- Email: [email protected]
The unusual nature of the components of a strain of pea enation mosaic virus
VmOLOGY
66, 1-13 (1973)
The Unusual Nature of the Components of a Strain of Pea Enation Mosaic Virus It HULL
AND
L. C. LANE
John Innes Institute, Golney L an e, Norwich, England Accepted A pri l 10, 1973
Pea en ut.ion mosaic virus (PEMV) has spherical components sedi ment ing at 99S an d 1128. Th e larger contains an R NA of molecul ar weight 1.6 X 106 and the smaller an R NA of molecular weight 1.3 X 106 • Th e viral comp onents contain simil ar percentages of RNA and separate according to size by gel elect rophoresis. The larger vi ral component cont ains abou t 180 coat protein subunits; the smaller one contains abo ut 150 coat pr ot ein sub units and apparent ly h as an u nusual struct ure . Both viral components are required for infectivity and th e l arger determines the coat protein. The di fferences betwe en the results obtained with this PEMV strain and those ob t ained by Gonsalves and Shep herd (1972, Vi rology 48 ,709) are discussed. INTRODUCTION
Pea enation mosaic virus (PEMV), the only circulative aphid-borne plant virus which has been purified in sufficient quantities for chemical and physical studies, consists of two nucleoprotein components (Izadpanah and Shepherd, 1966b; Gibbs et ol., 1966;Bozarth and Chow, 1966;Shepherd et al., 1968). These components have been called upper and lower (Gibbs ei al., 1966; Bozarth and Chow, 1966) or top and bottom (Izadpanah and Shepherd, 1966b; Shepherd et al., 1968). Since both components are nueleoproteins and since "top" component s suggests a nucleic acid-free component, in this paper we designat e the component s middle and bott om. Recent work on multicomponent plant viruses has shown that the information necessary for infection is commonly distributed between two or more components but the situation for PEMV is not clear. Izadpanah and Shepherd (196Gb) and Gibbs et al. (1966) reported that both components of PENrY were infectious whereas Bozarth and Chow (1966) found infectivity only in the bottom nucl eoprotein component . We att empted to resolve this discrepancy using gel electrophoresis and we report that PEMV has a divided genome and com-
ponents which differ in size rather than RNA percentage. D uring th e preparation of this paper Gonsalves and Shepherd (1972) published a report which disagreed substantially with some of our observations and conclusions. We have extended our study to deal wit h the points of disagreement. MATERIALS AND METHODS
V irus isolates, assay and purification . Wildtype PEMV was obtained from A. J. Cockbain (Rothamsted Experiment al Station) who isolated it from nat urally infected broad beans (Vicia jaba L.) . The variant arose during repeated subcultu ring of the wild type under glasshouse conditions. It was recognised as being different from wild type on characters to be described later and was separated from wild type by single lesion culture. Bioassays of PEMV have previously been made using local lesion production on Chenepodium amaraniicolor Coste and R eyn (Gibbs et al., 1966) C. quinoa L. (Hagedorn et al., 1964; Gonsalves and Shepherd, 1972) or Galactia sp. (I zadpanah and Sheph erd, 1966a) or by determination of dilution end point on peas (Bozarth and Chow, 1966; 1
Copyright © 1073 by Acude mic P ress, Inc. All rights of repro duc t ion in any form reserved .
2
HULL AND LANE
Izadpanah and Shepherd, 1966b). Lesion production on C. quinoa was the most reliable bioassay under our conditions. No lesions were produced in C. capitalum L., C. hybriclum L., C. murale L., Gomphrenaglobosa L., Momordica balsamina L., or 'I'eiraqona expensa Murr. Virus was purified from peas (Pisum sativum L.) c.v, Meteor which had been infected for 10-14 days. Each 100 g of infected leaves was homogenized at 4 C with 100 ml 0.2 M sodium acetate adjusted to pH 6 with acetic acid (acetate buffer), containing 0.1 M ascorbic acid and 5% sucrose. After rehomogenizing with 100 mIl: 1 butanol! chloroform for 1 min, the emulsion was broken by centrifugation for 5 min at 5000 rpm. The aqueous phase was subjected to two cycles of differential centrifugation. The high speed pellets were resuspended in 0.1 M acetate pH 6.0 containing 5% sucrose. Analytical techniques. Analytical sedimentation and density gradient equilibrium studies were performed in a Spinco model E as described in Hull et al. (1969). The extinction coefficient at 260 nm was taken as 7.5/mg/cm (Shepherd et al., 19(8). For difference spectroscopy the absorbancies of the two samples in the sample and reference beams, respectively, of a Unicam SP 800A spectrophotometer, were matched at 260 nm and the spectrum was recorded over the range 225-325 nm. Virus preparations, negatively stained in either saturated uranyl acetate, saturated uranyl formate, or 2% sodium tungstate adjusted to pH 6 with formic acid were examined in a Siemens IA electron microscope calibrated using catalase. Preparative techniques. Equilibrium density gradient centrifugation in CsCl was done by mixing 5 ml of the virus sample in 0.1111 acetate with 3.26 g CsCl and centrifuging for 16-24 hr at 45,000 rpm in a Spinco R50 rotor. Two-droplet fractions collected from the bottom of the tube were diluted with 1 ml 0.1 M acetate pH 6.0 and the absorbance was measured at 260 nm. Polyethylene glycol (PEG) solubility-concentration gradient centrifugations (Clark and Lister, 1971) were performed in 5-0% reverse concentration gradients of :PEG stabilized in 5-30% positive sucrose gradi-
ents in Spinco SW40 tubes centrifuged for 30 min at 12,000 rpm. Sucrose/D 20 gradients were made by layering 0.5 ml each of 70, 65, 60, 55, 50, and 45% sucrose dissolved in buffered D 20 (9 parts D 20 , 1 part 0.5 M acetate pH 6.0) in Spinco SW39 tubes which were then left for 24 hr at room temperature. The virus samples were made 30% in respect to sucrose, and 0.5 ml (about 1.5-3.0 mg virus) was layered on the gradient and then overlaid with paraffin oil. The gradients 'were centrifuged for 72 hr at 25 C at either 33,000 rpm in a SW39 rotor or 44,000 rpm in a SW50.1rotor. Two-drop fractions (0.1 ml) were taken from the bottom of the tubes, and 1 rnl 0.1 M acetate pH 6.0 'was added to alternate fractions which were then monitored at 260 nm. Refractive indices were measured on the other fractions, and the densities were determined from a calibration curve using data from Sober (1968) and taking a density of 1.10 g/cc for the buffered D 20 . Gel electrophoresis. Virus was electrophoresed in gels 7.5 em long and 0.5 em in diameter consisting of' 2.8 or 3.4% polyacrylamide gels (5% cross-linked), containing 3% sucrose, and buffered with 30 mM Tris, 3 mM Ca(OH)2 adjusted to pH 4.4 with lactic acid (virus gel buffer) (Lane and Kaesberg, 1971). Gels were prerun at constant voltage (16 V/cm) until current remained constant (approx 1 hr). Samples of 10-20 J.lg of virus in 25 J.lliters gel buffer containing .5 mg /rnl protamine sulfate, 0.1 M mercaptoethylamine hydrochloride and 20% sucrose (PMS) were applied to the anodic end of the gel. Electrophoresis was carried out at 4 C at 15-17 V/cm (about 2 mA per tube) for about 4 hr with 2.8% gels and 16 hr with 3.4% gels. Buffer was circulated between the upper and lower electrode chambers. The gels were scanned at 265 nm in a Joyce-Loebl "Polyfrac" scanner. RNA was electrophoresed on 2.5 or 2.8% polyacrylamide (5% cross-linked) gels containing 0.1% sodium dodecyl sulfate (SDS) and 3% sucrose. The buffer system was modified from that of Peacock and Dingman (1967) and contained 0.089 M Tris, 0.089 M boric acid, and 1 mM EDTA pH 8.3; the upper reservoir also contained 0.05% SDS.
PEA ENATION MOSAIC VilLUS COMPONENTS
3
Virus RNA was prepared in two ways: The RNA was obtained by phenol ext raction in t he pr esence of 1% w / v bentonite and 2% w/v SDS. After et ha nol precipitation it was heated for 10 min at ,50 C in 0.2% SDS, 1 ill urea, 5% sucrose, and 0.05 M mercaptoetha nol. Alternatively t he virus was heat ed for 10 min at 50 C in electrophoresis buffer containing 1% SDS, 1 ?vI ur ea, 5% sucrose, and 0.05 1If mercaptoethan ol. About 10 J.Lg RNA was applied to each gel and electroph oresis was at room temperat ure and 16 V Icm (about 3 rnA per tube) for 1.5 or 4.5 hr. For formaldehyde modification of the RNA, virus was heated to 60 C for 5 min in 0 .05 M phosphate buffer pH 7.5 containing 1% SDS, 0.5% mercapt oethanol, 1 mM EDTA, and 10% sucrose before adding Fro. 1. Schlieren patterns of unfractionat ed 710 vol of 40% formaldehyde solution and PEMV ; upper, wild type; lower, vari an t. Photofurther heating to 80 C for 10 min. The graph ta kcn 12 min after reaching speed of 42,040 modified RNA was electrophoresed in 2.5% rpm; sedimentation is from left t o righ t. polyacrylamide gels for 2.5 hr using the Wild-t ype PEMV contained more bottom conditions given above for the untreated RNA. t han middle component (Fig. 1 top) whereas For infectivity studies, t he RNA species the variant contained more middle than were ext racte d from polyacrylamid e gels as bottom component (Fig. 1 bottom) . described by Hull (1972). Nucleoprotein Electrophoresis of nucleoprotein. Electrocomponents were locat ed by scanning the phoresis of the virus on polyacrylamide gels gels in t he Joyc e-Loebl "Polyfrac " scanner. resolves two components (Fig. 2). The agreeThe nucleoprotein band s were excised with ment between the component ratios esti raz or blades and homogenized in a volume mated using ultracentrifugation (Fig. 1) and of 0.1 M sodium acet at e adj usted to pH 6.0 gel electrophoresis (Fig. 2) of wild-t ype and with acetic acid containing 5% sucrose and variant PEMV indicates that the slow 0.25% bovine serum albumin (to prevent electrophoretic component corresponds to abs orption of virus to the glassware) to give the bottom centrifugal component and the an estimated concentration of 1 or 2,ug/ml fast electrophoretic component to middle virus. The homogenate was stored overnight component. Further evidence will be preat 4 C prior to bioassay. sented to support this. . At pH 4.4, the relative migration of the RESU LTS middle to bottom component of both strains S edimentation properties of the nucleopro- of PEMV was 1: 0.940 in 2.8% gels and tein. Analytical ultracent rifugation of puri- 1 :0.741 in 3.4% gels (Fig. 3) suggesting t hat fied preparations of PEMV showed t wo t he separat ion was on the basis of size rath er nu cleoprotein components (Fig . 1) with t han charge. The ratio of mobilities was independent of pH between 3.5 and 6.0 S 20.. values of 112 S (bottom component) and 99 S (middle component) at £ 260=1 again indicating that separation was not OD /ml both in 0.1 M aceta te pH 6 and in based on charge. The virus precipitat ed on virus gel electro phoresis buffer. These differ the surface of gels run at pH 8.0 and 8.6. To examine the possibility of orientation slightl y from the pr eviously reported values of 115 and 95 S (Gibbs et al., 1966),113 and or distortion of one of the components in the 94.5 S (Bozarth and Chow, 1966), and 122 electric field, wild-type virus was electroand 106 S (Izadpanah and Shepherd, 1966b). phoresed at low voltage (2 V/ cm for 72 hr
HULL AND LANE
4
a
ttl to N
W
a
L~ b
It)
lD N
W
.. Distance
Ir-----~.~
b
+
FIG. 2. Densitometer tra ces of PEMV nucleopro tein (a) wild t ype ; (b) variant, elec tr ophoresed in 3.4% p olyacrylamide gels at 16 VI cm for 16 hr. The two sm all peaks all th e right of t race (a) are of dimer particles .
on 3.4% gels); it was not feasible to use a voltage significantly less than this. Decreasing the voltage did not affect th e relative mobilities of the two components . Each component of the variant had an electrophoret ic mobilit y of 0.75-0.80 that of the corresponding wild-type component (Fig. 2). Since this ratio was independent of gel pore size, the two strains differ in charge. Electrophoresis of virus at different pHs indicated an isoelectric point of around pH 7 for wild-type PEMV and pH 6 for variant PEMV. Shepherd et al. (1968) showed that in free electrophoresis their strain migrat ed as a single component; they estimated an isoelectric point of pH 5- 6. At pH 4.4, the bottom component of wildty pe PEMV migrat ed identically with brome mosaic virus in both 2.8 and 3.4% gels indicating that the tw o viruses were of similar hydrodynamic size and charg e at th at pH. PEG solubility-concentration gradients. PEG solubility- concentrat ion gradient s can apparently separate virus particles on size or
Distance
+.
FIG. 3. Densitometer traces of va riant PEMV elect rophores ed in (a ) 2.8% poly acryl am ide gel for 9 hr and (b) 3.4% polyacrylamide gel for 16 hr, both at 15 V/cm.
charge (Clark and Lister, 1971). Wild -type PEM V centrifuged on 5-0% PEG solubility-concentration gradients gave a major and a minor band . (Fig. 4). Gel electrophoretic an alysis indicated that the minor band consisted predominantly of the variant strain, which was a contaminant in this preparation. However the minor band was not examin ed in detail and it may be an artifact of this t echnique. In the major band, analysis of fraction 9 plus 10 (Fig. 4) showed that it contained 2.5% middle component whereas fra cti on 16 plus 17 contained 13.8% middle component; the original preparation contained 5.7% middle component . Middle component is ap parently more soluble in PEG than bot tom component. Clark and Lister (1971) showed that t he solubilityconcentra tion of viru s particles was dependent on ionic conditions. Although varyi ng the ionic conditions altered the solubility-concentration for PEMV (Table 1), bottom and middle components could
P EA ENAT lON MOSAIC VIRUS COMPONENTS
5
separated by preparative CsCI density cent rifugat ion (Fig . 5). Th e ultraviolet absorption spect rum of the lighter band (fraction 42) had a peak at 278 nm and a trough at 250 nm (278/260 nm ratio = 1.30) indio cating that it was mainly protein. Fractions 5, 9, and 12 were precipitated from the CsCI by 10% polyethylene glycol and resuspended o in 0.1% 111 acetate pH 6.0. These fractions 1·0 ~ W showed a single 112 S component in the analyt ical ultracentrifuge and a single band in gel electrophoresis comigrating with the slow component of untreated virus (Fig. 6). This further confirms that the slow gel electrophoretic component corresponds to bottom component. A pellet collected from the bottom of the preparative CsCI gradients, had the spectral 25 20 15 10 5 chara cteristics of nucleic acid. Fractions PE:'.fV variant showed only a small amount of material at p = 1.435 gl cc but FIG. 4. Di stribution of 10 mg of wild -ty pe P EMV after cen trifuga ti on for 30 min a t 12,000 much at p = 1.282 gl ce in both analytical rpm in a Spineo SW40 rotor in 5-0% reverse con- and preparative CsCI gradients. Clearly cent rati on grad ient of PEG stabili zed in 5-30% middle component is degraded in CsCI p osi tive Sucr ose gradient. Fract ions of 20 drops gradients. were diluted with 1 ml 0 .1 M acetate pH (i .O before In attempts to stabilize middle comex tin c tion me asureme nts. T op of t ube is on th e ponent by fixation in buffered glutaraldelef t. hyde, th e viru s was precipitated by glutaral dchyde concentrations greater than 1%j TABLE 1 preparations fixed in 0.2 and 0.5% glutaralE ~'F ECT OF N ACL C O N C ~; N 'l'ItATION ON THI~ dehydo behaved sim.ilarly to unfixed ones. SOLUIlILITY CO NCI'; N'I'RATJO N OF TH ~ Two The degradation of middle component by B ANDS OF PEMV IN P EG G l{ADJIo;NTS CsCI was examined using gel elect rophoresis of wild-type PEMV which had been incuNnel molarity % PEG bat ed at room temperature with various Minor band Main band concentrati ons of CsCI (Table 2). From Table 2 it can be seen that middle com1.05 0.05 2 .10 ponent was com.pletely degraded by 1.0 JYI 0.10 0 .95 1.90 CsCI and that Mg2+ appeared to have no 0.92 1. 73 0.20 effect on the degradation. Centrifugation of both wild-typ e and th e not be separat ed. The P E G solubility-con- vari ant st rain of PEJ~dV in isopycnic gradicentration of PEMV is unusually low com- ents of sucrose in D 20 each gave a single pared with that of oth er spherical virus es band at p = 1.36 glcc (Fig. 7). In th e same run (in sister tubes) broad bean mottle virus (Clark and Lister, 1971). Equilibrium centrifugation of nucleoprotein. layered on t he top of one gradient and the Wh en wild-type PEMV was centrifuged to bottom of another, banded in similar posiequilibrium in CsCI densit y gradients two tions (p = 1.33 g/cc) indicating th at the bands were found ; one at p = 1.435 gl ee and syst em was close to equilibrium. The ratios the ot her at p = 1.282 glee. These are close of t he components in fractions 3, 5, 7, 9, 11, to t he valu es reported by Bozarth and Chow 13, and 15 shown in Fig. 7 were estimated by (1966) (for the denser one alone) and by gel electrophoresis. Since this ratio did not Sehgal et al. (1970). The t wo bands were vary mark edly over the various fractions,
6
HULL AND LANE 1-50 1-45
1'40 1·35
Z' CJ
'iJj
-!:!.
~ S
o
o 1'0
'" W N
40
35 25
10
5
FIG. 5. Distribution of 12 mg of wild-type PEMV after centrifugation for 23 hr at 45,000 rpm in a Spin co 50 rotor in 5 ml CsCI p = 1.42 g/cm2 overlaid with light paraffin oil. Two-drop fractions, collected from the bottom of the tube were diluted with 1 ml 0.1 Jli[ acetate pH 6.0 for extinction measure ments after refractive index measurements on samples from every fifth fraction. TABLE 2 DIWRADATION OF MIDDLI" COMPONENT WILD-TYPE PEMV BY GBeL"
a
CsCI concentration
I
~
10
Ul
C'I
w
b
I
l
0 0.5M l.OM
OF
Components as % total virus+ M g2+
+EDTA
E'
M
79.0 87.3 100.0
21.0 12.7 0.0
B
79.0 91.5 100.0
M
21.0 8.5 0.0
a Virus treated with CsCI in presence of either O.OIM EDTA or 0.05 M Mg SO.l . b Estimated from scans of polyacrylamide gels. 'B = bottom component, M = middle component.
...
Distance
FIG. 6. Densitometer traces of wild-type PEMV nucleoprotein (a) from fraction 12 of the CsCI gradient shown in Fig. 5 b) fraction 12 control electrophoresed in 3.4% polyacrylamide gels for 16 hr at 16 VIcm (control is illustrated in Fig. 2a).
+
the two components of PEMV banded at similar densities. This was confirmed by measuring the S20w values of the components in suerose/Dsu solutions of known densities (Fig. 8). The extrapolated flotation density
of p = 1.37 glee was close to that estimated from equilibrium sucrose/Dati gradients (Fig. 7). Ultraviolet absorption. Preparations of wild-type virus (predominantly bottom component), variant isolate (predominantly middle component), and CsCl treated wildtype virus (bottom component) were compared by ultraviolet absorption spectroscopy. The samples exhibited no difference spectrum over the range 225-325 nm. To test the sensitivity of the technique, RNA was added to samples of broad bean mottle virus (22% RNA) to give 23.5 and 25% RNA. With the 22% RNA sample in the reference cuvette and the 23.5% RNA sample in
7
PEA ENATION MOSAIC VIRUS COMPONENTS
1'38 >0..... ...
1.34
(.J
·0..g COl
1·30 ~ ....
20
15
10
5
Fractions FIG. 7. Distribution of variant PEMV in a gradient of 45-70% sucrose made up in D,O buffered with 0.1111 acetate pH 6.0 and centrifuged for 72 hr at 33,000 rpm in a Spinco SW39 rotor. Two-drop fractions were taken from the light scattering zone and refractive index measurements were made from alternate ones. Then 1.0 ml a.livE acetate pH 6.0 was added to each fraction and the extinction was measured. The virus from fractions 3, 5, 7, g, 11, 13, and 15 was concentrated by PEG precipitation and analysed for component ratio (bottom component/middle component) by electrophoresis on 3.4% polyacrylamide gels. 120 • Bottom +Middle 80
40 20
',0
1,'
1·2
1·3
Density D/CC
FIG. 8. Sedimentation coefficients of bottom and middle components of wild-type PEMV in various concentrations of sucrose dissolved in D,O buffered with 0.1 M. acetate pH 6 measured in a Spineo Model E run at 40,000 rpm.
the sample cuvette, a decrease in absorbancy could be detected below 230 nm. When the sample cuvette contained 25% RNA, a marked absorbancy decrease was noted. Since the protein makes a greater relative contribution to the total absorbance of the virus below 230 nm than it does at 280 nm, this method is much more sensitive to variation in protein to RNA ratio than the
more commonly used comparison of absorptions at 260 and 280 nm. Since this technique can detect a 2% difference in percentage of RNA between virus components with RNA contents similar to PEMV, we conclude that the nucleic acid contents of the two PEMV components are similar-certainly within 2%. Electron microscopy. No size differences could be detected by electron microscopy in samples of bottom and middle components of either wild-type or variant isolates of PEMV, separated on polyacrylamide gels (Fig. 9). Measurements from micrographs of 50 particles of each component negatively stained with uranyl acetate gave diameters of 29.5 (±O.6) nm and 29.2 (±1.1) nm for bottom and middle components, respectively. Neither component exhibited structural details when negatively stained in uranyl acetate, uranyl formate, or sodium tungstate/formic acid. Bottom component particles, however, tended to have hexagonal outlines and to aggregate in hexagonal packing. Gibbs et al. (1966) and Farro and Rassel (1971) also noted that bottom component particles were more regular than
8
HULL AND LANE
Fr o. 9. El ectron mi crogruph s of wild -type P EMV components (a) b ottom compo nent , (b) middle c omp onen t , fr a etionut ed 0 11 3.4 % poly acrylamid e gels , negatively stai ned in uranyl acetate ; bar = 100 n m,
those of middle compon ent . The protubera nces on particles, r eported by Bozarth and C how (l9G6 ) , wer e not ob ser ved . It is pr oba ble t hat, as Farro and Rassel (1971) suggested , t hese were effect s of degrad ati on of t he p arti cles in negative stain . Nucl eic acid . E lectrop h oresis of the nu cleic a cid on polya crylamide gels resolv ed t hree nucleic acid compo nents (Fig. IDa-c) as re cently r eported by Gons alves and Shepherd (1972). Thoro were no appare nt differ ences in t he component com posit ions of samples prepared by the phenol te chniq ue a nd t h ose prepared by d ir ect disso ciation of the virus immediately prior to elect rophoresis. The molecular weight s of the three s pecies (1, 2, 3), estim ated by comparison with the E . coli ribosom al R NAs (1.07 and 0.56 X lOG) were 1.42, 1. 15 and abo ut 0.20 X lOG daltons, respectively . R NA species :3 was usually rather het erogeneous and varia ble in appa rent molecular weigh t (0.1100.270 X 10 6) . On exte nd ed elect rophores is, n ucleic acid s pecies 1 divided int o t wo bands (estim at ed m olecul ar w eights ] .42 and 1.45 X 10 6 daltons), B ecau se this did n ut happen with form aldeh yde-m odified RN A, these bands a ppar ently r epresent different
configurations of a single species. T he bands of R NA species 1 and 2 are broad compared wit h bands of R NA species of othe r viruses (Fig. 10). El ect rophoresis of form ald ehyde-modified R NA also gave t wo ma jor bands in proportio ns sim ilar to t hose of un t reat ed RNA (Fig, lOd, e) bu t which were much sharper. It is likely that t he sprea ding of t he ba nds of native R NA refiects configuratio nal het erogeneit y. Es timates of t he molecular weights of formaldehyd e-treated UNA species (using R. coli rib osomal RNAs and TMV RNA as standards) were 1.60 and l.;~O X lOB. These differ subs tan tially from the values reported by Gon salves and Shepherd (1972). I t is well kno wn t hat conditions of electrophoresis affect estimates of molecular weights of unmodified RNA. I t is however difficult t o explain t ho 20-25% differen ces in estima t ed molecular weights for formaldehyde- modifie d R NA. R eiaiionship of RN A species 10 nucleoprolein components. Gonsa lves and Sh epherd ( 972) not ed that t he R N A from virus pr epar ations which had about :~ O% of middle (t heir t op ) componen t contain ed about 60% of species 2. From this and from other d atn ,
PEA ENATIOl\; ",10:-;.-\1 C VIr tU,.; 3
a
21
C O M P ONE NT~
9
phoresis , the proportion of tot al R NA whi ch w as RNA s pecies :3 being estima ted from
short period electrophoresis (1.5 hr at 8 V Icrn) and those of RNA species ] and 2 from long period electrophoresis (5 hr at 14 Vl em). The close correlation of bottom component with H.NA speci es 1 (Table 3) indi cates that bottom nu cleoprotein componen t does not contain appreciable amount s 2 1 of R NA spe cies 2. Since our preparations b contain very lit tle R NA species 3 we are
(\j
\
I\JU \
l~
;
;
~
10
HULL AND LANE TABLE 3 RELATlONSHIP BETwlmN NUCLEOI'ROTEIN COMPO/'lJeN'NI AND RNA SPECIES Preparation
Var. b control Val'. fraction 1
2 4 5 6 7 Variant prep. Wt prep Wt prep Wt prep
RNA species-
Nucleoprotein components' % of total
% of total
Bb
M
1b
2
3
16.6 35.1 34.8 30.4 22.1 8.0 2.0 22.5 59.4 59.6 65.4
83.4 64.9 65.2 69.6 77.9 92.0 98.0 77.5 40.6 40.4 34.6
11.2 32.3 38.3 32.2 21.9 8.8 2.6 20.3 55.9 58.0 66.2
86.0 65.6 60.5 67.8 78.1 91.2 97.4 73.8 38.6 35.6 33.8
2.8 2.1 1.2 _e
5.9 5.5 6.4
• Measured from ultraviolet scans of polyacrylamide gels. b Wt and var. signify wild-type and variant isolates, respectively. Band M signify bottom and middle nucleoprotein components, respectively: RNA species 1, 2, and 3 as shown in Fig. 10. c No peak found. TABLE 4 INFECTIVITIES OF MIXTURES OF PEMV-RNA SPECIES RNA species«
1
+2+
1
+2+
1 3e 2 3 3
1+2+3 1
+
2
+2 +3 1 + 3 1 +2 +3
1
1 a b
2 + 3 +2+3
Lesion numbers"
24.6 54.9 12.1 60.6 0.3 69.0 115.3 63.9 24.2 67.1 24.8 58.8
Species numbers refer to those in Fig. 10. Average lesion numbers on 10 half-leaves of
Chenopodium quinoa. e Mixtures made from equal volumes of extracts of RNA species from polyacrylamide gels.
port our ideas that bottom component does not contain RNA species 2. To determine which component contained the information for coat protein, pea plants were inoculated with homologousand heterologous mixtures of the two nucleoprotein components from the two strains. Mixing of components enhanced infectivity in
heterologous as well as homologous mixtures (Table 5). Virus was prepared from single pea plants by grinding each in 2.5 ml 0.2 M acetate pH 6.0 containing 5% sucrose, shaking the sap with 2.5 ml L:1 n-butanolchloroform, and centrifuging at low speed. The aqueous phase was removed and PEG 6000 was added to it to give 10%; after 3-4 hr at 4 C the extracts were centrifuged. Each pellet was suspended in 0.2 ml virus gel buffer and a 25 ,ulitcr sample (to which was added 25,uliters P.MS) was electrophoresed in a 2.8% polyacrylamide gel. Electrophoretic mobility and the component composition of the heterologous mixtures corresponded to that of parent bottom component. While Gonsalves and Shepherd (1972) associated biological activity with PElVIV RNA species 2, they were unable to associate any activity with RNA species 1. We have found that species 1 enhances infectivity and also that it carries a genetic marker. Our evidence clearly shows that our strain of PEMV has a divided genome. Relationship between our strain of PEMV and that of Shepherd. Our data clearly differ from those of Gonsalves and Shepherd (1972). A double diffusion test utilizing antiserum to a Wisconsin isolate of PEMV (Izadpanah and Shepherd, 1966b) and a
11
PEA ENATION MOSAIC VIRUS COMPONENTS TABLE 5 INFECTIVITIES OF MIXTURES OF PEMV NUCLEOPROTEIN COMPONENTS Experiment
i-
2'
Isolate
and
Nucleoprotein component-
B B+M
Wt Wt Wt Wt Wt Wt Wt Wt Val'. Val'. Val'. Val'. Wt B Wt B Val'. B Val'. B
M B+M B B+M
M
+ val'. M + wt M + wt M + val'. M
B+M B B+M M B+M
Lesion numbers" 1.8 137.8 8.2 162.6
0.8 12.6 4.2 12.6
0.0
5.0 0.8 8.8 5.6
10.0
15.0 5.0
a Wt and val'. signify wild-type and variant isolates, respectively and Band M signify bottom and middle nucleoprotein components, respectively. o Average numbers of lesions on five half-leaves of Chenopodium quinoa. c Experiment 1 inoculum at 2 fLg/rol and Expt 2 inoculum 1 "g/m!.
TABLE 6 ESTIMATED NUMBERS OF PROTEIN SUBUNITS IN PEMV COMPONENTS Componenl
Method of estimation
Molecular weight X 106
Number of protein subunits'
B M
Svedberg equation Svedberg equation
5.57 4.38
182 143
B
Nucleic acid weight and content
5.71
187
4.64
152
M
a Based on a protein content of 72% and a subunit molecular weight of 22,000.
sample of the Davis isolate of PElVIV (Gonsalves and Shepherd, 1972), kindly provided by Dr. R. J. Shepherd, failed to distinguish our isolate from the Davis isolate. We could find no obvious differences in symptomatology. However, our attempts to purify the Davis isolate have resulted in extremely poor yields and a complex pattern on gel electrophoresis. In view of the poor yield the purified material may not be comparable to that of Gonsalves and Shepherd.
If the gel electrophoretic complexity per-
sists with improved purification procedures a comparison of strains will be an extensive undertaking. DISCUSSION
Our strain of PEMV has several unusual features. The two nucleoprotein components have similar nucleic acid contents but yet contain RNA molecules of different sizes. This is reflected by a size difference between the nucleoprotein components which can be detected by gel electrophoresis. Lister ei al. (1972) recently reported that the nucleoprotein components of tobacco streak virus (TSV) are of different sizes. Like PEMV, the components of TSV have similar percentages of RNA contained in capsids of different sizes. However one component of PEMV is degraded by 0.5 M CsCI whereas all those of TSV can withstand CsCI concentrations greater than 2 M. PElVIV differs from the other known multiple genetic component viruses in having the coat protein gene in the largest RNA. All the others have the coat protein gene in the smallest genetically independent RNA molecule.
12
HULL AND LANE
There are t wo major differences between the results obtained wit h our isolates of PEMV and those found with the Davis isolat e by Gonsalves and Shepherd (1972). These are first, the distribution of RNA species in nucleoprotein components, and second, the nature of the infective genome. Relatively simple mutations in the coat protein could produce more complex st rains with greater varieties of part icle ty pes which could obscure the multipartite nature of the genome. Conversion of our strain to a single genetic component virus (or vice versa) would involve a catas trophic mutation. The existence of different PE~/IV strains requiring different numb ers of RNA components for infectivity is therefore unlikely. The molecular weights of the two nucleoprotein component s of PEMV can be estimated by two methods. The first utilizes the Svedberg equation. The sedimentation coefficient s are 1128 for bottom component and 998 for middle component. Diffusion coefficients were estimated indirect ly in the absence of sufficient quanti ties of separ ate components for direct measurement. Bottom component of wild-type PEMV coelectrophoresed at pH 4.4 both in 2.8% and 3.4% acrylarnide gels with brome mosaic virus indicati ng that they had identical hydrat ed diamet ers (and hence identical diffusion coefficients). The diffusion coefficient for brome mosaic virus is 1.55 X 10-7 cm-/sec (Bockst ahler and K aesberg, 1962) and this value was taken for PE MV bottom component. The diffusion coefficient of th e dimer particle will be 0.7 tim es thi s value [molecular weight is twice that of the monomer and the 8 value is 1.4 times that of the monomer (Markham, 1962)]. Since the retardation by the polyacrylamide gel is related to t he diameter (and hence the diffusion coefficient) of the virus parti cle, t he diffusion coeffi cient (D ) for middle component of PEMV was estimat ed from a plot of log l iD against t he ratio of dist ance migrated on a 3.4% gel to that migrated on a 2.8% gel. With monomer and dimer particles of bottom component as standards , t he estimat e of D for middle component is 1.76 X 10-7 cm-/ sec. These values of D are lower than that given by Bozarth and Chow (1966) for unfractionated virus. The components of PEMV have similar
nucleic acid contents, and the value of 28% (Shepherd et al., 1968) was used. The partial specific volume of th e prot ein was calculat ed to be 0.72 cc/ g from the amino acid composition (Shepherd et al., 1968), and that of the R NA was taken as 0.55 cc/g ; this gave a value of 0.672 cc/g for the virus. Fr om these values, the Svedberg equation indi cated molecular weight s of 5.37 X 106 and 4.38 X 10 6 dalt ons for bot tom and middl e components, respectively. Molecular weights were estim at ed to be 5.71 X 106 and 4.64 X 10 6 claltons for bottom and middle components, respectively, from the percentage of RNA and the gel electro phoretic molecular weights of the formaldehyde-treated RNA species. Formaldehyde treatment removed secondary structure in the RNA and produced no app arent crosslinking (Lane, unpublished; Bo edtker, personal communication). The gel electro phoretic molecular weight of modified RNA should reflect the true molecular weight. Th e molecular weights estimate d for bot tom component are close to that suggested by Shepherd ei al. (1968). PEMV has a single coat protein with a molecular weight of 22,000, estimate d by amino acid analysis and SDS gel electrophoresis (Shepher d at al., 1968; Hill and Shepherd, 1972) and confirmed for both isolat es of our strain by SDS gel electro phoresis (Hull , 1971). Using this figure and the estimate d particle molecular weights, bottom component cont ains ab out 180 subunit s (Table 6) consist ent with a particle of icosahedr al symmetry having a triangulation number T = 3. As shown in T able 6, particles of middle component contain less than 180 subunits but more than th e 60 subunits of the icosahedron T = 1. All isometri c viruses so far studied have their prot ein subunits quasiequivalently arr anged in shells of icosah edral symmet ry t he prin ciples of constr uction of which dictat e that t he t riangulation number should be either T = 1,3, 4, 7, etc . (Caspar and Klug, 1962, 1963). It is possible to construct models with icosah edral symmetry but in which the chemical subunit s arc not quasiequivalently arranged. One such model, in which chemical subunits are shared between adj acent hexamer morphological subunits comprises
PEA ENATION MOSAIC VIRUS COMPONENTS
150 chemical subunits, a number consistent with that calculated in Table 6. However in the absence of positive evidence on the structure of middle component, all that can be said is that it appears, from its salt sensitivity and from its size difference, to be structurally different from bottom component. ACKNOWLEDGMENT L. C. L. acknowledges support by Grant number PF 756 from the American Cancer Society. REFERENCES BOCKsTAHLlm, L. E. and KAJ>SBIcltG, P. (1962). The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophys. J. 2,1-9. BClJoJD'l'KKR, H. (1960). Configurat.ional properties of tobacco mosuic virus ribonucleic acid. J. Mol, Bio!. 2, 171-178. BOZARTH, R. F. AND CHOW, C. C. (1966). Pea enation mosaic virus: Purification and properties. Contrib, Boyce Thompson Inet. 28, 301309. CASPAR, D. L. D. and KLUG, A. (1962). Physical principles in the construction of regular viruses, Cold Spring Harbor Symp. Quant. Bioi. 27,1-24. CASPAR, D. L. D. and KLUG, A. (1963). Structure and assembly of regular virus particles. In "Viruses, Nucleic Acids and Cancel'," pp. 27-39. Univ. of Texas M.D. Anderson Hospital and Tumor Institute. The Williams and Wilkins Co. CLARK, M. F. and LISTER, R. M. (1971). The application of polyethene glycol solubility-concentration gradients in plant virus research. Virology 43, 338-351. DINGMAN, C. W., FISHER, M. P., and KAKEFUDA, T. (1972). Role of molecular conformation in determining the electrophoretic properties of polynucleotides in agarosc-acrylamide gels. II. Biochemistry 11, 1242-1250. FARHa, A. and RASSEL,A. (1971). Caraeteristiques en microscopie 6lectronique d'un isolement du virus de la mosaique "enation" du pois. Ann. Phytopathol. 3,53-61. GIBBS, A. J., HARRISON, B. D., and WOODS, R, D. (1966). Purification of pea enation mosaic virus. Virology 29,348-351. GONSALVES, D. and SHlcPlllcRO, R. J. (1972). Biological and biophysical properties of the
13
two nucleoprotein components of pea enation mosaic virus and their associated nucleic acids. Virology 48, 709-723. HAGEDORN, D. J., LAYNE, R. E. C., and RUPPEL, E. G. (1964). Host range of pea enation mosaic virus and use of Chenopodium albl~m as a locallesion host. Phytopathology 64, 843-848. HILL, J. H. and SmcPHEHD, R. J. (1972). Molecular weights of plant virus coat proteins by polyacrylamide gel electrophoresis. Virology 47,817822. HUl'L, H. (1971). Examination of alfalfa mosaic virus protein on polyacrylamide gels. Virology 46,767-772. HULL, R. (1972). The multicomponent nature of broad bean mottle virus and its nucleic acid. J. Gen. Virol. 17,111-117. HULL, H.., HILLS, G. J., and MAHKHAM, R. (1969). Studies on alfalfa mosaic virus. II. The structure of the virus components. Virology 37,416-428. IZADPAHAH, K. and SJ:mnmHD, IL J. (1966a). Gouiciia sp. as a local lesion host for the pea enation mosaic virus. Phytopathology 66, 458459. IZADPANAH, K. and SHEl'HlmD, R. J. (1966b). Purification and properties of the pea enation mosaic virus. Vi'rology 28, 463-476. LANg, L. C. and KAESBEIW, P. (1971). Multiple genetic components in bromegrass mosaic virus. Naiure (London) New Biol, 232,40-43. LISTER, R. M., GJ:Ll.BHI AL, S. A., and SAKSENA, K. N. (1972). Evidence that particle size heterogeneity is the cause of centrifugal heterogeneity in tobacco streak virus. Virology 49, 290-299. MAHKHAM, R. (1962). The analytical ultracentrifuge as a tool for the investigation of plant viruses. Aclvan. 111:1'1I.S Res. 9, 241-270. PEACOCK, A. C. and DINGMAN, C. W. (1967). Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6, 1818-1827. SgHGAL, O. P., JICAN, J-H., BHALLA, R. B., SOONG, M. M., and KRAUSE, G. F. (1970). Correlation between buoyant density and ribonucleic acid content in viruses. Phytopathology 60,1778-1784. SJ:mPHlmD, R. J., WAKEMAN, R. J., and GHABlUAI" S. A. (1968). Preparation and properties of the protein and nucleic acid components of pea enation mosaic virus. Vil'olog1l36, 255-267. SOBEH, H. A. (ed). (1968). "Handbook of biochemistry. Selected data for molecular biology." The Chemical Rubber Co. Cleveland, Ohio.
66, 1-13 (1973)
The Unusual Nature of the Components of a Strain of Pea Enation Mosaic Virus It HULL
AND
L. C. LANE
John Innes Institute, Golney L an e, Norwich, England Accepted A pri l 10, 1973
Pea en ut.ion mosaic virus (PEMV) has spherical components sedi ment ing at 99S an d 1128. Th e larger contains an R NA of molecul ar weight 1.6 X 106 and the smaller an R NA of molecular weight 1.3 X 106 • Th e viral comp onents contain simil ar percentages of RNA and separate according to size by gel elect rophoresis. The larger vi ral component cont ains abou t 180 coat protein subunits; the smaller one contains abo ut 150 coat pr ot ein sub units and apparent ly h as an u nusual struct ure . Both viral components are required for infectivity and th e l arger determines the coat protein. The di fferences betwe en the results obtained with this PEMV strain and those ob t ained by Gonsalves and Shep herd (1972, Vi rology 48 ,709) are discussed. INTRODUCTION
Pea enation mosaic virus (PEMV), the only circulative aphid-borne plant virus which has been purified in sufficient quantities for chemical and physical studies, consists of two nucleoprotein components (Izadpanah and Shepherd, 1966b; Gibbs et ol., 1966;Bozarth and Chow, 1966;Shepherd et al., 1968). These components have been called upper and lower (Gibbs ei al., 1966; Bozarth and Chow, 1966) or top and bottom (Izadpanah and Shepherd, 1966b; Shepherd et al., 1968). Since both components are nueleoproteins and since "top" component s suggests a nucleic acid-free component, in this paper we designat e the component s middle and bott om. Recent work on multicomponent plant viruses has shown that the information necessary for infection is commonly distributed between two or more components but the situation for PEMV is not clear. Izadpanah and Shepherd (196Gb) and Gibbs et al. (1966) reported that both components of PENrY were infectious whereas Bozarth and Chow (1966) found infectivity only in the bottom nucl eoprotein component . We att empted to resolve this discrepancy using gel electrophoresis and we report that PEMV has a divided genome and com-
ponents which differ in size rather than RNA percentage. D uring th e preparation of this paper Gonsalves and Shepherd (1972) published a report which disagreed substantially with some of our observations and conclusions. We have extended our study to deal wit h the points of disagreement. MATERIALS AND METHODS
V irus isolates, assay and purification . Wildtype PEMV was obtained from A. J. Cockbain (Rothamsted Experiment al Station) who isolated it from nat urally infected broad beans (Vicia jaba L.) . The variant arose during repeated subcultu ring of the wild type under glasshouse conditions. It was recognised as being different from wild type on characters to be described later and was separated from wild type by single lesion culture. Bioassays of PEMV have previously been made using local lesion production on Chenepodium amaraniicolor Coste and R eyn (Gibbs et al., 1966) C. quinoa L. (Hagedorn et al., 1964; Gonsalves and Shepherd, 1972) or Galactia sp. (I zadpanah and Sheph erd, 1966a) or by determination of dilution end point on peas (Bozarth and Chow, 1966; 1
Copyright © 1073 by Acude mic P ress, Inc. All rights of repro duc t ion in any form reserved .
2
HULL AND LANE
Izadpanah and Shepherd, 1966b). Lesion production on C. quinoa was the most reliable bioassay under our conditions. No lesions were produced in C. capitalum L., C. hybriclum L., C. murale L., Gomphrenaglobosa L., Momordica balsamina L., or 'I'eiraqona expensa Murr. Virus was purified from peas (Pisum sativum L.) c.v, Meteor which had been infected for 10-14 days. Each 100 g of infected leaves was homogenized at 4 C with 100 ml 0.2 M sodium acetate adjusted to pH 6 with acetic acid (acetate buffer), containing 0.1 M ascorbic acid and 5% sucrose. After rehomogenizing with 100 mIl: 1 butanol! chloroform for 1 min, the emulsion was broken by centrifugation for 5 min at 5000 rpm. The aqueous phase was subjected to two cycles of differential centrifugation. The high speed pellets were resuspended in 0.1 M acetate pH 6.0 containing 5% sucrose. Analytical techniques. Analytical sedimentation and density gradient equilibrium studies were performed in a Spinco model E as described in Hull et al. (1969). The extinction coefficient at 260 nm was taken as 7.5/mg/cm (Shepherd et al., 19(8). For difference spectroscopy the absorbancies of the two samples in the sample and reference beams, respectively, of a Unicam SP 800A spectrophotometer, were matched at 260 nm and the spectrum was recorded over the range 225-325 nm. Virus preparations, negatively stained in either saturated uranyl acetate, saturated uranyl formate, or 2% sodium tungstate adjusted to pH 6 with formic acid were examined in a Siemens IA electron microscope calibrated using catalase. Preparative techniques. Equilibrium density gradient centrifugation in CsCl was done by mixing 5 ml of the virus sample in 0.1111 acetate with 3.26 g CsCl and centrifuging for 16-24 hr at 45,000 rpm in a Spinco R50 rotor. Two-droplet fractions collected from the bottom of the tube were diluted with 1 ml 0.1 M acetate pH 6.0 and the absorbance was measured at 260 nm. Polyethylene glycol (PEG) solubility-concentration gradient centrifugations (Clark and Lister, 1971) were performed in 5-0% reverse concentration gradients of :PEG stabilized in 5-30% positive sucrose gradi-
ents in Spinco SW40 tubes centrifuged for 30 min at 12,000 rpm. Sucrose/D 20 gradients were made by layering 0.5 ml each of 70, 65, 60, 55, 50, and 45% sucrose dissolved in buffered D 20 (9 parts D 20 , 1 part 0.5 M acetate pH 6.0) in Spinco SW39 tubes which were then left for 24 hr at room temperature. The virus samples were made 30% in respect to sucrose, and 0.5 ml (about 1.5-3.0 mg virus) was layered on the gradient and then overlaid with paraffin oil. The gradients 'were centrifuged for 72 hr at 25 C at either 33,000 rpm in a SW39 rotor or 44,000 rpm in a SW50.1rotor. Two-drop fractions (0.1 ml) were taken from the bottom of the tubes, and 1 rnl 0.1 M acetate pH 6.0 'was added to alternate fractions which were then monitored at 260 nm. Refractive indices were measured on the other fractions, and the densities were determined from a calibration curve using data from Sober (1968) and taking a density of 1.10 g/cc for the buffered D 20 . Gel electrophoresis. Virus was electrophoresed in gels 7.5 em long and 0.5 em in diameter consisting of' 2.8 or 3.4% polyacrylamide gels (5% cross-linked), containing 3% sucrose, and buffered with 30 mM Tris, 3 mM Ca(OH)2 adjusted to pH 4.4 with lactic acid (virus gel buffer) (Lane and Kaesberg, 1971). Gels were prerun at constant voltage (16 V/cm) until current remained constant (approx 1 hr). Samples of 10-20 J.lg of virus in 25 J.lliters gel buffer containing .5 mg /rnl protamine sulfate, 0.1 M mercaptoethylamine hydrochloride and 20% sucrose (PMS) were applied to the anodic end of the gel. Electrophoresis was carried out at 4 C at 15-17 V/cm (about 2 mA per tube) for about 4 hr with 2.8% gels and 16 hr with 3.4% gels. Buffer was circulated between the upper and lower electrode chambers. The gels were scanned at 265 nm in a Joyce-Loebl "Polyfrac" scanner. RNA was electrophoresed on 2.5 or 2.8% polyacrylamide (5% cross-linked) gels containing 0.1% sodium dodecyl sulfate (SDS) and 3% sucrose. The buffer system was modified from that of Peacock and Dingman (1967) and contained 0.089 M Tris, 0.089 M boric acid, and 1 mM EDTA pH 8.3; the upper reservoir also contained 0.05% SDS.
PEA ENATION MOSAIC VilLUS COMPONENTS
3
Virus RNA was prepared in two ways: The RNA was obtained by phenol ext raction in t he pr esence of 1% w / v bentonite and 2% w/v SDS. After et ha nol precipitation it was heated for 10 min at ,50 C in 0.2% SDS, 1 ill urea, 5% sucrose, and 0.05 M mercaptoetha nol. Alternatively t he virus was heat ed for 10 min at 50 C in electrophoresis buffer containing 1% SDS, 1 ?vI ur ea, 5% sucrose, and 0.05 1If mercaptoethan ol. About 10 J.Lg RNA was applied to each gel and electroph oresis was at room temperat ure and 16 V Icm (about 3 rnA per tube) for 1.5 or 4.5 hr. For formaldehyde modification of the RNA, virus was heated to 60 C for 5 min in 0 .05 M phosphate buffer pH 7.5 containing 1% SDS, 0.5% mercapt oethanol, 1 mM EDTA, and 10% sucrose before adding Fro. 1. Schlieren patterns of unfractionat ed 710 vol of 40% formaldehyde solution and PEMV ; upper, wild type; lower, vari an t. Photofurther heating to 80 C for 10 min. The graph ta kcn 12 min after reaching speed of 42,040 modified RNA was electrophoresed in 2.5% rpm; sedimentation is from left t o righ t. polyacrylamide gels for 2.5 hr using the Wild-t ype PEMV contained more bottom conditions given above for the untreated RNA. t han middle component (Fig. 1 top) whereas For infectivity studies, t he RNA species the variant contained more middle than were ext racte d from polyacrylamid e gels as bottom component (Fig. 1 bottom) . described by Hull (1972). Nucleoprotein Electrophoresis of nucleoprotein. Electrocomponents were locat ed by scanning the phoresis of the virus on polyacrylamide gels gels in t he Joyc e-Loebl "Polyfrac " scanner. resolves two components (Fig. 2). The agreeThe nucleoprotein band s were excised with ment between the component ratios esti raz or blades and homogenized in a volume mated using ultracentrifugation (Fig. 1) and of 0.1 M sodium acet at e adj usted to pH 6.0 gel electrophoresis (Fig. 2) of wild-t ype and with acetic acid containing 5% sucrose and variant PEMV indicates that the slow 0.25% bovine serum albumin (to prevent electrophoretic component corresponds to abs orption of virus to the glassware) to give the bottom centrifugal component and the an estimated concentration of 1 or 2,ug/ml fast electrophoretic component to middle virus. The homogenate was stored overnight component. Further evidence will be preat 4 C prior to bioassay. sented to support this. . At pH 4.4, the relative migration of the RESU LTS middle to bottom component of both strains S edimentation properties of the nucleopro- of PEMV was 1: 0.940 in 2.8% gels and tein. Analytical ultracent rifugation of puri- 1 :0.741 in 3.4% gels (Fig. 3) suggesting t hat fied preparations of PEMV showed t wo t he separat ion was on the basis of size rath er nu cleoprotein components (Fig . 1) with t han charge. The ratio of mobilities was independent of pH between 3.5 and 6.0 S 20.. values of 112 S (bottom component) and 99 S (middle component) at £ 260=1 again indicating that separation was not OD /ml both in 0.1 M aceta te pH 6 and in based on charge. The virus precipitat ed on virus gel electro phoresis buffer. These differ the surface of gels run at pH 8.0 and 8.6. To examine the possibility of orientation slightl y from the pr eviously reported values of 115 and 95 S (Gibbs et al., 1966),113 and or distortion of one of the components in the 94.5 S (Bozarth and Chow, 1966), and 122 electric field, wild-type virus was electroand 106 S (Izadpanah and Shepherd, 1966b). phoresed at low voltage (2 V/ cm for 72 hr
HULL AND LANE
4
a
ttl to N
W
a
L~ b
It)
lD N
W
.. Distance
Ir-----~.~
b
+
FIG. 2. Densitometer tra ces of PEMV nucleopro tein (a) wild t ype ; (b) variant, elec tr ophoresed in 3.4% p olyacrylamide gels at 16 VI cm for 16 hr. The two sm all peaks all th e right of t race (a) are of dimer particles .
on 3.4% gels); it was not feasible to use a voltage significantly less than this. Decreasing the voltage did not affect th e relative mobilities of the two components . Each component of the variant had an electrophoret ic mobilit y of 0.75-0.80 that of the corresponding wild-type component (Fig. 2). Since this ratio was independent of gel pore size, the two strains differ in charge. Electrophoresis of virus at different pHs indicated an isoelectric point of around pH 7 for wild-type PEMV and pH 6 for variant PEMV. Shepherd et al. (1968) showed that in free electrophoresis their strain migrat ed as a single component; they estimated an isoelectric point of pH 5- 6. At pH 4.4, the bottom component of wildty pe PEMV migrat ed identically with brome mosaic virus in both 2.8 and 3.4% gels indicating that the tw o viruses were of similar hydrodynamic size and charg e at th at pH. PEG solubility-concentration gradients. PEG solubility- concentrat ion gradient s can apparently separate virus particles on size or
Distance
+.
FIG. 3. Densitometer traces of va riant PEMV elect rophores ed in (a ) 2.8% poly acryl am ide gel for 9 hr and (b) 3.4% polyacrylamide gel for 16 hr, both at 15 V/cm.
charge (Clark and Lister, 1971). Wild -type PEM V centrifuged on 5-0% PEG solubility-concentration gradients gave a major and a minor band . (Fig. 4). Gel electrophoretic an alysis indicated that the minor band consisted predominantly of the variant strain, which was a contaminant in this preparation. However the minor band was not examin ed in detail and it may be an artifact of this t echnique. In the major band, analysis of fraction 9 plus 10 (Fig. 4) showed that it contained 2.5% middle component whereas fra cti on 16 plus 17 contained 13.8% middle component; the original preparation contained 5.7% middle component . Middle component is ap parently more soluble in PEG than bot tom component. Clark and Lister (1971) showed that t he solubilityconcentra tion of viru s particles was dependent on ionic conditions. Although varyi ng the ionic conditions altered the solubility-concentration for PEMV (Table 1), bottom and middle components could
P EA ENAT lON MOSAIC VIRUS COMPONENTS
5
separated by preparative CsCI density cent rifugat ion (Fig . 5). Th e ultraviolet absorption spect rum of the lighter band (fraction 42) had a peak at 278 nm and a trough at 250 nm (278/260 nm ratio = 1.30) indio cating that it was mainly protein. Fractions 5, 9, and 12 were precipitated from the CsCI by 10% polyethylene glycol and resuspended o in 0.1% 111 acetate pH 6.0. These fractions 1·0 ~ W showed a single 112 S component in the analyt ical ultracentrifuge and a single band in gel electrophoresis comigrating with the slow component of untreated virus (Fig. 6). This further confirms that the slow gel electrophoretic component corresponds to bottom component. A pellet collected from the bottom of the preparative CsCI gradients, had the spectral 25 20 15 10 5 chara cteristics of nucleic acid. Fractions PE:'.fV variant showed only a small amount of material at p = 1.435 gl cc but FIG. 4. Di stribution of 10 mg of wild -ty pe P EMV after cen trifuga ti on for 30 min a t 12,000 much at p = 1.282 gl ce in both analytical rpm in a Spineo SW40 rotor in 5-0% reverse con- and preparative CsCI gradients. Clearly cent rati on grad ient of PEG stabili zed in 5-30% middle component is degraded in CsCI p osi tive Sucr ose gradient. Fract ions of 20 drops gradients. were diluted with 1 ml 0 .1 M acetate pH (i .O before In attempts to stabilize middle comex tin c tion me asureme nts. T op of t ube is on th e ponent by fixation in buffered glutaraldelef t. hyde, th e viru s was precipitated by glutaral dchyde concentrations greater than 1%j TABLE 1 preparations fixed in 0.2 and 0.5% glutaralE ~'F ECT OF N ACL C O N C ~; N 'l'ItATION ON THI~ dehydo behaved sim.ilarly to unfixed ones. SOLUIlILITY CO NCI'; N'I'RATJO N OF TH ~ Two The degradation of middle component by B ANDS OF PEMV IN P EG G l{ADJIo;NTS CsCI was examined using gel elect rophoresis of wild-type PEMV which had been incuNnel molarity % PEG bat ed at room temperature with various Minor band Main band concentrati ons of CsCI (Table 2). From Table 2 it can be seen that middle com1.05 0.05 2 .10 ponent was com.pletely degraded by 1.0 JYI 0.10 0 .95 1.90 CsCI and that Mg2+ appeared to have no 0.92 1. 73 0.20 effect on the degradation. Centrifugation of both wild-typ e and th e not be separat ed. The P E G solubility-con- vari ant st rain of PEJ~dV in isopycnic gradicentration of PEMV is unusually low com- ents of sucrose in D 20 each gave a single pared with that of oth er spherical virus es band at p = 1.36 glcc (Fig. 7). In th e same run (in sister tubes) broad bean mottle virus (Clark and Lister, 1971). Equilibrium centrifugation of nucleoprotein. layered on t he top of one gradient and the Wh en wild-type PEMV was centrifuged to bottom of another, banded in similar posiequilibrium in CsCI densit y gradients two tions (p = 1.33 g/cc) indicating th at the bands were found ; one at p = 1.435 gl ee and syst em was close to equilibrium. The ratios the ot her at p = 1.282 glee. These are close of t he components in fractions 3, 5, 7, 9, 11, to t he valu es reported by Bozarth and Chow 13, and 15 shown in Fig. 7 were estimated by (1966) (for the denser one alone) and by gel electrophoresis. Since this ratio did not Sehgal et al. (1970). The t wo bands were vary mark edly over the various fractions,
6
HULL AND LANE 1-50 1-45
1'40 1·35
Z' CJ
'iJj
-!:!.
~ S
o
o 1'0
'" W N
40
35 25
10
5
FIG. 5. Distribution of 12 mg of wild-type PEMV after centrifugation for 23 hr at 45,000 rpm in a Spin co 50 rotor in 5 ml CsCI p = 1.42 g/cm2 overlaid with light paraffin oil. Two-drop fractions, collected from the bottom of the tube were diluted with 1 ml 0.1 Jli[ acetate pH 6.0 for extinction measure ments after refractive index measurements on samples from every fifth fraction. TABLE 2 DIWRADATION OF MIDDLI" COMPONENT WILD-TYPE PEMV BY GBeL"
a
CsCI concentration
I
~
10
Ul
C'I
w
b
I
l
0 0.5M l.OM
OF
Components as % total virus+ M g2+
+EDTA
E'
M
79.0 87.3 100.0
21.0 12.7 0.0
B
79.0 91.5 100.0
M
21.0 8.5 0.0
a Virus treated with CsCI in presence of either O.OIM EDTA or 0.05 M Mg SO.l . b Estimated from scans of polyacrylamide gels. 'B = bottom component, M = middle component.
...
Distance
FIG. 6. Densitometer traces of wild-type PEMV nucleoprotein (a) from fraction 12 of the CsCI gradient shown in Fig. 5 b) fraction 12 control electrophoresed in 3.4% polyacrylamide gels for 16 hr at 16 VIcm (control is illustrated in Fig. 2a).
+
the two components of PEMV banded at similar densities. This was confirmed by measuring the S20w values of the components in suerose/Dsu solutions of known densities (Fig. 8). The extrapolated flotation density
of p = 1.37 glee was close to that estimated from equilibrium sucrose/Dati gradients (Fig. 7). Ultraviolet absorption. Preparations of wild-type virus (predominantly bottom component), variant isolate (predominantly middle component), and CsCl treated wildtype virus (bottom component) were compared by ultraviolet absorption spectroscopy. The samples exhibited no difference spectrum over the range 225-325 nm. To test the sensitivity of the technique, RNA was added to samples of broad bean mottle virus (22% RNA) to give 23.5 and 25% RNA. With the 22% RNA sample in the reference cuvette and the 23.5% RNA sample in
7
PEA ENATION MOSAIC VIRUS COMPONENTS
1'38 >0..... ...
1.34
(.J
·0..g COl
1·30 ~ ....
20
15
10
5
Fractions FIG. 7. Distribution of variant PEMV in a gradient of 45-70% sucrose made up in D,O buffered with 0.1111 acetate pH 6.0 and centrifuged for 72 hr at 33,000 rpm in a Spinco SW39 rotor. Two-drop fractions were taken from the light scattering zone and refractive index measurements were made from alternate ones. Then 1.0 ml a.livE acetate pH 6.0 was added to each fraction and the extinction was measured. The virus from fractions 3, 5, 7, g, 11, 13, and 15 was concentrated by PEG precipitation and analysed for component ratio (bottom component/middle component) by electrophoresis on 3.4% polyacrylamide gels. 120 • Bottom +Middle 80
40 20
',0
1,'
1·2
1·3
Density D/CC
FIG. 8. Sedimentation coefficients of bottom and middle components of wild-type PEMV in various concentrations of sucrose dissolved in D,O buffered with 0.1 M. acetate pH 6 measured in a Spineo Model E run at 40,000 rpm.
the sample cuvette, a decrease in absorbancy could be detected below 230 nm. When the sample cuvette contained 25% RNA, a marked absorbancy decrease was noted. Since the protein makes a greater relative contribution to the total absorbance of the virus below 230 nm than it does at 280 nm, this method is much more sensitive to variation in protein to RNA ratio than the
more commonly used comparison of absorptions at 260 and 280 nm. Since this technique can detect a 2% difference in percentage of RNA between virus components with RNA contents similar to PEMV, we conclude that the nucleic acid contents of the two PEMV components are similar-certainly within 2%. Electron microscopy. No size differences could be detected by electron microscopy in samples of bottom and middle components of either wild-type or variant isolates of PEMV, separated on polyacrylamide gels (Fig. 9). Measurements from micrographs of 50 particles of each component negatively stained with uranyl acetate gave diameters of 29.5 (±O.6) nm and 29.2 (±1.1) nm for bottom and middle components, respectively. Neither component exhibited structural details when negatively stained in uranyl acetate, uranyl formate, or sodium tungstate/formic acid. Bottom component particles, however, tended to have hexagonal outlines and to aggregate in hexagonal packing. Gibbs et al. (1966) and Farro and Rassel (1971) also noted that bottom component particles were more regular than
8
HULL AND LANE
Fr o. 9. El ectron mi crogruph s of wild -type P EMV components (a) b ottom compo nent , (b) middle c omp onen t , fr a etionut ed 0 11 3.4 % poly acrylamid e gels , negatively stai ned in uranyl acetate ; bar = 100 n m,
those of middle compon ent . The protubera nces on particles, r eported by Bozarth and C how (l9G6 ) , wer e not ob ser ved . It is pr oba ble t hat, as Farro and Rassel (1971) suggested , t hese were effect s of degrad ati on of t he p arti cles in negative stain . Nucl eic acid . E lectrop h oresis of the nu cleic a cid on polya crylamide gels resolv ed t hree nucleic acid compo nents (Fig. IDa-c) as re cently r eported by Gons alves and Shepherd (1972). Thoro were no appare nt differ ences in t he component com posit ions of samples prepared by the phenol te chniq ue a nd t h ose prepared by d ir ect disso ciation of the virus immediately prior to elect rophoresis. The molecular weight s of the three s pecies (1, 2, 3), estim ated by comparison with the E . coli ribosom al R NAs (1.07 and 0.56 X lOG) were 1.42, 1. 15 and abo ut 0.20 X lOG daltons, respectively . R NA species :3 was usually rather het erogeneous and varia ble in appa rent molecular weigh t (0.1100.270 X 10 6) . On exte nd ed elect rophores is, n ucleic acid s pecies 1 divided int o t wo bands (estim at ed m olecul ar w eights ] .42 and 1.45 X 10 6 daltons), B ecau se this did n ut happen with form aldeh yde-m odified RN A, these bands a ppar ently r epresent different
configurations of a single species. T he bands of R NA species 1 and 2 are broad compared wit h bands of R NA species of othe r viruses (Fig. 10). El ect rophoresis of form ald ehyde-modified R NA also gave t wo ma jor bands in proportio ns sim ilar to t hose of un t reat ed RNA (Fig, lOd, e) bu t which were much sharper. It is likely that t he sprea ding of t he ba nds of native R NA refiects configuratio nal het erogeneit y. Es timates of t he molecular weights of formaldehyd e-treated UNA species (using R. coli rib osomal RNAs and TMV RNA as standards) were 1.60 and l.;~O X lOB. These differ subs tan tially from the values reported by Gon salves and Shepherd (1972). I t is well kno wn t hat conditions of electrophoresis affect estimates of molecular weights of unmodified RNA. I t is however difficult t o explain t ho 20-25% differen ces in estima t ed molecular weights for formaldehyde- modifie d R NA. R eiaiionship of RN A species 10 nucleoprolein components. Gonsa lves and Sh epherd ( 972) not ed that t he R N A from virus pr epar ations which had about :~ O% of middle (t heir t op ) componen t contain ed about 60% of species 2. From this and from other d atn ,
PEA ENATIOl\; ",10:-;.-\1 C VIr tU,.; 3
a
21
C O M P ONE NT~
9
phoresis , the proportion of tot al R NA whi ch w as RNA s pecies :3 being estima ted from
short period electrophoresis (1.5 hr at 8 V Icrn) and those of RNA species ] and 2 from long period electrophoresis (5 hr at 14 Vl em). The close correlation of bottom component with H.NA speci es 1 (Table 3) indi cates that bottom nu cleoprotein componen t does not contain appreciable amount s 2 1 of R NA spe cies 2. Since our preparations b contain very lit tle R NA species 3 we are
(\j
\
I\JU \
l~
;
;
~
10
HULL AND LANE TABLE 3 RELATlONSHIP BETwlmN NUCLEOI'ROTEIN COMPO/'lJeN'NI AND RNA SPECIES Preparation
Var. b control Val'. fraction 1
2 4 5 6 7 Variant prep. Wt prep Wt prep Wt prep
RNA species-
Nucleoprotein components' % of total
% of total
Bb
M
1b
2
3
16.6 35.1 34.8 30.4 22.1 8.0 2.0 22.5 59.4 59.6 65.4
83.4 64.9 65.2 69.6 77.9 92.0 98.0 77.5 40.6 40.4 34.6
11.2 32.3 38.3 32.2 21.9 8.8 2.6 20.3 55.9 58.0 66.2
86.0 65.6 60.5 67.8 78.1 91.2 97.4 73.8 38.6 35.6 33.8
2.8 2.1 1.2 _e
5.9 5.5 6.4
• Measured from ultraviolet scans of polyacrylamide gels. b Wt and var. signify wild-type and variant isolates, respectively. Band M signify bottom and middle nucleoprotein components, respectively: RNA species 1, 2, and 3 as shown in Fig. 10. c No peak found. TABLE 4 INFECTIVITIES OF MIXTURES OF PEMV-RNA SPECIES RNA species«
1
+2+
1
+2+
1 3e 2 3 3
1+2+3 1
+
2
+2 +3 1 + 3 1 +2 +3
1
1 a b
2 + 3 +2+3
Lesion numbers"
24.6 54.9 12.1 60.6 0.3 69.0 115.3 63.9 24.2 67.1 24.8 58.8
Species numbers refer to those in Fig. 10. Average lesion numbers on 10 half-leaves of
Chenopodium quinoa. e Mixtures made from equal volumes of extracts of RNA species from polyacrylamide gels.
port our ideas that bottom component does not contain RNA species 2. To determine which component contained the information for coat protein, pea plants were inoculated with homologousand heterologous mixtures of the two nucleoprotein components from the two strains. Mixing of components enhanced infectivity in
heterologous as well as homologous mixtures (Table 5). Virus was prepared from single pea plants by grinding each in 2.5 ml 0.2 M acetate pH 6.0 containing 5% sucrose, shaking the sap with 2.5 ml L:1 n-butanolchloroform, and centrifuging at low speed. The aqueous phase was removed and PEG 6000 was added to it to give 10%; after 3-4 hr at 4 C the extracts were centrifuged. Each pellet was suspended in 0.2 ml virus gel buffer and a 25 ,ulitcr sample (to which was added 25,uliters P.MS) was electrophoresed in a 2.8% polyacrylamide gel. Electrophoretic mobility and the component composition of the heterologous mixtures corresponded to that of parent bottom component. While Gonsalves and Shepherd (1972) associated biological activity with PElVIV RNA species 2, they were unable to associate any activity with RNA species 1. We have found that species 1 enhances infectivity and also that it carries a genetic marker. Our evidence clearly shows that our strain of PEMV has a divided genome. Relationship between our strain of PEMV and that of Shepherd. Our data clearly differ from those of Gonsalves and Shepherd (1972). A double diffusion test utilizing antiserum to a Wisconsin isolate of PEMV (Izadpanah and Shepherd, 1966b) and a
11
PEA ENATION MOSAIC VIRUS COMPONENTS TABLE 5 INFECTIVITIES OF MIXTURES OF PEMV NUCLEOPROTEIN COMPONENTS Experiment
i-
2'
Isolate
and
Nucleoprotein component-
B B+M
Wt Wt Wt Wt Wt Wt Wt Wt Val'. Val'. Val'. Val'. Wt B Wt B Val'. B Val'. B
M B+M B B+M
M
+ val'. M + wt M + wt M + val'. M
B+M B B+M M B+M
Lesion numbers" 1.8 137.8 8.2 162.6
0.8 12.6 4.2 12.6
0.0
5.0 0.8 8.8 5.6
10.0
15.0 5.0
a Wt and val'. signify wild-type and variant isolates, respectively and Band M signify bottom and middle nucleoprotein components, respectively. o Average numbers of lesions on five half-leaves of Chenopodium quinoa. c Experiment 1 inoculum at 2 fLg/rol and Expt 2 inoculum 1 "g/m!.
TABLE 6 ESTIMATED NUMBERS OF PROTEIN SUBUNITS IN PEMV COMPONENTS Componenl
Method of estimation
Molecular weight X 106
Number of protein subunits'
B M
Svedberg equation Svedberg equation
5.57 4.38
182 143
B
Nucleic acid weight and content
5.71
187
4.64
152
M
a Based on a protein content of 72% and a subunit molecular weight of 22,000.
sample of the Davis isolate of PElVIV (Gonsalves and Shepherd, 1972), kindly provided by Dr. R. J. Shepherd, failed to distinguish our isolate from the Davis isolate. We could find no obvious differences in symptomatology. However, our attempts to purify the Davis isolate have resulted in extremely poor yields and a complex pattern on gel electrophoresis. In view of the poor yield the purified material may not be comparable to that of Gonsalves and Shepherd.
If the gel electrophoretic complexity per-
sists with improved purification procedures a comparison of strains will be an extensive undertaking. DISCUSSION
Our strain of PEMV has several unusual features. The two nucleoprotein components have similar nucleic acid contents but yet contain RNA molecules of different sizes. This is reflected by a size difference between the nucleoprotein components which can be detected by gel electrophoresis. Lister ei al. (1972) recently reported that the nucleoprotein components of tobacco streak virus (TSV) are of different sizes. Like PEMV, the components of TSV have similar percentages of RNA contained in capsids of different sizes. However one component of PEMV is degraded by 0.5 M CsCI whereas all those of TSV can withstand CsCI concentrations greater than 2 M. PElVIV differs from the other known multiple genetic component viruses in having the coat protein gene in the largest RNA. All the others have the coat protein gene in the smallest genetically independent RNA molecule.
12
HULL AND LANE
There are t wo major differences between the results obtained wit h our isolates of PEMV and those found with the Davis isolat e by Gonsalves and Shepherd (1972). These are first, the distribution of RNA species in nucleoprotein components, and second, the nature of the infective genome. Relatively simple mutations in the coat protein could produce more complex st rains with greater varieties of part icle ty pes which could obscure the multipartite nature of the genome. Conversion of our strain to a single genetic component virus (or vice versa) would involve a catas trophic mutation. The existence of different PE~/IV strains requiring different numb ers of RNA components for infectivity is therefore unlikely. The molecular weights of the two nucleoprotein component s of PEMV can be estimated by two methods. The first utilizes the Svedberg equation. The sedimentation coefficient s are 1128 for bottom component and 998 for middle component. Diffusion coefficients were estimated indirect ly in the absence of sufficient quanti ties of separ ate components for direct measurement. Bottom component of wild-type PEMV coelectrophoresed at pH 4.4 both in 2.8% and 3.4% acrylarnide gels with brome mosaic virus indicati ng that they had identical hydrat ed diamet ers (and hence identical diffusion coefficients). The diffusion coefficient for brome mosaic virus is 1.55 X 10-7 cm-/sec (Bockst ahler and K aesberg, 1962) and this value was taken for PE MV bottom component. The diffusion coefficient of th e dimer particle will be 0.7 tim es thi s value [molecular weight is twice that of the monomer and the 8 value is 1.4 times that of the monomer (Markham, 1962)]. Since the retardation by the polyacrylamide gel is related to t he diameter (and hence the diffusion coefficient) of the virus parti cle, t he diffusion coeffi cient (D ) for middle component of PEMV was estimat ed from a plot of log l iD against t he ratio of dist ance migrated on a 3.4% gel to that migrated on a 2.8% gel. With monomer and dimer particles of bottom component as standards , t he estimat e of D for middle component is 1.76 X 10-7 cm-/ sec. These values of D are lower than that given by Bozarth and Chow (1966) for unfractionated virus. The components of PEMV have similar
nucleic acid contents, and the value of 28% (Shepherd et al., 1968) was used. The partial specific volume of th e prot ein was calculat ed to be 0.72 cc/ g from the amino acid composition (Shepherd et al., 1968), and that of the R NA was taken as 0.55 cc/g ; this gave a value of 0.672 cc/g for the virus. Fr om these values, the Svedberg equation indi cated molecular weight s of 5.37 X 106 and 4.38 X 10 6 dalt ons for bot tom and middl e components, respectively. Molecular weights were estim at ed to be 5.71 X 106 and 4.64 X 10 6 claltons for bottom and middle components, respectively, from the percentage of RNA and the gel electro phoretic molecular weights of the formaldehyde-treated RNA species. Formaldehyde treatment removed secondary structure in the RNA and produced no app arent crosslinking (Lane, unpublished; Bo edtker, personal communication). The gel electro phoretic molecular weight of modified RNA should reflect the true molecular weight. Th e molecular weights estimate d for bot tom component are close to that suggested by Shepherd ei al. (1968). PEMV has a single coat protein with a molecular weight of 22,000, estimate d by amino acid analysis and SDS gel electrophoresis (Shepher d at al., 1968; Hill and Shepherd, 1972) and confirmed for both isolat es of our strain by SDS gel electro phoresis (Hull , 1971). Using this figure and the estimate d particle molecular weights, bottom component cont ains ab out 180 subunit s (Table 6) consist ent with a particle of icosahedr al symmetry having a triangulation number T = 3. As shown in T able 6, particles of middle component contain less than 180 subunits but more than th e 60 subunits of the icosahedron T = 1. All isometri c viruses so far studied have their prot ein subunits quasiequivalently arr anged in shells of icosah edral symmet ry t he prin ciples of constr uction of which dictat e that t he t riangulation number should be either T = 1,3, 4, 7, etc . (Caspar and Klug, 1962, 1963). It is possible to construct models with icosah edral symmetry but in which the chemical subunit s arc not quasiequivalently arranged. One such model, in which chemical subunits are shared between adj acent hexamer morphological subunits comprises
PEA ENATION MOSAIC VIRUS COMPONENTS
150 chemical subunits, a number consistent with that calculated in Table 6. However in the absence of positive evidence on the structure of middle component, all that can be said is that it appears, from its salt sensitivity and from its size difference, to be structurally different from bottom component. ACKNOWLEDGMENT L. C. L. acknowledges support by Grant number PF 756 from the American Cancer Society. REFERENCES BOCKsTAHLlm, L. E. and KAJ>SBIcltG, P. (1962). The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophys. J. 2,1-9. BClJoJD'l'KKR, H. (1960). Configurat.ional properties of tobacco mosuic virus ribonucleic acid. J. Mol, Bio!. 2, 171-178. BOZARTH, R. F. AND CHOW, C. C. (1966). Pea enation mosaic virus: Purification and properties. Contrib, Boyce Thompson Inet. 28, 301309. CASPAR, D. L. D. and KLUG, A. (1962). Physical principles in the construction of regular viruses, Cold Spring Harbor Symp. Quant. Bioi. 27,1-24. CASPAR, D. L. D. and KLUG, A. (1963). Structure and assembly of regular virus particles. In "Viruses, Nucleic Acids and Cancel'," pp. 27-39. Univ. of Texas M.D. Anderson Hospital and Tumor Institute. The Williams and Wilkins Co. CLARK, M. F. and LISTER, R. M. (1971). The application of polyethene glycol solubility-concentration gradients in plant virus research. Virology 43, 338-351. DINGMAN, C. W., FISHER, M. P., and KAKEFUDA, T. (1972). Role of molecular conformation in determining the electrophoretic properties of polynucleotides in agarosc-acrylamide gels. II. Biochemistry 11, 1242-1250. FARHa, A. and RASSEL,A. (1971). Caraeteristiques en microscopie 6lectronique d'un isolement du virus de la mosaique "enation" du pois. Ann. Phytopathol. 3,53-61. GIBBS, A. J., HARRISON, B. D., and WOODS, R, D. (1966). Purification of pea enation mosaic virus. Virology 29,348-351. GONSALVES, D. and SHlcPlllcRO, R. J. (1972). Biological and biophysical properties of the
13
two nucleoprotein components of pea enation mosaic virus and their associated nucleic acids. Virology 48, 709-723. HAGEDORN, D. J., LAYNE, R. E. C., and RUPPEL, E. G. (1964). Host range of pea enation mosaic virus and use of Chenopodium albl~m as a locallesion host. Phytopathology 64, 843-848. HILL, J. H. and SmcPHEHD, R. J. (1972). Molecular weights of plant virus coat proteins by polyacrylamide gel electrophoresis. Virology 47,817822. HUl'L, H. (1971). Examination of alfalfa mosaic virus protein on polyacrylamide gels. Virology 46,767-772. HULL, R. (1972). The multicomponent nature of broad bean mottle virus and its nucleic acid. J. Gen. Virol. 17,111-117. HULL, H.., HILLS, G. J., and MAHKHAM, R. (1969). Studies on alfalfa mosaic virus. II. The structure of the virus components. Virology 37,416-428. IZADPAHAH, K. and SJ:mnmHD, IL J. (1966a). Gouiciia sp. as a local lesion host for the pea enation mosaic virus. Phytopathology 66, 458459. IZADPANAH, K. and SHEl'HlmD, R. J. (1966b). Purification and properties of the pea enation mosaic virus. Vi'rology 28, 463-476. LANg, L. C. and KAESBEIW, P. (1971). Multiple genetic components in bromegrass mosaic virus. Naiure (London) New Biol, 232,40-43. LISTER, R. M., GJ:Ll.BHI AL, S. A., and SAKSENA, K. N. (1972). Evidence that particle size heterogeneity is the cause of centrifugal heterogeneity in tobacco streak virus. Virology 49, 290-299. MAHKHAM, R. (1962). The analytical ultracentrifuge as a tool for the investigation of plant viruses. Aclvan. 111:1'1I.S Res. 9, 241-270. PEACOCK, A. C. and DINGMAN, C. W. (1967). Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6, 1818-1827. SgHGAL, O. P., JICAN, J-H., BHALLA, R. B., SOONG, M. M., and KRAUSE, G. F. (1970). Correlation between buoyant density and ribonucleic acid content in viruses. Phytopathology 60,1778-1784. SJ:mPHlmD, R. J., WAKEMAN, R. J., and GHABlUAI" S. A. (1968). Preparation and properties of the protein and nucleic acid components of pea enation mosaic virus. Vil'olog1l36, 255-267. SOBEH, H. A. (ed). (1968). "Handbook of biochemistry. Selected data for molecular biology." The Chemical Rubber Co. Cleveland, Ohio.