Reconstitution of turnip yellow mosaic virus RNA with TMV protein subunits

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 phosphate buffer at pH values near 7.0 at 30° for several hours, some of the RNA is coated with protein 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 than obtained with TMV RNA near pH 7.0. The reconstitution reaction (as judged by chemical criteria) has a second pH optimum near pH 4.8 for both TYMV RNA and TMV RNA. At this pH, the reaction is much more rapid and higher yields of coated RNA are obtained. However, as judged by infectivity of TMV, very few complete rods are formed at this pH. TYMV RNA coated with TMV protein at pH 6.7 or pH '1.8 and isolated by high speed sedimentation may retain some infectivity for Chinese cabbage, but all, or almost all, this infectivity is lost on treatment with ribonuclease. The infectivity in the reconstituted preparations probably resides in particles consisting of an intact TYMV RNA complement partially coated with TMV protein. INTRODUCTION

Fraenkel-Conrat and Williams (1955) showed that infectious tobacco mosaic virus (TMV) rods could be reconstituted from T1VIV RNA and the viral protein subunits. Hart and Smith (1956) obtained evidence suggesting that rods could be formed using TMV protein and yeast RNA or various synthetic polyribonucleotides. FraenkelConrat and Singer (1964) obtained reconstitution with certain artificial polymers, but not with RNA from wheat germ or ascites cells. Holoubek (1962) found that the RNA from various strains of TJ'vIV could be reconstituted into infectious rods with protein subunits from a type strain. In his early work Fraenkel-Conrat used the term reconstitution in a strict sense, for the in vitro formation of infectious TlVlV rods. For convenience the term will be used here for the formation of rods of TMV protein containing RNA, without necessarily implying biological activity. In the work described below it has been found that under 82

appropriate conditions of incubation near pH 7.0 or pH 4.8 TMV protein subunits will coat turnip yellow mosaic virus (TYMV) RNA to form rods. A low level of infectivity for Chinese cabbage may be retained by such rods. Most of the TYMV RNA is not completely coated in such preparations since all or 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 cabbage iBraeeica pekinensis (Lour.) Rupr. val'. Wong Bok). TMV was isolated by three cycles of high speed sedimentation from 0.1 M phosphate buffer pH 7.2 containing 0.2111 ethylenediaminetetraacetic 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

purified viruses by a modification of the phenol procedure of Ralph and Bellamy (1964). Virus in 0.01 M phosphate buffer pH 7.2 containing 0.02 111 EDTA was extracted twice with 3 volumes of water-saturated phenol containing 0.1 % 8-hydroxyquinoline. Phenol was removed with ether, and the RNA was precipitated as the cetyltrimethylammonium salt. This was converted to the sodium salt in 70 % ethanol and taken to a dry powder as described by Ralph and Bellamy (1964). RNA was stored in a desiccator over phosphorous pentoxide. TMV protein. TMV protein subunits were isolated by the acetic acid method of Fraenkel-Conrat (1957) from freshly prepared TMV and stored in 0.01 MpH 6.7 phosphate buffer at 2°. Radiochemical methods. 32P-Iabeled virus was prepared as described previously (Matthews, 1960). Radioactivity was measured on planchettes using Millipore-type filters and a Phillips thin-end window ONI tube (PM 18515). Spectrophotometry. Ultraviolet absorption measurements were made with a Zeiss PMQ II spectrophotometer using cells of J-cm path length. Suctose density gradient fractionation. Samples were layered over a 5-20 % linear sucrose gradient. The sucrose contained 0.14 M NaCI and 0.01 M phosphate buffer pH 7.3. The gradients were then centrifuged for 4 hours at 35,000 rpm in a Spinco SW 39 rotor at about 4°. Samples were collected by a drip out procedure. Assay for reconetiuuion. To detect and estimate reconstitution three methods were used: (a) Sedimentation for 1 hour at 3.5,000 rpm at pH 6.7-7.3 followed by ultraviolet absorption measurements on the redissolved pellets, as used by Fraenkel-Conrat and Singer (1964). After incubations near pH 7.0 for several hours at 300 virtually no free RNA remained sedimentable, However, after short incubations near pH 4.8 some free UNA may sediment. For this reason mixtures incubated at pH 4.8 were sometimes treated with pancreatic ribonuclease at pH 6.7 (1-2 J-lgjml, 20 minutes at room temperature) before isolation of any reconstituted rods. (b) Electron microscopy. In some experiments solutions containing re-

83

constituted material were mixed with an equal volume of phosphotungstic acid (2.0 % at pH 6.7) and the mixture was placed on carbon films, dried, and examined. In others a standard solution of polystyrene latex particles was mixed with the reconstituted material, and the mixture was sprayed onto grids. After shadowing with palladiumplatinum, appropriate small drops were photographed for estimating size distribution of any rods present. (c) Resistance to ribonuclease. RNA coated with TlVIV protein subunits is resistant to attack by ribonuclease. We used the increase in resistance to ribonuclease as a rapid method for assay of reconstitution. Aliquots of the incubation mixture containing 32P-Iabeled RNA were diluted in saline or buffer and incubated at pH 6.6-7.3 with ribonuclease (2 J-lg/ml) for about 20 minutes at room temperature. Carrier protein (0.5 mg per sample) and trichloroacetic acid to a final concentration of 5 % were then added. The sample was Millipore-filtered and washed with 5 % trichloroacetic acid and the radioactivity was measured. Under these conditions about 1.4 % of the radioactivity in free TMV RNA and 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 the results reported. In most experiments these quantities would not have been significantly above background in the samples measured. Infectivity assays. Infectivity of TMV preparations 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 the resistance of infectivity to ribonuclease, samples in 0.01 M phosphate buffer, pH 6.7, were treated with the enzyme at 0.1 J.lg/ml for 20-30 minutes at room temperature, RESULTS Heconeiitnuiot: with TY MV RNA near pH 7.3

As a starting point, the conditions that Fraenkel-Conrat and Singer (1959, 1964) had found to be optimal for reconstitution with TlVIV RNA were used. U nless otherwise stated 1 mg TMV protein subunits and .50 p.g of RNA was used per milliliter in all experiments.

84

MATTHEWS

Effect of teniperoiure. F igur e 1 sh ows results of an experiment t o determine the temperature optimum for reconstit ution with TYMV R NA at pH 7.3 . All three methods noted above were used t o detect reconsti tuton, Aliquot s of the incubati on mixtures were used to determine the proportion of R NA tha t h ad become resist an t to ribonuclease. The bulk of each incubation mixtur e was cen t rifuged at 38,000 rpm for 1 hour in a Spineo No. 40 rotor . Any pelleted mate rial was redissolved in buffer at pH 7.3 and centriguged a second time. Yields of sediment able nucleoprot ein followed closely the p attern shown for ribonuclease resistance (Fig . 1). Examination of samples by electron microscopy revealed rods (mostly short) only in the 24°, 30°, and 40° sam ples. Protein subunits incubated at pH 7.3 and 30° without R NA prod u ced no rod s. I t was conclude d tha t TlVIV protein subuni ts can pack around T Y i\IV R NA to form rods and t hat the t emperature optimum for t he process at pH 7.3 is close t o 30°, as found for T MV RN A by Fraenkel-Conrat a nd Singer (1959) . 3.0

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Temperature FlO. 1. Temperature optimum for reconst.it ution at pH 7.3. Aliquots of u mixture contain ing per m illilitre 1 m.g TMV pr ote in subuni ts and 50 I'g of ~2P -label e d TTIvIV RNA in 0.1 M ph osphate buffer were in cubate d a t vari ous te mperatures for 6 ho urs. P ercentage of reconstitution was est ima ted by ribo n uclease resis t anc e (X ----- X ) and by yield of sedimen ta ble nucleoprot ein


50

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cf.

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0.01 Molarity

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buffer

F IG. 2. Effect of m olarity of phosp h ate b uffer on re cons t i tu tion at pH 7.3. The mix lure COli t ai ued TMV p ro tein sub unit s at 1 mg /rnl and UP-labeled R)\ A at 50 pg/ml; it was inc ub at ed for 24 ho urs a t 30 0 • Reconstit ut ion was estima ted by ribonu clease resistance . 0 --0 = TM V R NA; X -·· -- X = T Yi\IV RNA.

M olal'l:ty of buffer. Figure 2 sh ows the marked dependence of reconsti tution of T YMV R NA on molarity of the pH 7.3 phosphat e buffer used, as found by F raenkelConrat and Singer (1964) for T l\>fV. In our tests virtu ally no reconstitut ion occurred for eit her T YMV or T MV RNA at 0.033 M or below . Time cow'se of reconstitu tion. F igure 3 shows t he increase in reconst itut ion of TY1VIV RNA with time. After () hours a t 30° and pH 7.3 in 0.1 111 phosphate buffer some 4 o/cl of the RNA had become resistant t o ribonuclease. Figure 3 illustrates a m aj or difficulty with TYl,IV RKA under t hese conditions. More and more of the R N A not coate d with Tl\IV protein becomes so fur degraded that it is no longer insoluble in 5 % trichloroacetic acid. This is presumably due t o traces of nuclease present in t he ine ubation mixt ure, E:O'ecl of pH. Figure 4 shows tha t in the range pH 5.0-8.0 the opt imum for reconsti tution of T YMV R NA is neal' pH 7.0 , as it is for T l\IV. In va rious experiments with T YMV R N A using different batches of mat erials the pH

85

RECONSTITUTION OF TYMV RNA WITH TMV PROTEIN

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70

60 c

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co(J

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Hours of incubohon

FIG. 3. Time course of rec oustitution and RNA breakdown at pH 7.3. The mix tu re con tained TMV pr otein subunits at 1 mg /ml and siP-labeled TYMV RNA a t 50 /Lg/ml it, was in cuba t ed in 0.1 ill phosphate hulf'er at 30°. X -----X = per cen tage of RNA r endered r ibnllllel eas e res is tnn t. , 0 - -0 '= percentage RNA rend ered so luble in 5% tr ichlor oacetic acid.

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op timum varied some what and frequently wa s as low as pH 6.7. Y ields. Under optimum couditions at pH 6.7-7.3 (0.2-0.2G JlI phos pha te buff er , at 300 for 0-24 hours) th e yields of reconsti t uted m ateri al have v aried widely, rungi ng from abou t If} to 50 (1,) for T MV UN A a nd 1 to 30 % for T Y i\lV UNA .

6 .0

7.0

6.5

7.5

8.0

ph

F IG. .1. Effect of pH 0 11 recons ti tut.i ou near pH 7.0 TMV. T ile mixtu re cont ained p rotein sub uni ts a t 1.0 rng yml and 82p -lah el ed RNA at 50 Jig/mI., it was in cub a ted for 23 hours a t. 30° in 0 .25 M phosphate buffer. Recona titu ti ou was estimated by rib ouuolease r esistance. 0 - - 0 TMV RNA; X ----- X = TYMV RNA.

R econsntuiion. neal' pH 1,.8. Unless otherwise stated T lVIV protein subunit s wer e used at 1 mg /ml and RNA at 50,ug/ml. EjJect oj pH. The lower pH values tested in the experiment of Fig. 4 suggested that there might be a second, lower pH optimum at least for TYlVIV. The experim ent to test this showed that the re was a second pH optimum near 4 .5-5.0 for both TlVIV RKA and TYMV RNA (F ig. 5), as judged by increased ribonuclease resis tan ce of the R NA. In th is experiment aliq uots of the incub ation mixture were dilu ted in sa line and incuba ted with ribonuclease. Subsequ en t t ests showed t hat if the pH of th e samples was brought to 6.7 before ribo nu clease tre atm en t ab out one-third of th e apparent ly ribonucleaseresistan t RNA be cam e suscep tibl e t o digestion . It is well-known that at these lower pH

values T MV protein sub units aggrega t e to form rod s of indeterminate length and that t~le.se rod s aggre gat e to form visible precipitates, The RNA protected from ribonuclease attack at pH 4.5-5.0 but not at pH 6.7 was probably trapped nonspecifically in the aggregates of rod s formed a t the low er pH, and thus given prot ection from rib onuclease at tack. To avoid t his complicat ion in furt h er experiments aliquots of sam ples incubat ed in the low pH range were brought to pH 6.7- 7.0 before being tested for resistance to ribonuclease. Temperature op timum at pH 4.8. The experiment summarised in Fig. 6 sh ows t hat t he t emperature optimum for re consti tutio n as judge d by resistance of the RNA t o ribonucl ease, is ab out 5.5- 50° for b oth T l\'[V R NA and TYMV R NA . The differenee in

86

MATTHEWS

80

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FIG. 5. Effect of pH on reconstitution near pH 5.0. Conditions as for experiment of Fig. 4 except for pH. 0 - 0 TMV RNA; X-----X TYMV RNA.

yield between TYMV RNA and TIVIV is much less at pH 4.8 and GO° than at pH values near 7.0 and 30°. Ti'llw course at pH 4.8. The experiment of Fig. 7 shows that the rate of increase in resistance to ribonuclease is very much more rapid at pH 4.8 and 60° than at pH 7.0 and 30°. The reaction was essentially complete for both TlVIV RNA and TYMV RNA after 5 minutes' incubation. Effect of ratio of protein s'ubunits to RNA at pH 4.8. The experiment of Fig. 8 shows that at pH 4.8, using an incubation period of 5 minutes at 60°, doubling the amount of protein subunits in the mixture over the standard "molar" ratio increased the amount of RNA protected from ribonuclease attack. Increasing the "molar" ratio of protein to RNA above 2: 1 gave no further increase. Ellect of phoephatemolariui at pH 4.8. The effect of increasing molarity of phosphate in the incubation medium on reconstitution at

pH 4.8 (Fig. 9) was generally similar to the effect at pH 7.3 (Fig. 2), However, some reconstitution took place at pH 4.8 at 0.01 and 0.03 M whereas none occurred at these molarities at pH 7.3. Yields at pH 4.8. Yields of reconstituted material in repeated experiments under the same conditions at pH 4.8 varied quite widely, as they do at pH values neal' 7.0. However, as shown in Figs. 6-9, the yields of reconstituted material both for TMV RNA and TYlVIV RNA are much higher at pH 4.8 than at pH values near 7.0. In these experiments reconstitution was measured by the ribonuclease resistance at pH 6.7 of aliquots taken from the incubation mixtures. Yielcls based on amount of nucleoprotein material isolated by two cycles of high speed sedimentation at pH 6.7 (1 hour at 38,000 rpm) were always lower. They have ranged in various experiments from about 15 to 60 % of that expected from ribonuclease resistance assays on aliquots of the incubation mixture. The lower yield was probably due in part at least to the high proportion of very short rods formed in incubation mixtures at pH 4.8, much of this material being left in the supernatant fluids. A second centrifugation of the supernatant fluid gave a further yield of nucleoprotein containing RNA resistant to ribonuclease. Properties of the Product Produced by Reconstitution. of T111V RNA and TY 111 V RNA with 'l'MV Protein Subunits

For examination of various properties, reconstituted material was isolated by high speed sedimentation from the incubation mixture. For material incubated near pH 7,0, reconstituted rods were sedimented at 38,000 rpm for 1 hour in a Spinco No. 40 rotor. The water-clear pellets were resuspended in 0.01 JI{ phosphate buffer at pH 6.7-7.0 and resedimented under the same conditions. The final pellets were taken up in a small volume of the same buffer. Incubation mixtures at pH 4.8 were first sedimented at 30,000 rpm for .5 minutes. Because of the gross aggregation at this pH all reconstituted material was sedimented under these conclitions. The pellets were then taken up in 0.01 111 phosphate buffer pH 6.7 and the reconstituted rods were sedimented twice as for

RECONSTITUTION OF TiNIY RAN WITH TMV PROTEIN

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

material in cubated near pH 7.0 . This pro cedure gave v ariable but low yields of mat erial with a protein-like UV absorption sp ectrum when RNA was omitted from the incubation mixt ure. In some experiments, the pellets resusp ended aft er 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 not coated with protein. UV absorption spectrum . Tl\ IV RNA reconstituted at pH value s neal' 7.0 had a UV absorption spect r um indist inguishable from the parent Tl\fV. TYl\IV RNA recoustituted neal' pH 7.0 or at pH 4.8 and isolated as des cribed abov e had a UV absorption spectrum wit h a peak or plat eau in the region 2Gii-270 m u , A typical spe ctrum is shown 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 they probably include (1) the difference in base composition of T lvIV RNA and T YM V RNA, the lat ter having a much h igher cytidylic acid content; (2) differences in and variability in the length distribu t ion of t he TYiVIV RNA rods, leading to differences in t he cont ribut ion of light scattering to the absorption spectrum ; and (3) variation in t he content of RNA in the TYJ.\IV R NA rod prepar ations. Size distribution. TY:.\lV RNA is ve ry similar in length t o T?dV R NA , so complete rods formed by TYl\lV RNA and TMV pro tein would be expected to be about the same length as TMV. Precise data concerning th e size dist rib ution of T MV R NA and TYl\IV RNA reconstituted under various condit ions has not yet been obtained. H owever , a number of pr eparations, isolated as

88

MATTHEWS 100. .

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Time of incubation (minutes)

FIG. 7. Time course of reconstitution at pH 4.8 ancl60°. Mixture contained TMV protein subunits at 1 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 the of full-length rods. (5) Reconstituted TYMV electron microscope using PTA staining or RNA preparations examined by the spray sprayed droplets followed by metal shadow- drop procedure show a lower proportion of ing. From these observations the following long rods than when examined by PTA staingeneral statements can be made: (1) TMV ing. Further work may show the TYMV RNA reconstituted near pH 7.0 gives a high RNA rods to have points of weakness that proportion of rods about the length of nat- are broken by the shear forces involved in ural TlVIV, as found by Fraenkel-Conrat and the spray drop procedure. Singer. (2) TlVIV-RNA reconstituted at pH Figure 11 shows rods produced from 4.8, at least in the temperature range 30-60°, TYMV RNA and TIVrV protein at pH 6.7 consists of numerous rods much shorter than and 4.8. For comparison a TMV preparation intact TMV, with very few full-length rods. used for making subunit protein is shown (3) TYlVIV RNA preparations reconstituted (Fig. l l A): Fig. llD shows rods produced at at pH 6.7-7.0 and 30° and examined by PTA pH 4.8 in the absence of RNA. Under these staining show a small proportion of rods (a conditions long rods of indeterminate length few per cent) about the length expected for were formed, as others previously have the full RNA complement (ca. 290 A). Most found. of the rods are substantially shorter than Proportion of RNA. Very approximate estithis. (4) TYMV RNA preparations recon- mations of the percentage of RNA in TYMV stituted at pH 4.8 also have a low proportion RNA preparations reconstituted at pH 6.7

89

HECONSTITUTION OF TThIV RNA WITH TMV PROTEIN

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FIG . 8 . Effect of inc reasing the ratio of pr otein to RNA on ree oust.i t ut ion at pH 4.8. Samples were incubated for 5 minutes a t GO° in 0.25 IH ph osphate pH 4.8. 32P-1l1heled RNA at 50 ,ug/ml was mi xed with TMV pro tein subu n its at vari ous conceutrat ions . Fo r the " mo la r" ra tio 1: 1 pr otein was at 1 mg /ml, Recons ti tu ti on me asured by ribonuclea se resi stance. 0--0 = TMV RNA ; X ----- X = TYMVRNA .

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FIG . 10. Ultraviol et absorpti on spectrum of r ods formed from TYMV RNA and TMV pro tein s ubunits . A mix ture conta in ing TMV prot ein subunits at 1 mg /ml and TYMV RNA a t 50 1'g/ml in 0.25 iII pho sphate pH 4.8 was incubated at 0° for 15 m inu tes , t he n at 37° for 50 minutes. Reconet it uted material was isolated by two cycles of hi gh speed sedimentation at pH 6.7. Y ield of sedimentable nucleoprotein was 13%. Absorption measurements were made in 0.001 ivI phosphate buffer, pH 6.7.

Molority of buff er

FIG . 9. Effect of molari ty of phosphate at pH 4.8 . TMV protein subunits at 1 mg/rnl and "Plab eled RN A at 50 ug/rnl in phospha te , incubated for 5 minu t es at GO°. R econs ti t utio n measured by rib on ucle ase resistance . 0 - - 0 = TM V R NA; X--- --X = TYMV R NA .

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

Stability to high er pH. TYMV RNA reconstituted with TMV protein remained stable to ribonucl eas e attack at higher pH v alues. For exam ple, a preparation wa s 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 incub ated in 0.05 M ph osphate or Tris buffer at various pH's, with 1 }.lg/ml ribonuclease for 15 minutes at room temperature. Percentages of RNA resistant to ribonuclease were as follows: pH 7.3 ; 74 %; pH 7.6, 74 % j pH 7.9, 69 %; pH 8.5, 67 %; pH 8.9, 72 %.

90

i\I.-\TTH E\VS

FIG. llA

F IG. us Ero. 11. Elcctron mier og rnphs of rod s neg atively stained with PTA . (A) A '1'M V pre para tio n used for the m aking 'I'M \' preorein s ub u nits . (B) Rods for mcd from 3' P- labclled TY MV RN A (50 Jlgjml) and T MV prote in su b u nit s (1 nig /rnl ) follo win g incu bation in 0.25 M phos phate buller pH G.7 for (i houra al. 30°. Yield of reco ns t it u t ed mate rial was 4.1% based Oil J1NA res istunt to ri bonu clease in rods scdi mc nted for 1 hour a t 38,000 rpm at p H n.7. T he prepurution con t ained ap pro ximate ly ()% H.NA based on ubsor hancy and rud ioact ivi ty measur em ent s . (e ) Hods formed Irnm 32P -labelled TYMV R NA (50 j.lgj ml) and 'fMV protei n s ub u ni ts (1 mgj ml) follow ing incub a tion in 0.25 M phosphat e p H 4.8 for 24 hours aL30°. Y ield of r econstitut ed mu t.eri al wus 4.7(70 based on I1N A reaist nut to ri bo nucleas e in ro ds isolated by t wo cycles of high speed se di me nt ution at pH (i.7. The prep aration con tained appro xima tely 5.5% ItN 11. b ased on ab s orba ucy a nd rad ioactivity mcusurcmeuts . (D ) Rods formed from TMV pr otein subunits (1 mgj ml ) wi thout HN A after incuba t.ion us for (C ). A sam ple of th e iucubutiou mixture was d iluted a t pH 4.8 M I d pl a ced 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 nucleoprotein material obtained at pH 4.1:> had a higher infectivity than free H.NA as found by Fraenkel-Conrat and Singer (19,1)9) for Tl\fV reconstituted 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 of Fraenkel-Conrat and Singer (1959). Although the yields of nucleoprotein were higher under two conditions of incubation at pH 4.8, the material had only about the same infectivity as the free UNA used, and most of this infectivity was lost on treatment with ribonuclease. Infectivity of reconstituted 1'J!M V. The clif-

92

MATTHEWf; TABLE 1

INFEC1'IVITY OF TMV RNA RECONSTITUTED

UNDER

Per cent yield of reconstituted material

Preparation

VARlOUS CONDITIONS·

Number of local lesions per half-leaf Concentration (f-lg RNA/ml) - - - - - - - - with Untreated Treated ribonuclease 10

TMV RNA RNA 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

14 50 33

0.13 218 1.2 2.5

5.5 289 57 22 0.2

o TMV protein subunits at 1 rng/rnl were incubated with TMV RNA at 50 Jlg/ml in 0.25 AI phosphate under the conditions noted in the table. Reconstituted material was isolated by one cycle of high speed sedimentation at pH 6.7. Yields were estimated from absorbancy measurement.s on the redissolved pellets. 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

Preparation

Per cent yield of reconstituted material

Concentrution (Ilg RNA/ml)

Number of local .esions per half-leaf Untreated

Treated with ribonuclease

Experiment A TYMVRNA RNA reconstituted at pH 67 for 2 hours at 30 0 RNA reconstituted at pH 4.8 for 5 minutes at 60 0 RNA reconstituted at p'H 4.8 for 2 hours at 37 0

30 4.4

58 3.2

0 0

106

99

27

0

75

70

3.6

3.9

0

11.6 0

0 0

Experiment B TYMV RNA RNA reconstituted at pH 6.7 for 22 hours at 30 0 RNA reconstituted at pH 4.8 for 22 hours at 30° RNA reconstituted at pH 4.8 for 22 hours at 0°

31

100 B4 (95%)"

42

136 (80%)

0

33

135 (70%)

13.6

0 0.17

a TMY protein subunits at 1 mg/rnl were incubated with TYMV UN A at 50 Ilg/ml in 0.25 M phosphate under the conditions noted ill the table. Reconstituted material was isolated by one cycle of high speed sedimentation at pH G.7. Yields were estimated from absorbancy measurements on the redissolved pellets. 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 ribonuclease digestion at pH 6.7.

RECONSTITUTION OF TYMV RNA WITH TMV PROTEIN

ficulties in carrying out precise and reproducible infectivity assays with TYMV using the chlorotic local lesions produced in Chinese cabbage are well known. They are clue to wide variability in individual plants, and in leaves on the same plant, in their response to infection. Many leaves may show no clear lesions although they are quite heavily infected, Numerous attempts have been made to determine the effect of coating with TMV protein on the infectivity of TYMV RNA. Details of three experiments will be given. In experiment A of Table 2 the three conditions of incubation tested were the same as those used for TMV in Table 1. All preparations contained some infectious material but

93

there was no increase in specific infectivity over the free RNA, and all infectivity was lost on treatment with ribonuclease. In experiment B of Table 2 longer times of incubation were tested. No infectivity remained in preparations incubated at pH 6.7 or pH 4.8 at 30° for 22 hours. The material produced during incubation at pH 4.8 for 22 hours at 0° had a specific infectivity about equal to the free viral RNA. Nearly all this infectivity was lost on treatment with ribonuclease. The possibility that the infectivity of the reconstituted preparations was due to small amounts of contaminating free viral RNA was tested by subjecting the reconstituted sample to sucrose density gradient fractionation under concli tions where most of the re-

6

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U

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2

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0.6

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12

Fro. 12. Sucrose density fractionation of TYlVIV ENA reconstituted at pH 4.8 and of untreated TYMV RNA. A mixture containing TMV protein subuni ts at 1.3 mg/ml and 32P-labeled TYMV RNA at 50 ,ug/ml ill 0.25 M phosphate pH 4.8 was incubated for 22 hours at 0°. Reconstituted RNA W83 isolated by two cycles of high speed sedimentation at pH 6.7. Of the RNA in the preparation, 70% was resistant to ribonuclease digestion. Yield of ribonuclease resistant RNA was 7%. The reconstituted material was fractionated on a sucrose density gradient as described under Materials and Methods. Untreated TYMV HNA 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 reconstituted sample.

94

MATTHEWS

constituted material would be in the pellet, under the same conditions at pH 4.8, the while free viral RNA would sediment about average rocllength is quite short (Figs. He), half way down the gradient. Such an experi- suggesting that the RNA may control rod length at pH 4.8 as it does near pH 7.0. ment is illustrated in Fig. 12. There was no indication of any contamina- Lauffer (1962) has shown that at pH values tion of the reconstituted rods with free intact near 4.8 the spontaneous aggregation of the viral RNA. Of the RNA in the pellet 84 % protein subunits to form rods in the absence was resistant to ribonuclease as judged by of RNA is a reversible process. Stacking insolubility in 5 % trichloracetic acid follow- around an RNA molecule presumably gives ing treatment with the enzyme. The pelleted a more stable structure, ancl this process may material was inoculated (at an RNA concen- be assisted by the tendency of the subunits to tration of 26 ,ltg/ml) to 14 half-leaves of aggregate spontaneously. Chinese cabbage, and gave an average of 1.0 It is unfortunate that we do not yet have local lesion per leaf. Another sample treated reliable size distributions for TYl\IIV RNA with ribonuclease gave an average of 0.14 and TlVlV RNA reconstituted under various conditions. Measurements on PTA-stained lesion per leaf. In another sucrose gradient experiment preparations placed directly on the grid may like that of Fig. 12, the pelleted material in- not be representative. On the other hand, we oculated at 22 flg RNA/ml gave 0.17 lesion have evidence suggesting that the spray drop per half-leaf (mean of 18 leaves). Treated procedure as we have used it may lead to with ribonuclease it gave 0.06. The un- fracture of many of the reconstituted TYl\IV treated TYMV RNA inoculated at 30 t-tg/rnl RNA rods. Nevertheless with the incubation gave 6.8 lesions per half-leaf (mean of 18 conditions tested so far, only TMV RNA at leaves). After treatment with ribonuclease it pH values near 7.0 gives a high proportion of full-length rods. With TYlVlV RNA at pH's gave no lesions. neal' 7.0, incubation times must be several DISCUSSION hours at 30° to give significant yields. This The appearance in mixtures of TYlVIV gives opportunity for traces of nuclease in RNA and TMV protein subunits incubated the incubation mixture to partially degrade at pH values near 7.0 of sedimentable nucleo- the R~A. There is another factor that may be operprotein consisting of rods, and in which most of the RNA is resistant to ribonuclease ating for TYJVIV RNA at pH 7.0 and for digestion, demonstrate that TYlVIV RNA both RNA's at pH 4.8. Witz et al. (1965) can be coated by TlVIV protein to produce from a study of small-angle X-ray scattering rod-shaped particles. Reconstitution at pH of RNA in solution at pH 6.8 concluded that 4.8 is potentially complicated by the fact under these conditions both TMV RNA and that the protein subunits aggregate spon- TYJVIV RNA exist as slightly flexible moletaneously to form RNA-free rods at this pH cules possessing rodlike base-paired segments and that such rods aggregate to form visible joined end to encl. TYMV RNA had a more precipitates which could nonspecific ally trap compact structure than TMV RNA. Thus and protect the TYiVIV RNA. Nevertheless RNA under the conditions of incubation for formation of stable nucleoprotein rods does reconstitution is partly in an internally baseoccur at this pH since the RNA in the prod- paired state, alternating with single-stranded uct isolated by high speed sedimentation at regions. Under these conditions coating with pH 6.7 is stable to ribonuclease digestion at protein subunits may proceed simultanepH values up to 8.9. Furthermore such R.NA ously in more than one region of the molecule has a sedimentation rate at pH 7.3 very on single-stranded regions. Base-paired remuch greater than the original viral RNA gions may slow the rate of, or create a block to, further reconstitution. Forces acting on (Fig. 12). On incubation at pH 4.8, TiVIV protein rigid reconstituted sections may disrupt the alone forms rods of indeterminate length, the RNA chain. Another factor increasing the proportion and many of these are very long (Fig. llD). In contrast when TY1VIV RNA is present of short rods with TYMV RNA may be the

RECONSTITUTION OF TYMV RNA WITH TMV PROTEIN

inherent instability of this R NA within its protein shell (Hase lkorn, 1962 j K aper and H alperin, 1965 ; Matthews and Ralph, 1966). RNA preparations, unless made from freshly isolate d virus from recently infected plants, may contain a significant pr oportion of breaks even before the incubation for reconstit ution. Further testing of conditions for reconstitution at pH 4.8 and 0°, particularly of the nature 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 ion mixtures is unlikely to come from the RNA since viral RNA isolated by the procedure used retains undiminished infectivity aft er incubation alone at 30° for ma ny hours (A. R. Bellamy, personal communi cation) . It is mu ch more likely to be a contaminant of the protein subunit preparations. T he nuclease most likely to cont aminate TMV protein subunit preparations, at least those that are freshly made from fresh virus, is t he toba cco leaf ribonuclease described by Fri sch-Niggemeyer and Reddi (19,">7). This enzyme was some 5 times more active at pH 5.0 th an at pH 7.0, and in citrate-phosphate buffer the pH optimum was close to pH 4.8. T he enzyme 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 and higher temperatures may well be the majo r reason for the short reconstituted rods produced under these conditions. T he number of local lesions obtained so far have been too low to estab lish whet her or not TYl\IV RNA fully protected by Tl\1V protein from ribonuclease action is still infectious for Chinese cabbage. Most if not all the infectivity in th e reconsti tuted TYl\lV rods is probably due to complete RNA complements partially coated with protein. Sander and Schramm (1963) have described a strain of TY:.\IV which infects tobacco with the production of chlorotic local lesions. It would be of some interest to test the infectivity for tobacco and Chi nese cabhuge of the RNA from such a strai n coated with TMV protein. If the RNA of other rod-shaped plant viruses has a more open stru cture in solution

95

than TYMV RNA, it may be that they will reconstitute more readily t han TYMV R NA with T.MV prot ein. Several such viruses can multiply in mixed infections with T MV in tobacco (e.g., potato virus X) . The possibility may then exist for some degree of miscoating or " phenotypic mixing" under in vivo condi tions, It is unlikely that many message RNA's differ more widely in base composition from Tl\IV RKA than TYl\1V RKA, It may thus be possible to use TMV protein subunits to prepare cellular message R NA in a biologically active state but protected from nuclease attack. I n preliminary experiments at pH 4.8 we have obtained reconsti tut ed r ods in low yield from calf spleen nu cleic acids. Naked R NA injected into an int act animal is almost certainly rap idly degraded by nucleases. In pre liminary experiments with TYld V RNA in mice I'll' have found (Matthews and .J. Marbrook, unpublished observations) that 32P-labeled TYlVIV RNA coated with Tl\'lV protein has a markedly different distribution in the animal following foot pad injection than does free RNA, and that preimmunization of the mice with TMV further changes thi s distribut ion. ACKNOWLEDGMENTS T his work was supported in part by USPHS grant AI-04973 .

REFERENCES II., (1957). Deg radation of tobacco mosaic v ir us wit h a cetic aci d . Viroloyy 4-, 1- 4. FRA E N K E L·C o N R A T , II., and SINGE R, E ., (1959) . R econs t it ution of tobacco mosaic v irus. I II . Improv ed methods and th e use of mixed n ucleic acids. Biochim , Biophys. Ac/a 33, 359-370. FRAENKEL-CO NRAT, H ., and SINGER, E ., (19M). Re constitution of tob acco mosaic virus . IV . I nhibit ion by en zymes and other proteins, and use of polynucleotides. Vi1'ology 23, 354-362 . FRAENKEL-CoNRAT, I-1., and \VILLIAMS, R . C . , (1955) . Re cons t itution of active t oba cco mosaic vi rus from its in activ e protein and nucleic a cid comp onents. P roc. N ail. .'lead. Sci . U .S . ,n , 690- G98 . FnrSCH-NIGGEMEYER , 'trV., an d REDDl , K . K ., (1957) . Studies on ri bonuclease in tobacco leav es. I. Purification and proper- ties. Biochini , Bi oph ys . A cia 26,40-46. FRAENKEL-CONRAT,

96

MATTHEWS

HART, R. G. and SMITH, J. D., (1956). Interactions of ribonucleotide polymers with tobacco mosaic virus protein to form virus-like particles. NatU1'e 178, 739-740. HASELKORN, R., (1962). Studies on infectious RNA from turnip yellow mosaic virus. J. Mol. Bioi. 4. 357-367. HOLOUBEJK, V., (1962). Mixed reconstitution between protein from common tobacco mosaic virus and ribonucleic acid from other strains. Virology 18, 401-404. KAPEJR, J. M., and HALPERIN, J. E., (19G5). Alkaline degradation of turnip yellow mosaic virus. II. In siht breakage of the ribonucleic acid. Biochemistry 4, 2434--2441. LAUFFER, M. A" (1962). In "The Molecular Basis of Neoplasia," pp. 180-206. University of Texas Press, Austin. MATTHEWS, R. E. F., (1960). Properties of nucleo-

protein fractions isolated from turnip yellow mosaic virus preparations. Virology 12,521-539. MATTHEWS, R. E. F., and HARDIE, J. D., (1966). Reconstitution of RNA from spherical viruses with tobacco mosaic virus protein. Virology 28, 165-168. MATTHEWS, R. E. F., and RALPH, R. K, (1966). Turnip yellow mosaic virus. Advan. Virus Res. 12, in press. RALPH, R. K., and BELLAMY, A. R., (1964). Isolation and purification of unclegracled ribonucleic acids. Biochim, Biophys. Acta 87, 9-16. SANDER, V. E., and SCHRAMM, G., (1963). Die Bedeutung cler Proteinhalle fur die Wirtsspezifitiit von Pflanzenuiren. Z. Nalu1jo1'sch. 18b, 199-202. WITZ, J., HIRTH, L., and LUZZATI, V" (1965). La structure des acides ribonucleiques en solution; des RNA de virus de plantes. J. Mol. Bioi. 11, 613-619.