show a small proportion of rods (afew per cent) about the length expected forthe full RNA complement (ca. 290 A). Mostof the rods are substantially shorter thanthis. (4) TYMV RNA preparations recon­stituted at pH 4.8 also have a low proportion

of full-length rods. (5) Reconstituted TYMVRNA preparations examined by the spraydrop procedure show a lower proportion oflong rods than when examined by PTA stain­ing. Further work may show the TYMVRNA rods to have points of weakness thatare broken by the shear forces involved inthe spray drop procedure.

Figure 11 shows rods produced fromTYMV RNA and TIVrV protein at pH 6.7and 4.8. For comparison a TMV preparationused for making subunit protein is shown(Fig. l l A): Fig. llD shows rods produced atpH 4.8 in the absence of RNA. Under theseconditions long rods of indeterminate lengthwere formed, as others previously havefound.

Proportion of RNA. Very approximate esti­mations of the percentage of RNA in TYMVRNA preparations reconstituted at pH 6.7

HECONSTITUTION OF TThIV RNA WITH TMV PROTEIN 89

2.0_- - - - - - - - - - - - .....

1.0

1.5

QlUCoD

isen.0et

--~X__ ----x--'".;'

'"'",-,'"

0---"""""'--_...__....1:1

~ 40oc:Ql

~

~ 20

c;

.2

.~Vi 60c82!

"Mol or" rat io protein /RNA

FIG . 8 . Effect of increasing the ratio of pr oteinto RNA on reeoust.i t ut ion at pH 4.8. Samples wereincubated for 5 minutes a t GO° in 0.25 IH phosphatepH 4.8. 32P-1l1heled RNA at 50 ,ug/ml was mi xedwith TMV protein subu n its at vari ous conceutra- 0.5t ions . Fo r the " mola r" ra tio 1: 1 pr otein was at1 mg /ml, Recons ti tu ti on me asured by ribonucleaseresi stance. 0--0 = TMV RNA; X -----X =TYMVRNA.

0 ....- .....----"----....1.-.....280240

Stability to high er pH. TYMV RNA re­constituted with TMV protein remained sta­ble to ribonuclease attack at higher pHvalues. For example, a preparation was re­consti tuted at pH 4.8 and 30° for 24 hours,and isolated by two cycles of high speed sedi­mentation at pH 6.7. Aliquots were incu­bated in 0.05 M ph osphate or Tris buffer atvarious pH's, with 1 }.lg/ml ribonuclease for15 minutes at room temperature. Percent­ages of RNA resistant to ribonuclease wereas follows: pH 7.3 ; 74 %; pH 7.6, 74 % j pH7.9, 69 %; pH 8.5, 67 %; pH 8.9, 72 %.

260Wav elength (m,u.)

FIG. 10. Ultraviol et absorption spectrum ofrods formed from TYMV RNA and TMV proteinsubunits . A mix ture conta in ing TMV proteinsubunits at 1 mg /ml and TYMV RNA a t 50 1'g/mlin 0.25 iII phosphate pH 4.8 was incubated at 0°for 15 minu tes , t he n at 37° for 50 minutes. Re­conet ituted material was isolated by two cycles ofhi gh speed sedimentation at pH 6.7. Y ield ofsedimentable nucleoprotein was 13%. Absorptionmeasurements were made in 0.001 ivI phosphatebuffer, pH 6.7.

~ 40oCQl

~

~ 20

c.9

~Vi 60coue!

80

and pH 4.8 were made, using the known spe­cific radioactivity of the RNA and the ex­perimentally determined factor, A 260 = 3.3for a solution of TMV containing 1 mg/rnl,Values have ranged from 3 to 5 % RNA forvarious preparations.

0.01 0.03 0.1 0.3 1.0

Molority of buff er

FIG . 9. Effect of molari ty of phosphate at pH4.8 . TMV protein subunits at 1 mg/rnl and "P­lab eled RNA at 50 ug/rnl in phospha te , incubatedfor 5 minu tes at GO°. R econs ti t ution measured byrib on ucle ase resistance . 0--0 = TMV R NA;X-----X = TYMV RNA .

90 i\I.-\TTH E\VS

FIG. llA

F IG. usEro. 11. Elcctron mierogrnphs of rods neg atively stained with PTA . (A) A '1'MV pre para tio n used for

the m aking 'I'M\' preorein subunits . (B) Rods for mcd from 3' P- labclled TY MV RNA (50 Jlgjml) and T MVprote in su bu nit s (1 nig /rnl ) follo wing incu bation in 0.25 M phos phate buller pH G.7 for (i houra al. 30°.Yield of recons t it u t ed material was 4.1% based Oil J1NA res istunt to ribonu clease in rods scdimc nted for1 hour a t 38,000 rpm at p H n.7. T he prepurution con tained approximate ly ()% H.NA based on ubsorhancyand rud ioact ivi ty measurem ent s . (e ) Hods formed Irnm 32P-labelled TYMV RNA (50 j.lgj ml) and 'fMVprotei n sub uni ts (1 mgj ml) follow ing incub a tion in 0.25 M phosphat e p H 4.8 for 24 hours aL30°. Yield ofreconstitut ed mut.eri al wus 4.7(70 based on I1N A reaist nut to ribo nuclease in rods isolated by t wo cyclesof high speed se diment ution at pH (i.7. The preparation con tained approxima tely 5.5% ItN 11. based onab sorbaucy a nd rad ioactivity mcusurcmeuts . (D ) Rods formed from TMV pr otein subunits (1 mgj ml )wi thout HNA after incuba t.ion us for (C ). A sample of th e iucubutiou mixture was diluted a t pH 4.8M Id pl aced directly on th e grid .

HECONSTITUTION OF TYMV RNA WITH TM"PHOTEIN

FIG.llC

FIG. lID

Infecl7'vity ofl'econstituted TMV. The ex­periment summarized in Table 1 was de­signed to test whether the nucleoproteinmaterial obtained at pH 4.1:> had a higherinfectivity than free H.NA as found byFraenkel-Conrat and Singer (19,1)9) for Tl\fVreconstituted near pH 7.0. TJ\IV RNA re­constituted at pH G.7 had the greatly in­creased iufect.ivity and the high resistance to

ribonuclease expected from the results ofFraenkel-Conrat and Singer (1959). Al­though the yields of nucleoprotein werehigher under two conditions of incubation atpH 4.8, the material had only about the sameinfectivity as the free UNA used, and most ofthis infectivity was lost on treatment withribonuclease.

Infectivity of reconstituted 1'J!M V. The clif-

92 MATTHEWf;

TABLE 1

INFEC1'IVITY OF TMV RNA RECONSTITUTED UNDER VARlOUS CONDITIONS·

Number of local lesions perhalf-leaf

Treated withribonucleaseUntreated

Concentration(f-lg RNA/ml) ---------

Per centyield ofreconsti-

tutedmaterial

Preparation

TMV RNARNA reconstituted at p H G.7; 2 hours at 30°HNA reconstituted at pH 4.8 for 5 minutes at 60°RNA reconstituted at pH 4.8 2 hom'S at 37°Protein subunits alone at 28 jLg/ml

10145033

5.528957220.2

0.13218

1.22.5

o TMV protein subunits at 1 rng/rnl were incubated with TMV RNA at 50 Jlg/ml in 0.25 AI phosphateunder the conditions noted in the table. Reconstituted material was isolated by one cycle of high speedsedimentation at pH 6.7. Yields were estimated from absorbancy measurement.s on the redissolvedpellets. Infectivity was measured on half-leaves of N. qlutinosa (8-15 half-leaves per assay). Ribonu­clease digestion was carried out with 0.1 !J.g of enzyme pel' milliliter, pH G.7 0.01111 phosphate buffer,30 minutes at room temperature.

TABLE 2

INFECTIVITY OF TYMV RNA RECONSTI'I'UTED UNDER VARIOUS CONDITIONS

Per cent yield ofNumber of local .esions per

Concentrution half-leafPreparation reconstituted (Ilg RNA/ml)material Untreated Treated with

ribonuclease

Experiment A

TYMVRNA 30 58 0RNA reconstituted at pH 67 for 3.6 4.4 3.2 0

2 hours at 300

RNA reconstituted at pH 4.8 for 106 99 27 05 minutes at 60 0

RNA reconstituted at p'H 4.8 for 75 70 3.9 02 hours at 370

Experiment B

TYMV RNA 100 11.6 0RNA reconstituted at pH 6.7 for 31 B4 (95%)" 0 0

22 hours at 300

RNA reconstituted at pH 4.8 for 42 136 (80%) 0 022 hours at 30°

RNA reconstituted at pH 4.8 for 33 135 (70%) 13.6 0.1722 hours at 0°

a TMY protein subunits at 1 mg/rnl were incubated with TYMV UN A at 50 Ilg/ml in 0.25 M phosphateunder the conditions noted ill the table. Reconstituted material was isolated by one cycle of high speedsedimentation at pH G.7. Yields were estimated from absorbancy measurements on the redissolvedpellets. Infectivity was measured OIl chluesc cabbage (13-25 half-leaves per assay).

h Figures in parentheses are the percentage or RNA in the preparution resistant to ribonucleasedigestion at pH 6.7.

RECONSTITUTION OF TYMV RNA WITH TMV PROTEIN 93

ficulties in carrying out precise and repro­ducible infectivity assays with TYMV usingthe chlorotic local lesions produced inChinese cabbage are well known. They areclue to wide variability in individual plants,and in leaves on the same plant, in their re­sponse to infection. Many leaves may showno clear lesions although they are quiteheavily infected, Numerous attempts havebeen made to determine the effect of coatingwith TMV protein on the infectivity ofTYMV RNA. Details of three experimentswill be given.

In experiment A of Table 2 the three con­ditions of incubation tested were the same asthose used for TMV in Table 1. All prepara­tions contained some infectious material but

there was no increase in specific infectivityover the free RNA, and all infectivity waslost on treatment with ribonuclease. In ex­periment B ofTable 2 longer times of incuba­tion were tested. No infectivity remained inpreparations incubated at pH 6.7 or pH 4.8at 30° for 22 hours. The material producedduring incubation at pH 4.8 for 22 hours at0° had a specific infectivity about equal tothe free viral RNA. Nearly all this infectiv­ity was lost on treatment with ribonuclease.

The possibility that the infectivity of thereconstituted preparations was due to smallamounts of contaminating free viral RNAwas tested by subjecting the reconstitutedsample to sucrose density gradient fractiona­tion under concli tions where most of the re-

6

1.0

Q 0.8, \I ,

I \, 0I \ '", '", ,0.6 <:(

~ ~,I

,,, \, ,,~

0.4,, \I ,

P ,\, \ 0.2, ,, a...;j

2 3 4 5 6 7 8 9 10 II 12Fraction no.

p

1 5x

f;!o..

.; 4cEQj0-

-E 3::>o

U

2

Fro. 12. Sucrose density fractionation of TYlVIV ENA reconstituted at pH 4.8 and of untreatedTYMV RNA. A mixture containing TMV protein subuni ts at 1.3 mg/ml and 32P-labeled TYMV RNA at50 ,ug/ml ill 0.25 M phosphate pH 4.8 was incubated for 22 hours at 0°. Reconstituted RNA W83 isolatedby two cycles of high speed sedimentation at pH 6.7. Of the RNA in the preparation, 70% was resistantto ribonuclease digestion. Yield of ribonuclease resistant RNA was 7%. The reconstituted material wasfractionated on a sucrose density gradient as described under Materials and Methods. Untreated TYMVHNA was sedimented in a second tube. 0-----0 A2&o of TYMV RNA; .--. = total counts pCI'minute in reconstituted sample; X--X = ribonuclease resistant counts pel' minute in reconstitutedsample.

94 MATTHEWS

constituted material would be in the pellet,while free viral RNA would sediment abouthalf way down the gradient. Such an experi­ment is illustrated in Fig. 12.

There was no indication of any contamina­tion of the reconstituted rods with free intactviral RNA. Of the RNA in the pellet 84 %was resistant to ribonuclease as judged byinsolubility in 5 % trichloracetic acid follow­ing treatment with the enzyme. The pelletedmaterial was inoculated (at an RNA concen­tration of 26 ,ltg/ml) to 14 half-leaves ofChinese cabbage, and gave an average of 1.0local lesion per leaf. Another sample treatedwith ribonuclease gave an average of 0.14lesion per leaf.

In another sucrose gradient experimentlike that of Fig. 12, the pelleted material in­oculated at 22 flg RNA/ml gave 0.17 lesionper half-leaf (mean of 18 leaves). Treatedwith ribonuclease it gave 0.06. The un­treated TYMV RNA inoculated at 30 t-tg/rnlgave 6.8 lesions per half-leaf (mean of 18leaves). After treatment with ribonuclease itgave no lesions.

DISCUSSION

The appearance in mixtures of TYlVIVRNA and TMV protein subunits incubatedat pH values near 7.0 of sedimentable nucleo­protein consisting of rods, and in which mostof the RNA is resistant to ribonucleasedigestion, demonstrate that TYlVIV RNAcan be coated by TlVIV protein to producerod-shaped particles. Reconstitution at pH4.8 is potentially complicated by the factthat the protein subunits aggregate spon­taneously to form RNA-free rods at this pHand that such rods aggregate to form visibleprecipitates which could nonspecific ally trapand protect the TYiVIV RNA. Neverthelessformation of stable nucleoprotein rods doesoccur at this pH since the RNA in the prod­uct isolated by high speed sedimentation atpH 6.7 is stable to ribonuclease digestion atpH values up to 8.9. Furthermore such R.NAhas a sedimentation rate at pH 7.3 verymuch greater than the original viral RNA(Fig. 12).

On incubation at pH 4.8, TiVIV proteinalone forms rods of indeterminate length,and many of these are very long (Fig. llD).In contrast when TY1VIV RNA is present

under the same conditions at pH 4.8, theaverage rocllength is quite short (Figs. He),suggesting that the RNA may control rodlength at pH 4.8 as it does near pH 7.0.Lauffer (1962) has shown that at pH valuesnear 4.8 the spontaneous aggregation of theprotein subunits to form rods in the absenceof RNA is a reversible process. Stackingaround an RNA molecule presumably givesa more stable structure, ancl this process maybe assisted by the tendency of the subunits toaggregate spontaneously.

It is unfortunate that we do not yet havereliable size distributions for TYl\IIV RNAand TlVlV RNA reconstituted under variousconditions. Measurements on PTA-stainedpreparations placed directly on the grid maynot be representative. On the other hand, wehave evidence suggesting that the spray dropprocedure as we have used it may lead tofracture of many of the reconstituted TYl\IVRNA rods. Nevertheless with the incubationconditions tested so far, only TMV RNA atpH values near 7.0 gives a high proportion offull-length rods. With TYlVlV RNA at pH'sneal' 7.0, incubation times must be severalhours at 30° to give significant yields. Thisgives opportunity for traces of nuclease inthe incubation mixture to partially degradethe R~A.

There is another factor that may be oper­ating for TYJVIV RNA at pH 7.0 and forboth RNA's at pH 4.8. Witz et al. (1965)from a study of small-angle X-ray scatteringof RNA in solution at pH 6.8 concluded thatunder these conditions both TMV RNA andTYJVIV RNA exist as slightly flexible mole­cules possessing rodlike base-paired segmentsjoined end to encl. TYMV RNA had a morecompact structure than TMV RNA. ThusRNA under the conditions of incubation forreconstitution is partly in an internally base­paired state, alternating with single-strandedregions. Under these conditions coating withprotein subunits may proceed simultane­ously in more than one region of the moleculeon single-stranded regions. Base-paired re­gions may slow the rate of, or create a blockto, further reconstitution. Forces acting onrigid reconstituted sections may disrupt thethe RNA chain.

Another factor increasing the proportionof short rods with TYMV RNA may be the

RECONSTITUTION OF TYMV RNA WITH TMV PROTEIN 95

inherent instability of this RNA within itsprotein shell (Hase lkorn, 1962 j Kaper andHalperin, 1965 ; Matthews and Ralph, 1966).RNA preparations, unless made from freshlyisolate d virus from recently infected plants,may contain a significant proportion ofbreaks even before the incubation for re­constitution.

Further testing of conditions for reconsti­tution at pH 4.8 and 0°, particularly of thenature and concentration of ions present,may give improved conditions for the recon­stitutionof "foreign" RKA. The low tempera ­ture should minimize nuclease attack.

Contaminating nuclease in the incubat ionmixtures is unlikely to come from the RNAsince viral RNA isolated by the procedureused retains undiminished infectivity afterincubation alone at 30° for ma ny hours (A.R. Bellamy, personal communication) . It ismuch more likely to be a contaminant of theprotein subunit preparations. T he nucleasemost likely to contaminate TMV proteinsubunit preparations, at least those that arefreshly made from fresh virus, is t he tobaccoleaf ribonuclease described by Fri sch-Nigge­meyer and Reddi (19,">7). This enzyme wassome 5 times more active at pH 5.0 th an atpH 7.0, and in citrate-phosphate buffer thepH optimum was close to pH 4.8. Theenzyme lost only 3 % of its activity on heat­ing at pH 4.5 for 10 minutes at 60°. This en­hanced nuclease activity at pH '1.8 andhigher temperatures may well be the majorreason for the short reconstituted rods pro­duced under these conditions.

The number of local lesions obtained so farhave been too low to establish whether ornot TYl\IV RNA fully protected by Tl\1Vprotein from ribonuclease action is stillinfectious for Chinese cabbage. Most if notall the infectivity in the reconsti tutedTYl\lV rods is probably due to completeRNA complements partially coated withprotein. Sander and Schramm (1963) havedescribed a strain of TY:.\IV which infectstobacco with the production of chlorotic locallesions. It would be of some interest to testthe infectivity for tobacco and Chinese cab­huge of the RNA from such a strai n coatedwith TMV protein.

If the RNA of other rod-shaped plantviruses has a more open structure in solut ion

than TYMV RNA, it may be that they willreconstitute more readily than TYMV R NAwith T.MV protein. Several such viruses canmultiply in mixed infect ions with T MV intobacco (e.g., potato virus X) . The possi­bility may then exist for some degree of mis­coating or " phenotypic mixing" under in vivocondi tions,

It is unlikely that many message RNA'sdiffer more widely in base composition fromTl\IV RKA than TYl\1V RKA, It may thusbe possible to use TMV protein subunits toprepare cellular message R NA in a biologi­cally active state but protected from nu­clease attack. I n preliminary experiments atpH 4.8 we have obtained reconsti tut ed rodsin low yield from calf spleen nucleic acids.Naked R NA injected into an int act animalis almost certainly rap idly degraded by nu­cleases. In pre liminary experiments withTYld V RNA in mice I'll' have found(Matthews and .J. Marbrook, unpublishedobservations) that 32P-labeled TYlVIV RNAcoated with Tl\'lV protein has a markedlydifferent distribution in the animal followingfoot pad injection than does free RNA, andthat preimmunization of the mice with TMVfurther changes thi s distribut ion.

ACKNOWLEDGMENTS

T his work was supported in part by USPHSgrant AI-04973 .

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FRAE NK E L·C o NRA T , II., and SINGER, E ., (1959) .R econst it ution of tobacco mosaic v irus. I II .Improved methods and the use of mixed n ucleicacids. Biochim , Biophys. Ac/a 33, 359-370.

FRAENKEL-CONRAT, H ., and SINGER, E ., (19M).Re constitution of tob acco mosaic virus . IV .Inhibit ion by en zymes and other proteins, anduse of polynucleotides. Vi1'ology 23, 354-362.

FRAENKEL-CoNRAT, I-1., and \VILLIAMS, R . C . ,

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96 MATTHEWS

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