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The 3′-termini of the genome RNA segments of silkworm cytoplasmic polyhedrosis virus
J. Mol. Biol. (1972) 64, 619-632
The 3’-Termini of the Genome RNA Segmentsof Silkworm Cytoplasmic Polyhedrosis Virus YASUHIRO
FWUICHI
National Institute
AND
KIN-ICHIRO
of Genetics, Mishima,
MIURAT
411, Japan
(Received 17 Februury 1971, and in revised form 15 September 1971) The double-stranded RNA genome of cytoplasmic polyhedrosis virus contains ten kinds of segments, namely twenty RNA terminals. The 3’-termini of these RNA segments were studied by 3H-labeling the terminal ribose moiety, with periodate oxidation and successive reduction using tritiated borohydride. None of the 3’-termini of ten RNA segments were phosphorylated as all were labeled to the same extent. The 3’4erminal nucleosides were determined as 50% cytidine (ten termini) and 50% uridine (ten termini) by analysis of nucleoside tri-alcohols obtained from the ribonuclease T, (or alkali) digest of the 3H-labeled RNA. In a 3H-labeled RNA preparation, radioactive material other than RNA was found. The data suggest that this material may be a complex consisting of polysaccharide and protein.
1. Introduction The cytoplasmic polyhedrosis virus of silkworm is known to contain a helical doublestranded RNA as its genetic material (Miura, Fujii, Sakaki, Fuke & Kawase, 1968). When this RNA is extracted from purified virus by a number of different procedures, a mixture of ten segments is obtained consistently (Fujii-Kawata, lUiura & Fuke, 1970). The presence of such segments has been observed in a number of doublestranded RNA viruses. This list now includes reovirus, wound tumor virus, rice dwarf virus and blue tongue virus (Watanabe & Graham, 1967; Bellamy, Shapiro, August & Joklik, 1967 ; Shatkin, Sipe & Loh, 1968 ; Kalmakoff, Lewandowski $ Black, 1969; Verwoerd, Louw & Oellermann, 1970; Fujii-Kawata et aE., 1970). The fact that none of the different segments of reovirus RNA shows any homolog:y suggests that these segments do not arise by random breakage, but are most likely segments of RNA produced by cleavage at specific weak points in the molecule (Watanabe, Prevec & Graham, 1967 ; Bellamy & Joklik, 1967). Recently, Millward & Graham (1970) found that the double-stranded RNA Iof reovirus exists as a discontinuous structure also inside the virus particle, and both strands of the duplex are interrupted at intervals. However, there is as yet no information about the arrangement or the type of linkage involved in their association. In order to elucidate the structure of the RNA segments in a cytoplasmic pol,yhedrosis virus, the distribution of 3’-terminal nucleoside residues was determined quantitatively. An end-labeling technique was used consisting of periodate oxidation for ribose moieties at 3’-terminals, reduction by tritiated borohydride (RajBhandary, 1968), and analysis of the radioactive nucleoside tri-alcohols released t
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reprint requests
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be sent. 619
620
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PURUICHI
AND
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MIURA
from labeled terminals of the RNA after hydrolysis by ribonuclease ‘I!, The results show that the 3’-termini are not phosphorylated as are those of reovirus RNA (Millward & Graham, 1970) and that the terminal nucleosides are predominantly cytidine (50%) and uridine (50%). It should be emphasized that no adenosine was found, though all the 3’-termini of the RNA viruses so far elucidated have adenosine. In the course of this work, a polysaccharide-protein complex was found in the purified CP virus? RNA.
2. Materials and Methods (a) Preparation
of cytoplasmic
polyhedrosis
viral
RNA
Double-stranded RNA was prepared from purified CP virus by phenol treatment followed by precipitation with 2.5 vol. of ethanol (Miura et al., 1968). When further purification was necessary, the RNA was chromatographed on a mothylated albuminkieselguhr column and/or benzoylated DEAE-cellulose column. The double-strandrd RNA fraction, eluted at 0.8 M-NaCl in the case of methylated albumin-kieselguhr column and 0.6 M in the case of benzoylated DEBE-cellulose column, was pooled and precipitated with 2 vol. of cold ethanol. (b) Prepratior~
of 32P-labeled
cytoplasm&
polyhedrosis
virus
RNA
32P-labeled viral RNA was prepared according to Kawase & Kawamori (1968). 260 silkworm larvae were injected with the CP virus suspension obtained by dissolving cytoplasmic polyhedra in 0.05 M-Na,COa-0.05 M-NaHCO, (pH 10.8). At 30 and 50 hr after infection, O.Olml. of 0.05 &r-phosphate buffer (pH 6.7) containing 32P-labeled phosphoric acid (1 mCi/ml.) was injected into the silkworm. After 20 hr the midguts of the diseased silkworms were removed. The midguts were homogenized in cold water with a Waring Blendor (2 min at high speed) and the homogenate was filtered through gauze and the sap filtrate centrifuged at 8000 g for 10 min, yielding a sediment which contained polyhedra. This sediment was washed several times with cold water until the supernatant fluid was clear. The wet pellet was suspended in 40 ml. of carbonate buffer (9 : 1 mixture of 0.2 or-NaaCO, and 0.2 M-NaHCO, (pH lO.S)), at 20°C. After 1 hr incubation at room temperature, the mixture was diluted with 3 vol. of 0.05 &r-acetate buffer (pH 6.0) and centrifuged at 8000 g for 30 min. The supernatant fraction, in which CP virus was suspended, was centrifuged at 65,000 g for 1 hr. The virus pellet was resuspended in 40 ml. of 0.05 Macetate buffer (pH 6.0) and centrifuged at 10,000 g for 10 mm to remove insoluble materials. The virus preparation thus obtained was almost homogeneous. The phenol treatment used for extraction of 3aP-labeled RNA was essentially the same as that described previously (Miura et al., 1968). The final yield of viral RNA was about 1 mg and the specific radioactivity was 400 cts/min/pg of RNA. (c) Strand
separatiorh
of double-stranded
RNA
In order to denature double-stranded RNA without chain scission, formamide was used as a solvent. Since denatured viral RNA has a tendency to renature and to aggregate, formaldehyde was added to the denaturation solvent as in the case of DNA (Thomas & Berns, 1962). The double-stranded RNA preparation was dissolved in 95% formamide containing 1.5% formaldehyde. After incubation at 60°C for 5 min, it was chilled rapidly to 0°C. Then 2.5 vol. of ethanol containing 1.5% formaldehyde was added and left in a freezer ( - 2O’C) for 4 hr. Denatured RNA precipitate was collected by centrifugation. (d) Preparation Nucleoside corresponding of the reaction 7 Abbreviation
of nucleoside
tri-alcoho1.s
tri-alcohols of adenosine, cytidine and uridine were prepared from the nucleosides as described by RajBhandary (1968), except for the separation products. The products were isolated by passing reaction mixtures through used: CP virus, cytoplasmic
polyhedrosis
virus.
DOUBLE-STRANDED
VIRAL
RNA
621
charcoal columns (1 cm x 5 cm), washing the columns with several column-volumes of water, followed by elution with a water-ethanol gradient (0 to 50%, v/v). The eluate containing nucleoside tri-alcohol was concentrated under reduced pressure, and fractions were chromatographed in solvent A (isopropanol-concentrated ammonium hydroxide-O.1 m-boric acid (7 : 1: 2, by vol.) ). In all cases, no parent nucleoside was left, and a single ultraviolet-absorbing spot was observed which had an increased mobility compared to the corresponding nucleoside. Guanosine tri-alcohol, which was prepared from guanosine R’-monophosphate according to the method of RajBhandary (1968), was kindly supplied by Y. Itano of Nagoya University. (e) 3H-labeling
of d’-terminal
groups
The reaction conditions were essentially the same as those described by Millward 6: units) was dissolved in 1 .O ml. of 0.08 M-sodium Graham (1970). CPviralRNA (14 0.D.26,,nm acetate-O.03 M-EDTA @H 5.0). The phosphatase-treated RNA was similarly dissolved. Phosphatase treatment of CP viral RNA was carried out as follows: about 20 o.D.~@,,~~ units of CP viral RNA were dissolved in 0.6 ml. of 0.5 M-TrisHCl (pH 8.2) and incubated. with 8 units of Escheri&a coli alkaline phosphatase (Worthington) at 37°C for 30 min. followed by phenol treatment. Sodium metaperiodate (0.02 M, 0.25 ml.) was added to the RNA solution and the mixture was kept at 0°C in the dark. After 60 min, 0.35 ml. of 10% (by vol.) solution of propylene glycol was added for a further 10 min to destroy excess periodate. The RNA was precipitated with 2.5 vol. of cold ethanol, washed with ether and dried in a stream of air. Then the precipitate was redissolved in 0.3 ml. of 0.5 M-phosphate buffer (pH 7*0), and mixed with 0.1 ml. of a freshly prepared 0.1 M-solution of 3H-labeled sodium borohydride (specific activity 600 mCi/m-mole; from The Radiochemical Centre, England). After a 4-hr period in the dark at O’C, 0.1 ml. of glacial acetic acid was added to decompose excess borohydride in the mixture. The mixture was filtered through a column of Sephadex G75 equilibrated with 0.01 M-TrisHCl-0.3 M-NaCl-0.001 M-EDTA-7 M-urea, and the ultraviolet-absorbing fractions excluded by the gel were recovered by precipitation with cold ethanol. (f) Polyacrylantide
gel electrophoresis
Preparation of polyacrylamide gel and the electrophoresis were as described previous15 that a solution containing 0.036 M-Tris-0.032 M(Fujii-Kawata et al., 1970), except potassium dihydrogen phosphate (pH 78) and 0.01 M-EDTA was used for preparation of the gel and electrophoresis (Loening, 1969). After electrophoresis, the gel was stained acid for 1 hr and destained in 1 M-acetic acid. with 0.4% acridine orange in 1 M-aCetiC For the estimation of activity, the gel was cut into 1 to 2-mm slices with a razor bladse and the pieces of sliced gel were solubilized with 0.3 ml. hydrogen peroxide (35%) in a vial at 60°C. To these samples were added 15 ml. Kinard’s scintillator (Kinard, 1957) and the radioactivities counted. (Kinard’s scintillator: 5 g PPO (2,5-diphenyloxazole), 0.05 1% dimethyl-POPOP (1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzol), 80 g naphthalene in 1 1. of xylene-1,4 dioxane-ethanol mixture (5 : 5 : 3, by vol.).) (g) Glycerol
gradient
sedimentation
Samples were layered onto 4.5-ml. linear density-gradients (5 to 20% glycerol, 0.02 MTris . HCl (pH 82), 0.1 M-NaCl and 0.001 M-EDTA), and centrifuged at 4°C in a Spinco SW39 rotor at 38,000 rev./min for 4.5 hr. (h) Hydrolysis
by RNase Ts and the analysis terminal nucleoside derivatives
of 3H-labeled
RNA precipitates were dissolved in water; 15 ‘~1. portions containing about 2 :o.D.~~,,~~ units of RNA were adjusted to pH 4.0 by the addition of 2 ~1. of 1 M-sodium acetate-O.25 MEDTA buffer (pH 4.0). To this mixture were added 15 units of ribonuclease T, (Sankyo Ltd.) in 30 ~1. of 0.005 M-acetate buffer (pH 4.0) and incubated at 37°C for 24 hr. A.s reference points for analysis, unlabeled nucleoside tri-alcohols, about 2 0.D.280nm units each, were added to the reaction mixture. The nucleoside derivatives were separated on paper by two-dimensional paper chromatography Whatman no. 1 chromatography
Y. FURUICHI
622
AND
K. MIURA
(ascending), first in solvent A and secondly in solvent B (isobutyric acid-05 M-ammonium hydroxide (10:6, v/v)). The spots were eluted from the paper with water, and the 3H radioactivity was determined in Kinard’s scintillator. Residual paper besides the spots was cut into 1.5-cm squares, and 3H radioactivity was counted iu toluene scintillator (4 g PPO, 0.05 g dimethyl-POPOP and toluene made up to 1 1.). No noteworthy radioactivity was observed, except for the origin; comment on this will be made later. (i) Radioactivity
All determinations Tricarb spectrometer,
of radioactivity model 3315.
assays
were made by scintillation
counting
in a Packard
3. Results (a)
Characterization of labeled cytoplasmic polyhedrosis virus RNA
(i) Sephadex gel jiltration Equivalent amounts of CP virus RNA and phosphatase-treated CP virus RNA were treated with periodate. The oxidized RNA was then reduced with [3H]borohydride, and the product isolated from the reaction mixture by Sephadex G75 gel filtration in the presence of 7 M-urea (Fig. 1). As shown in Figure 1, both samples were tritiated to the same extent, and ultraviolet-absorbing fractions were not observed other than those excluded by the gel, indicating that there was no significant
degradation during the oxidation-reduction
step.
(ii) Polyacrylumide gel electrophoresis The modified RNA was analyzed by polyacrylamide gel electrophoresis, together with 32P-labeled CP viral RNA not modified as a control, which resolved ten segments of CP viral RNA into nine peaks (Fig. 2(a)). It was found that the 3H and 32P coincided for all nine corresponding classes of the RNA resolved; in other words, the
Froct;on no
(b)
Fro. 1. Sephadex G75 gel filtration
of 3H-labeled CP virus RNA. Reaction mixture was layered on a 1.4 cm x 30 cm column of Sephadex G75 equilibrated with 0.01 M-Tris . HCl (pH 7.6) containing 0.3 an-NaCl-0.001 M-EDTA-7 M-urea and &ted with the same buffer.
-m-m-,
(a) CP virus RNA;
(b) phosphatase-treated
Absorbancy at 260 nm; --O-O--,
CP virus RNA.
radioactivity.
DOUBLE-STRANDED
VIRAL
623
RNA
modification did not alter the electrophoretic mobilities, i.e. molecular sizes, of these classes. Each peak in Figure 2(b) incorporated 3H to approximately the same extent, indicating that the termini of ten segments of CP viral RNA are almost uniformly labeled. In addition to the radioactive peaks described above, a large amount of 3H appeared near the origin. 3H in this fraction is considered not to be derived from virus RNA because (1) the origin of the gel was not stained well by acridine orange, (2) of the large amount of radioactivity in comparison with the following nine peaks and (3) no 32P was found at the origin when 32P-labeled CP viral RNA was treated similarly with periodate and non-radioactive borohydride. The nature of this radioactive substance will be described in section (c) below. (iii) Glycerol gradient centrifugation Modified RNA was analyzed by ultracentrifugation as described in Materials and Methods. Typical profiles of absorbancy td radioactivity are shown in Figure 3.
(a)
50(
-F ..g \ I ” I 10 Li 2
30(
I O( (b)
A ‘_ 1 ; .i
30(
Ii! 201
lOi
20
40
60 Dlstonce
00 migrated
100
120
140
(mm)
FIG. 2. Polyacrylamide gel electrophoresis of the sH-end-labeled CP virus RNA. About 1 O.D.Zg,,nm unit of 32P-labeled CP virus RNA and 3H-end-labeled CP virus RNA was applied to a 3’77 polyacrylamide gel (0.6 em x 14 cm) and eiectrophoresed at 6 mA for 12 hr. The gel was stained and then analyzed for radioactivity. A calibrated diagram of the stained gel at t,he top of the Figure demonstrates the correlation between the nine stained bands of double-stranded RNA segments and the radioactive peaks. The anode, cathode and the direction of electrophoresis are indicated. (a) 32P-labeled CP virus RNA; (b) 3H-end-labeled CP virus RNA.
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I.0
0.6
02
g
05
5
z cl 73 =‘ 03 2 D 2 :: 0.1
3
I
05
5
03
3
0.1
I IO
20
30
40
FIG. 3. Glycerol-gradient sedimentation analysis of sH-end-labeled CP virus RNA. CP virus RNA and 3H-end-labeled RNA were layered on a 5 to 20% glycerol gradient and centrifuged for 4.5 hr at 38,000 rev./min at 4°C in a Spinco SW39 rotor. Each fraction was diluted with 0.5 ml. of water and uItravioIet absorption was determined. For the assay of 3H radioactivity, samples of each fraction were dissolved in 10 ml. of Kinard’s scintillator and counted. The bottom of the tube is to the left. (a) Normal CP virus RNA. (b) sH-end-labeled CP virus RNA. (c) 3H-end-labeled CP virus RNA (phosphatase-treated before modification). -O-O--, Absorption at 0.D.260nm; -O-Q-, 3H radioactivity. The arrows indicate the position of E. coli 16 s ribosomal RNA and purified yeast tyrosine tRNA run in the same gradient.
The modified RNA sedimented at 10 to I6 s, as did the control CP virus RNA, when 16 s ribosomal RNA and purified yeast tyrosine tRNA (Puruichi, Wataya, Hayatsu & Ukita, 1970) were used as marker. Since no ultraviolet absorption was found except for 10 to 16 s components, it is suggested that no random breakage occurred during modification.
1st solvent
A
PLATE I. Two-dimensional paper chromat,ography of the products of hydrolysis of end-labeled CP virus RNA. Four nucleoside derivatives were added after hydrolysis as markers. diethylene glycol: U’, uracil-hydroxymet’hyl dbbreviations : C’, cytosine-hydroxymet,hyl diethylene glycol; d’, adenine-hydroxymethyl diethylene glycol: G’, guanine-hydrowymethyl diethylene glycol.
[facing
p. 625
DOUBLE-STRANDED
VIRAL
626
RNA
The profile of 3H radioactivit#y accompanied by ultraviolet absorption was taken as a reasonable indication that the labeling of the 3’-terminus had been almost homogeneous in all the segments, the molecular weights of which vary from 0.35 x IO6 to 2.6 x lo6 daltons (Fujii-Kawata et al., 1970). After these fractions were pooled, the efficiency of the modification was calculated using as reference [3H]uridine trialcohols prepared from uridine and the same lot of [3H]borohydride. About 30% of t’he theoretical number of 3’-termini of double-stranded RNA were labeled during the reduction step, and this is in agreement with recent findings on the reovirus RNA (Millward & Graham, 1970). 3H radioactivity observed at the bottom and near the top of the tube was not from the CP virus RNA (see section (c) below). (b) Analysis
of 3’-terminal
nucleosides
The 3’-terminal labeled RNA was hydrolyzed completely with ribonuclease T, or alkali, and the resulting four kinds of nucleoside derivatives were separated by two-dimensional paper chromatography (Plate I). The area corresponding to nucleoside tri-alcohols was extracted with water and radioactivities counted in Kinard’s scintillator. When the paper was cut into 1.5-cm square pieces and counted in toluene scintillator, little radioactivity was observed except for that at the origin. TABLE
Distributions
RNA subjected analysis
to
(1) CP virus RNA Preparation I Preparation II Preparation III (2) Phosphatasetreated CP virus RNA Preparation I Preparation II
of 3’-terminal
I
nucleosides of cytoplasmic
polyhedrosis
virus RNA
Total radioactivity of nucleoside derivatives (cts/min)
A’
C’
U’
G’
Radioactivity remaining at origin (&/mm)
RNase T, RNase T, Alkali
3526 3217 1275
2.5 0.8 0
43.4 46.6 46.9
44.4 46.6 53.1
9.8 6.0 0
24,600 2270 163
RNase T, RNase T,
293s 3498
0.4 0
45.6 49.1
45.5 48.6
9.0 2.3
16,357 1171
Hydrolysis
Nucleoside derivatives (mole %I
Conditions of hydrolysis by ribonuolease T, and terminal nucleoside determination are described in Materials and Methods. Preparation I: preparation of end-labeled RNA which was obtained by precipitation with ethanol after Sephadex G50 gel filtration. Preparation II : preparation of endlabeled RNA which was purified from preparation I by glycerol gradient sedimentation. Fractions designated in Fig. 3 were pooled and precipitated with 2.5 vol. of cold ethanol. Preparation III: preparation of end-labeled RNA which was purified by glycerol gradient sedimentation similar to preparation II. The starting viral RNA was purified by methylated albumin-kieselguhr column chromatography (Mandell & Hershey, 1960) before 3’terminal modification. Hydrolysis of this preparation was by incubation with 0.3 N-KOH, at 37OC, for 24 hr and the reaction mixture was neutralized with Dowex-50 (H+-form). Radioactive material remaining in each nucleoside derivative was determined in Kinard’s sointillator after extraction with water; radioactivity remaining at the origin was determined by soaking the paper in toluene scintillator. The counting efficiency of the latter was about a quarter of the former.
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The relative distribution of nucleoside derivatives is summarized in Table 1. It is obvious that the major terminal nucleosides are cytosine and uridine in equal amounts, whether the RNA was treated with phosphatase or not. The sum of these pyrimidine nucleosides reached 94 to 98% of the total terminal nucleosides in the purified peparations II and III, which were purified by glycerol gradient centrifugation. Adenosine, which was common in the 3’-terminus of RNA viruses analyzed to date, was scarcely detectable in these preparations. Since guanosine was reduced markedly after purification by glycerol gradient centrifugation, it is uncertain whether 10% of the radioactivity contained in preparation I was really derived from CP viral RNA or not. However, in the case of phosphatase-treated preparations II and III, the amounts of guanosine and adenosine were negligible. Therefore, it is likely that 20 3’-termini of CP viral RNA segments consist of cytidine (50%) and uridine (50%). Essentially the same results were obtained repeatedly when another preparation of CP viral RNA was used. Further, similar results were obtained when CP virus itself was oxidized with periodate, and the RNA extracted was reduced by 3H-labeled sodium borohydride (details to be published). In order to determine whether any deamination had occurred in these experiments, 32P-labeled CP viral RNA and nucleosides were exposed to the same treatments for end-labeling and analyzed as described above. With 32P-labeled CP viral RNA, after chemical modification and digestion with ribonuclease T, , two-dimensional paper chromatography was carried out and radioactivities in the nucleotide spots were determined. The results showed that the relative distribution of radioactivity coincided with the base composition of this RNA, and no selective loss of radioactivity of the nucleotide was observed. In the case of nucleosides, such as cytosine, adenosine and guanosine, when they were subjected to paper chromatography in solvent B after chemical modification and incubation with ribonuclease T,, no ultraviolet-absorbing spots were found except for their corresponding nucleoside derivatives. These facts show that, in this set of experiments there is no specific deamination which produces uridine from cytidine, inosine from adenosine, and xanthosine from guanosine. However, there arose doubt as to whether hydrolysis by ribonuclease T, was complete, since there were large amounts of radioactivity remaining at the origin on the paper. In order to test this possibility the origin on the paper (1.5 cm x 1.5 cm) was cut out and soaked in 3 ml. of 0.01 M-Tris*HCI (pH 7.6) containing 1 mM-EDTA and kept at room temperature with occasional stirring. Half of the radioactivity was extracted from the paper after 48 hr by means of a change of equivalent volume of buffer. When the extracts were passed through a Sephadex 650 column, all radioactivity was excluded by the column. After treatment with ribonuclease T,, paper chromatography was performed in solvent A. As shown in Figure 4 all the radioactive compounds remained at the origin, and no radioactivity was detected at the positions where nucleoside tri-alcohols migrated. These results show that radioactivity remaining at the origin is not unhydrolyzed RNA. Thus, irrespective of treatment with phosphatase, preparation II, which was purified by glycerol gradient centrifugation, contains a much smaller amount of radioactive material at the origin than preparation I. Further, when alkaline hydrolysis was substituted for ribonuclease T, digestion the same results were obtained, as is shown in the case of preparation III in Table 1. Therefore, the conditions for ribonuclease digestion were complete. In order to confirm that only 3’-termini of the genome RNA are labeled with
DOUBLE-STRANDED
VIRAL
RNA
Orlgln
G’
A’,C:U’
Front
t
I
t
t
627
FIG. 4. Paper chromatogram of the radioactive compound, which was eluted from the origin part on Plate I, after repetition of ribonucleasse Ta treatment. A calibrated diagram at the top of the Figure demonstrates the mobilities of four nucleoside derivatives, which were added as markers. The solvent was solvent A.
Fraction
no
Pm. 5. Sedimentation pattern of 3H-end-1abeled 3aP-labeled virus RNA. CP virus RNA uniformly labeled with saP was purified by benzoylated DEAE-cellulose column chromatography. RNA which was &ted by O-6 to 0.7 M-N&I concentration was pooled and precipitated with 2.6 vol. of cold ethanol. The RNA was labeled at the 3’-termini with 3H as described in the text and analyzed by glycerol gradient centrifugation. Preparation was layered on toh 12-ml. linear glycerol gradient (5 to 30%) containing 0.01 M-TrisHCl (pH 7.6), 0.1 M-N&I, 0.001 M-EDTA, and centrifuged in a Spinco SW41 rotor at 33,000 rev./min for 16 hr. 3H radioaotivity ; -O--O-, s2P radioactivity. -•-a-,
628
Y. FURUICHI
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[3H]borohydride, 32P-labeled CP viral RNA which was purified by benzoylated DEAE-cellulose column chromatography was subjected to periodate oxidation and 3H-labeledsodiumborohydridereduction,asdescribedinMaterialsandMet,hods.Doubly labeled CP viral RNA was then analyzed by glycerol density-gradient centrifugation. AS shown in Figure 5 the RNA is almost homogeneously labeled with 3H and the radioactivity observed near the top of the tube is not accompanied by 32P radioactivity. In contrast with Figure 3, no radioact,ivity was found at the bottom of the tube. The bottom material would be removed by the benzoylated DEAE-cellulose column chromatography. The 3H radioactive compound observed near the t,op of the tube is not RNA but some other polymer, because all the radioactivities were excluded by the Sephadex G50 column. The doubly labeled CP viral RNL4 was hydrolyzed by ribonuclease T, and analyzed by paper chromatography. In Figure 6 no 3H radioactivity is observed at the positions to which nucleotides and nucleosides migrated. 3H radioactivity was detected at the origin, where unhydrolyzed RN-4 was not detected, as mentioned above, or at the position where nucleoside tri-alcohols migrate. To eliminate the possibility that there is some internal nucleoside resulting from nicks in the duplex segment, CP viral RNA terminally labeled with 3H was analyzed by 5 to 30% glycerol density-gradient centrifugation after separation of the strands by denaturation with formamide containing formaldehyde. Figure 7 shows that, all the radioactive material sediments at peaks ranging from 18 s to 30 s of single strands corresponding to the full length of every segment and no small fragment wad observed in the centrifugation. We can conclude, therefore, that the RNL4 has no random nick in the strand. These facts indicate that only the 3’-termini of the genome RNA are labeled with [3H]borohydride, and no internal nucleoside residue is labeled.
00 4-
GP
2 5
5 2mv) ‘; I c) I -P
3- --
0 0 QG G’ A U C
Ap UP CP
---I
A’ U’ c’
‘7 i ’
I-J I I I 1 I -2
&L-A 4
-
k
_ _8
_ _- ---12 16
-20
--
24
D~stonce from owqn (cm1
Pm. 6. Paper chromatogram of the ribonuclease T, digest of CP virus RNA. sH-end-labeled CP virus RNA (uniformly labeled with azP) was hydrolyzed with ribonuclease Ta and the reaction mixture was chromatographed on a strip of Whatman no. 1 paper in solvent A. The paper was cut into pieces (I.0 cm x l-6 cm) and the radioactivity was counted in a vial with Kinard’s scintillator. An ultraviolet absorption diagram above the Figure demonstrates the mobilities of four nucleotides (Up, Cp, Ap, Gp), four nucleosides (U, C, A, G) and four nucleoside trialcohols (U’, C’, A’, G’) which were added as markers. , 3H radioactivity; - - - - -, 32P radi0activit.y.
DOUBLE-STRANDED
VIRAL
RNA
629
FIG. ‘7. Strand separation of the terminal labeled double-stranded RNA. (a) The 3H-end-labeled RNA, which was purified by glycerol gradient centrifugation as in Fig. e, (fractions 14 through 17), was denatured in 95% formamide containing 1.5% formaldehyde at, 6O’C for 5 min and cooled rapidly in ice water. Denatured RNA was recovered by ethanol precipi.. tation with carrier RNA obtained from E. coli ribosome. The RNA’s were layered on a 5 to 300/, glycerol gradient containing 1.5% formaldehyde and centrifuged for 15.5 hr at 28,000 rev./min at 4°C in a Spinco SW41 rotor. For the assay of 3H radioactivity, samples of each fraction were dis.. solved in 7 ml. of Kinard’s scintillator and counted. The bottom of the tube is to the left. (b) s2P-labeled double-stranded RNA of CP virus was centrifuged under the same conditions ----, Absorption at O.D.zeonm; -a--•--, 3H radioactivity; -O-O---, a2P radio.. activity.
(c) Characterization of radioactive compounds other than cytoplasmic polyhedrosis viral RNA In order to determine the nature of the radioactive compounds which were found at the bottom and near the top of the glycerol gradient cent’rifugation, each of these in vacuum below fractions was pooled, dialyzed against water and concentrated 30°C. As both of them are excluded by a Sephadex G50 column, their molecular weights would exceed 30,000 daltons. These t.wo compounds remained at the top of t,he polyacrylamide gel on electrophoresis under the conditions in which the double. stranded RNA segments were separated. These compounds did not show ultraviolet absorption at 260nm. When they were treated with ribonuclease T, and subjected t,o two-dimensional paper chromatography as described above, all the radioactive material remained at the origin. These facts indicate that these two radioactive frac.. tions are not RNA but some other polymers. The radioactive fraction obtained from the bottom of glycerol density-gradient; centrifugation was t’reated with (a) alkali, (b) acid, (c) a-amylase and (d) trypsin. The reaction mixture was chromatographed on a Sephadex G50 column and was compared with an untreated control sample. Treatment with alkali, which can hydrolyze CP viral RNA completely, did not have any effect on the elution pattern. However, treatment with acid, the condition which hydrolyzes glycogen to glucose
630
Y.
FURUICHI
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MIURA
(Hassid & Abraham, 1957), degrades the polymer completely to small components. Further, it was found that treatment with ol-amylase and with trypsin decomposed the compound. These facts suggest that the radioactive compound resulted from a polysaccharide-protein complex. In the course of oxidation and reduction for 3H labeling, the sugar moieties in this compound would be modified and tritiated. Hamilton & Smith (1956) showed that polysaccharide can be tritiated by the same method, giving a poly-alcohol. This would explain why t’he bottom fraction in a glycerol density-gradient showed extremely high radioactivities in spite of little ultraviolet absorption. The radioactive fractions observed near the top of the glycerol density gradient showed the same characteristics as the bottom fractions. This indicates that the top component could also be a kind of polysaccharide-protein complex. 4. Discussion It has been shown that genomes of reovirus and CP virus consist of ten doublestranded RNA segments. This raises many questions about their transcription replication and the association of one set of genome and viral coat protein. Elucidation of the terminal sequence of these segments would contribute to the resolution of these problems. In this paper we have studied the relative distribution of 3’-terminal nucleoside of CP viral RNA using a tritium end-labeling technique. It was found that the amount of tritium incorporated into RNA was similar whether or not E. coli alkaline phosphatase treatment was used before labeling. This indicates that the 3’-termini of CP virus RNA are not phosphorylated, as is the case for reovirus RNA (Millward & Graham, 1970). The efIiciency of reduction with [3H]borohydride was about 30%, assuming that RNA from a CP virus particle consists of twenty 3’-termini and 1 mg units in distilled water. The explanation for low efficiency of this RNA is 20 0.D.26,-,nm of the end-labeling is in agreement with the comments of Glitz, Bradley & FraenkelConrat (1968), who applied the same techniques with single-stranded phage MS2 RNA and f2 RNA. When 3H-labeled RNA was characterized by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the results suggested that the RNA was labeled to the same extent for all segments, and no random cleavage of RNA occurred. The results in Figures 6 and 7 indicate that only 3’-terminal nucleosides of the genome RNA were labeled with [3H]b orohydride and no internal nucleoside residues was labeled. Analysis of terminal nucleosides revealed that CP viral RNA contains 50% uridine and 50% cytosine as its 3’-terminus for both phosphatase-treated and non-treated CP viral RNA. Adenosine is common to the 3’-terminus of single-stranded viral RNA such as tobacco mosaic virus RNA (Whitfeld, 1965; Steinschnedier & FraenkelConrat, 1966; Mandeles, 1967), MS2 RNA (Sugiyama, 1965; De Wachter & Fiers, 1967), f2 RNA (Lee & Gilham, 1965; Glitz et al., 1968), RI7 RNA (Dahlberg, 1968) and QB RNA (Weith & Gilham, 1967; Dahlberg, 1968) ; terminal @dine of satellite tobacco necrosis virus RNA (Wimmer & Reichmann, 1969) is an exceptional case. However, in the 3’-termini of the double-stranded CP viral RNA, adenosine was not, found at all. In the case of guanosine, some radioactivity was observed in preparation I (Table 1). However, when the labeled RNA was further purified by glycerol gradient centrifugation before hydrolysis by ribonuclease T, , most of the radioactivity in the guanosine tri-alcohol part disappeared without concomitant loss of radioactivity
DOUBLE-STRANDED
VIRAL
RNA
631
from uridine and cytidine. Therefore, it did not result from CP viral RNA. Further, it’ was confirmed that no specific deamination occurred throughout the whole course of the experiments, as described in Results. From these observations, we conclude that half of the twenty 3’-termini of the RNA segments of CP virus are uridine and the remaining half are cytidine. When RNA. was modified in situ in the virion according t.o the technique of Millward & Graham (1970), the ten distinct RNA segments were equally labeled and analysis of the 3’-terminal nucleoside gave the same results as described above, namely, 500;;, cytidine and 50% uridine (unpublished data). With regard to the distribution of uridine and cytidine of the 3’-terminus of double-stranded RNA, there are three possibilities : (1) terminal uridine and cytidine are distributed at random over the ten segments of CP virus RNA; (2) each of the five segments contains only uridine at its 3’-termini and each of the residual five segments contains only cytidine; (3) ever> segment is composed of two complementary strands in an antiparallel relationship, one strand of which contains uridine and the other strand cytidine. At present it ia: uncertain which of these three possibilities is correct. Some complexes consisting of polysaccharide and protein were observed in the preparation of RNA extracted from the purified CP virus. Since these materials were found also in the RNA preparation which was purified further by methylated albumin-kieselguhr column chromatography (unpublished data), these are likely to originat’e from a virus particle in which these compounds form a complex with RNA. We are indebted to Mr Y. Itano of Nagoya University, for his help in the preliminary st,ages of this work, especially with the preparation of guanosine tri-alcohol. This work was partly the Naito Foundation.
supported
by grants
from
the Ministry
of Education
of Japan
and
REFERENCES Bellamy, A. R. & Joklik, W. K. (1967). J. Mol. Biol. 29, 19. Bellamy, A. R., Shapiro, L., August, J. T. & Joklik, W. K. (1967). J. Mol. Biol. 29, 1. Dahlberg, J. E. (1968). Nature, 220, 548. De Wachter, R. & Fiers, W. (1967). J. Mol. BioZ. 30, 507. Fujii-Kawata, I., Miura, K. & Fuke, M. (1970). J. Mol. BioZ. 51, 247. Furuichi, Y., Wataya, Y., Hayatsu, H. & Ukita, T. (1970). Biochem. Biophys. Res. Comm. 41, 1185. Glitz, D. G., Bradley, A. & Fraenkel-Conrat, H. (1968). Biochim. biophys. Acta, 161, 1. Hamilton, J. K. & Smith, F. (1956). J. Amer. Chem. Xoc. 78, 5907. ed. by S. P. Colowick & Hassid, W. Z. & Abraham, S. (1957). Methods in Enzymology, N. 0. Kaplan, vol. 3, p. 34. New York: Academic Press Inc. Kalmakoff, J., Lewandowski, L. J. & Black, R. D. (1969). J. VGoZ. 4, 851. Kawase, S. & Kawamori, I. (1968). J. Invert. Pathol. 12, 395. Kinard, F. K. (1957). Rew. Sci. In&. 28, 293. Lee, J. C. 8: Gilham, P. T. (1965). J. AmeT. Chem. Sot. 87, 4000. Loening, U. E. (1969). Biochem. J. 113, 131. Mandeles, S. (1967). J. BioZ. Chem. 242, 3103. Mandell, J. D. & Hershey, A. D. (1960). AlzaZyt. Biochem. 1, 66. Millward, S. & Graham, A. F. (1970). Proc. Nat. Acd Sci., Wash. 65, 442. Miura, K., Fujii, I., Sakaki, T., Fuke, M. & Kawase, S. (1968). J. VGoZ. 2, 1211. RajBhandary, U. L. (1968). J. BioZ. Chem. 243, 556. Shatkin, A. J., Sipe, J. D. & Loh, P. (1968). J. V&oZ. 2, 986. Steinschnedier, A. & Fraenkel-Conrat, H. (1966). Biochemistry, 5, 2735. Sugiyama, T. (1965). J. Mol. Biol. 11, 856. Thomas, C. A., Jr. & Berns, K. I. (1962). J. Mol. BioZ. 4, 309. 41
632
Verwoerd, Watanabe, Watanabe, Weith, H. Whitfeld, Wimmer,
Y. FURUICHI
AND
K.
MIURA
K. W., Louw, H. & Oellermann, R. A. (1970). J. Viirol. 5, 1. Y. & Graham, A. F. (1967). J. Vi’iroE. 1, 665. Y., Prevec, L. & Graham, A. F. (1967). Proc. Nut. Acad. Sci., Wa.sh. 58, 1040. & Gilham, P. T. (1967). J. Amer. Chem. Xoc. 89, 5473. P. R. (1965). Biochim. biophys. Acta, 108, 202. E. & Reichmann, M. E. (1969). Nature, 221, 1122.
The 3’-Termini of the Genome RNA Segmentsof Silkworm Cytoplasmic Polyhedrosis Virus YASUHIRO
FWUICHI
National Institute
AND
KIN-ICHIRO
of Genetics, Mishima,
MIURAT
411, Japan
(Received 17 Februury 1971, and in revised form 15 September 1971) The double-stranded RNA genome of cytoplasmic polyhedrosis virus contains ten kinds of segments, namely twenty RNA terminals. The 3’-termini of these RNA segments were studied by 3H-labeling the terminal ribose moiety, with periodate oxidation and successive reduction using tritiated borohydride. None of the 3’-termini of ten RNA segments were phosphorylated as all were labeled to the same extent. The 3’4erminal nucleosides were determined as 50% cytidine (ten termini) and 50% uridine (ten termini) by analysis of nucleoside tri-alcohols obtained from the ribonuclease T, (or alkali) digest of the 3H-labeled RNA. In a 3H-labeled RNA preparation, radioactive material other than RNA was found. The data suggest that this material may be a complex consisting of polysaccharide and protein.
1. Introduction The cytoplasmic polyhedrosis virus of silkworm is known to contain a helical doublestranded RNA as its genetic material (Miura, Fujii, Sakaki, Fuke & Kawase, 1968). When this RNA is extracted from purified virus by a number of different procedures, a mixture of ten segments is obtained consistently (Fujii-Kawata, lUiura & Fuke, 1970). The presence of such segments has been observed in a number of doublestranded RNA viruses. This list now includes reovirus, wound tumor virus, rice dwarf virus and blue tongue virus (Watanabe & Graham, 1967; Bellamy, Shapiro, August & Joklik, 1967 ; Shatkin, Sipe & Loh, 1968 ; Kalmakoff, Lewandowski $ Black, 1969; Verwoerd, Louw & Oellermann, 1970; Fujii-Kawata et aE., 1970). The fact that none of the different segments of reovirus RNA shows any homolog:y suggests that these segments do not arise by random breakage, but are most likely segments of RNA produced by cleavage at specific weak points in the molecule (Watanabe, Prevec & Graham, 1967 ; Bellamy & Joklik, 1967). Recently, Millward & Graham (1970) found that the double-stranded RNA Iof reovirus exists as a discontinuous structure also inside the virus particle, and both strands of the duplex are interrupted at intervals. However, there is as yet no information about the arrangement or the type of linkage involved in their association. In order to elucidate the structure of the RNA segments in a cytoplasmic pol,yhedrosis virus, the distribution of 3’-terminal nucleoside residues was determined quantitatively. An end-labeling technique was used consisting of periodate oxidation for ribose moieties at 3’-terminals, reduction by tritiated borohydride (RajBhandary, 1968), and analysis of the radioactive nucleoside tri-alcohols released t
To whom
reprint requests
should
be sent. 619
620
Y.
PURUICHI
AND
K.
MIURA
from labeled terminals of the RNA after hydrolysis by ribonuclease ‘I!, The results show that the 3’-termini are not phosphorylated as are those of reovirus RNA (Millward & Graham, 1970) and that the terminal nucleosides are predominantly cytidine (50%) and uridine (50%). It should be emphasized that no adenosine was found, though all the 3’-termini of the RNA viruses so far elucidated have adenosine. In the course of this work, a polysaccharide-protein complex was found in the purified CP virus? RNA.
2. Materials and Methods (a) Preparation
of cytoplasmic
polyhedrosis
viral
RNA
Double-stranded RNA was prepared from purified CP virus by phenol treatment followed by precipitation with 2.5 vol. of ethanol (Miura et al., 1968). When further purification was necessary, the RNA was chromatographed on a mothylated albuminkieselguhr column and/or benzoylated DEAE-cellulose column. The double-strandrd RNA fraction, eluted at 0.8 M-NaCl in the case of methylated albumin-kieselguhr column and 0.6 M in the case of benzoylated DEBE-cellulose column, was pooled and precipitated with 2 vol. of cold ethanol. (b) Prepratior~
of 32P-labeled
cytoplasm&
polyhedrosis
virus
RNA
32P-labeled viral RNA was prepared according to Kawase & Kawamori (1968). 260 silkworm larvae were injected with the CP virus suspension obtained by dissolving cytoplasmic polyhedra in 0.05 M-Na,COa-0.05 M-NaHCO, (pH 10.8). At 30 and 50 hr after infection, O.Olml. of 0.05 &r-phosphate buffer (pH 6.7) containing 32P-labeled phosphoric acid (1 mCi/ml.) was injected into the silkworm. After 20 hr the midguts of the diseased silkworms were removed. The midguts were homogenized in cold water with a Waring Blendor (2 min at high speed) and the homogenate was filtered through gauze and the sap filtrate centrifuged at 8000 g for 10 min, yielding a sediment which contained polyhedra. This sediment was washed several times with cold water until the supernatant fluid was clear. The wet pellet was suspended in 40 ml. of carbonate buffer (9 : 1 mixture of 0.2 or-NaaCO, and 0.2 M-NaHCO, (pH lO.S)), at 20°C. After 1 hr incubation at room temperature, the mixture was diluted with 3 vol. of 0.05 &r-acetate buffer (pH 6.0) and centrifuged at 8000 g for 30 min. The supernatant fraction, in which CP virus was suspended, was centrifuged at 65,000 g for 1 hr. The virus pellet was resuspended in 40 ml. of 0.05 Macetate buffer (pH 6.0) and centrifuged at 10,000 g for 10 mm to remove insoluble materials. The virus preparation thus obtained was almost homogeneous. The phenol treatment used for extraction of 3aP-labeled RNA was essentially the same as that described previously (Miura et al., 1968). The final yield of viral RNA was about 1 mg and the specific radioactivity was 400 cts/min/pg of RNA. (c) Strand
separatiorh
of double-stranded
RNA
In order to denature double-stranded RNA without chain scission, formamide was used as a solvent. Since denatured viral RNA has a tendency to renature and to aggregate, formaldehyde was added to the denaturation solvent as in the case of DNA (Thomas & Berns, 1962). The double-stranded RNA preparation was dissolved in 95% formamide containing 1.5% formaldehyde. After incubation at 60°C for 5 min, it was chilled rapidly to 0°C. Then 2.5 vol. of ethanol containing 1.5% formaldehyde was added and left in a freezer ( - 2O’C) for 4 hr. Denatured RNA precipitate was collected by centrifugation. (d) Preparation Nucleoside corresponding of the reaction 7 Abbreviation
of nucleoside
tri-alcoho1.s
tri-alcohols of adenosine, cytidine and uridine were prepared from the nucleosides as described by RajBhandary (1968), except for the separation products. The products were isolated by passing reaction mixtures through used: CP virus, cytoplasmic
polyhedrosis
virus.
DOUBLE-STRANDED
VIRAL
RNA
621
charcoal columns (1 cm x 5 cm), washing the columns with several column-volumes of water, followed by elution with a water-ethanol gradient (0 to 50%, v/v). The eluate containing nucleoside tri-alcohol was concentrated under reduced pressure, and fractions were chromatographed in solvent A (isopropanol-concentrated ammonium hydroxide-O.1 m-boric acid (7 : 1: 2, by vol.) ). In all cases, no parent nucleoside was left, and a single ultraviolet-absorbing spot was observed which had an increased mobility compared to the corresponding nucleoside. Guanosine tri-alcohol, which was prepared from guanosine R’-monophosphate according to the method of RajBhandary (1968), was kindly supplied by Y. Itano of Nagoya University. (e) 3H-labeling
of d’-terminal
groups
The reaction conditions were essentially the same as those described by Millward 6: units) was dissolved in 1 .O ml. of 0.08 M-sodium Graham (1970). CPviralRNA (14 0.D.26,,nm acetate-O.03 M-EDTA @H 5.0). The phosphatase-treated RNA was similarly dissolved. Phosphatase treatment of CP viral RNA was carried out as follows: about 20 o.D.~@,,~~ units of CP viral RNA were dissolved in 0.6 ml. of 0.5 M-TrisHCl (pH 8.2) and incubated. with 8 units of Escheri&a coli alkaline phosphatase (Worthington) at 37°C for 30 min. followed by phenol treatment. Sodium metaperiodate (0.02 M, 0.25 ml.) was added to the RNA solution and the mixture was kept at 0°C in the dark. After 60 min, 0.35 ml. of 10% (by vol.) solution of propylene glycol was added for a further 10 min to destroy excess periodate. The RNA was precipitated with 2.5 vol. of cold ethanol, washed with ether and dried in a stream of air. Then the precipitate was redissolved in 0.3 ml. of 0.5 M-phosphate buffer (pH 7*0), and mixed with 0.1 ml. of a freshly prepared 0.1 M-solution of 3H-labeled sodium borohydride (specific activity 600 mCi/m-mole; from The Radiochemical Centre, England). After a 4-hr period in the dark at O’C, 0.1 ml. of glacial acetic acid was added to decompose excess borohydride in the mixture. The mixture was filtered through a column of Sephadex G75 equilibrated with 0.01 M-TrisHCl-0.3 M-NaCl-0.001 M-EDTA-7 M-urea, and the ultraviolet-absorbing fractions excluded by the gel were recovered by precipitation with cold ethanol. (f) Polyacrylantide
gel electrophoresis
Preparation of polyacrylamide gel and the electrophoresis were as described previous15 that a solution containing 0.036 M-Tris-0.032 M(Fujii-Kawata et al., 1970), except potassium dihydrogen phosphate (pH 78) and 0.01 M-EDTA was used for preparation of the gel and electrophoresis (Loening, 1969). After electrophoresis, the gel was stained acid for 1 hr and destained in 1 M-acetic acid. with 0.4% acridine orange in 1 M-aCetiC For the estimation of activity, the gel was cut into 1 to 2-mm slices with a razor bladse and the pieces of sliced gel were solubilized with 0.3 ml. hydrogen peroxide (35%) in a vial at 60°C. To these samples were added 15 ml. Kinard’s scintillator (Kinard, 1957) and the radioactivities counted. (Kinard’s scintillator: 5 g PPO (2,5-diphenyloxazole), 0.05 1% dimethyl-POPOP (1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzol), 80 g naphthalene in 1 1. of xylene-1,4 dioxane-ethanol mixture (5 : 5 : 3, by vol.).) (g) Glycerol
gradient
sedimentation
Samples were layered onto 4.5-ml. linear density-gradients (5 to 20% glycerol, 0.02 MTris . HCl (pH 82), 0.1 M-NaCl and 0.001 M-EDTA), and centrifuged at 4°C in a Spinco SW39 rotor at 38,000 rev./min for 4.5 hr. (h) Hydrolysis
by RNase Ts and the analysis terminal nucleoside derivatives
of 3H-labeled
RNA precipitates were dissolved in water; 15 ‘~1. portions containing about 2 :o.D.~~,,~~ units of RNA were adjusted to pH 4.0 by the addition of 2 ~1. of 1 M-sodium acetate-O.25 MEDTA buffer (pH 4.0). To this mixture were added 15 units of ribonuclease T, (Sankyo Ltd.) in 30 ~1. of 0.005 M-acetate buffer (pH 4.0) and incubated at 37°C for 24 hr. A.s reference points for analysis, unlabeled nucleoside tri-alcohols, about 2 0.D.280nm units each, were added to the reaction mixture. The nucleoside derivatives were separated on paper by two-dimensional paper chromatography Whatman no. 1 chromatography
Y. FURUICHI
622
AND
K. MIURA
(ascending), first in solvent A and secondly in solvent B (isobutyric acid-05 M-ammonium hydroxide (10:6, v/v)). The spots were eluted from the paper with water, and the 3H radioactivity was determined in Kinard’s scintillator. Residual paper besides the spots was cut into 1.5-cm squares, and 3H radioactivity was counted iu toluene scintillator (4 g PPO, 0.05 g dimethyl-POPOP and toluene made up to 1 1.). No noteworthy radioactivity was observed, except for the origin; comment on this will be made later. (i) Radioactivity
All determinations Tricarb spectrometer,
of radioactivity model 3315.
assays
were made by scintillation
counting
in a Packard
3. Results (a)
Characterization of labeled cytoplasmic polyhedrosis virus RNA
(i) Sephadex gel jiltration Equivalent amounts of CP virus RNA and phosphatase-treated CP virus RNA were treated with periodate. The oxidized RNA was then reduced with [3H]borohydride, and the product isolated from the reaction mixture by Sephadex G75 gel filtration in the presence of 7 M-urea (Fig. 1). As shown in Figure 1, both samples were tritiated to the same extent, and ultraviolet-absorbing fractions were not observed other than those excluded by the gel, indicating that there was no significant
degradation during the oxidation-reduction
step.
(ii) Polyacrylumide gel electrophoresis The modified RNA was analyzed by polyacrylamide gel electrophoresis, together with 32P-labeled CP viral RNA not modified as a control, which resolved ten segments of CP viral RNA into nine peaks (Fig. 2(a)). It was found that the 3H and 32P coincided for all nine corresponding classes of the RNA resolved; in other words, the
Froct;on no
(b)
Fro. 1. Sephadex G75 gel filtration
of 3H-labeled CP virus RNA. Reaction mixture was layered on a 1.4 cm x 30 cm column of Sephadex G75 equilibrated with 0.01 M-Tris . HCl (pH 7.6) containing 0.3 an-NaCl-0.001 M-EDTA-7 M-urea and &ted with the same buffer.
-m-m-,
(a) CP virus RNA;
(b) phosphatase-treated
Absorbancy at 260 nm; --O-O--,
CP virus RNA.
radioactivity.
DOUBLE-STRANDED
VIRAL
623
RNA
modification did not alter the electrophoretic mobilities, i.e. molecular sizes, of these classes. Each peak in Figure 2(b) incorporated 3H to approximately the same extent, indicating that the termini of ten segments of CP viral RNA are almost uniformly labeled. In addition to the radioactive peaks described above, a large amount of 3H appeared near the origin. 3H in this fraction is considered not to be derived from virus RNA because (1) the origin of the gel was not stained well by acridine orange, (2) of the large amount of radioactivity in comparison with the following nine peaks and (3) no 32P was found at the origin when 32P-labeled CP viral RNA was treated similarly with periodate and non-radioactive borohydride. The nature of this radioactive substance will be described in section (c) below. (iii) Glycerol gradient centrifugation Modified RNA was analyzed by ultracentrifugation as described in Materials and Methods. Typical profiles of absorbancy td radioactivity are shown in Figure 3.
(a)
50(
-F ..g \ I ” I 10 Li 2
30(
I O( (b)
A ‘_ 1 ; .i
30(
Ii! 201
lOi
20
40
60 Dlstonce
00 migrated
100
120
140
(mm)
FIG. 2. Polyacrylamide gel electrophoresis of the sH-end-labeled CP virus RNA. About 1 O.D.Zg,,nm unit of 32P-labeled CP virus RNA and 3H-end-labeled CP virus RNA was applied to a 3’77 polyacrylamide gel (0.6 em x 14 cm) and eiectrophoresed at 6 mA for 12 hr. The gel was stained and then analyzed for radioactivity. A calibrated diagram of the stained gel at t,he top of the Figure demonstrates the correlation between the nine stained bands of double-stranded RNA segments and the radioactive peaks. The anode, cathode and the direction of electrophoresis are indicated. (a) 32P-labeled CP virus RNA; (b) 3H-end-labeled CP virus RNA.
624
Y. FURUICHI
AND
K.
MIURA
I.0
0.6
02
g
05
5
z cl 73 =‘ 03 2 D 2 :: 0.1
3
I
05
5
03
3
0.1
I IO
20
30
40
FIG. 3. Glycerol-gradient sedimentation analysis of sH-end-labeled CP virus RNA. CP virus RNA and 3H-end-labeled RNA were layered on a 5 to 20% glycerol gradient and centrifuged for 4.5 hr at 38,000 rev./min at 4°C in a Spinco SW39 rotor. Each fraction was diluted with 0.5 ml. of water and uItravioIet absorption was determined. For the assay of 3H radioactivity, samples of each fraction were dissolved in 10 ml. of Kinard’s scintillator and counted. The bottom of the tube is to the left. (a) Normal CP virus RNA. (b) sH-end-labeled CP virus RNA. (c) 3H-end-labeled CP virus RNA (phosphatase-treated before modification). -O-O--, Absorption at 0.D.260nm; -O-Q-, 3H radioactivity. The arrows indicate the position of E. coli 16 s ribosomal RNA and purified yeast tyrosine tRNA run in the same gradient.
The modified RNA sedimented at 10 to I6 s, as did the control CP virus RNA, when 16 s ribosomal RNA and purified yeast tyrosine tRNA (Puruichi, Wataya, Hayatsu & Ukita, 1970) were used as marker. Since no ultraviolet absorption was found except for 10 to 16 s components, it is suggested that no random breakage occurred during modification.
1st solvent
A
PLATE I. Two-dimensional paper chromat,ography of the products of hydrolysis of end-labeled CP virus RNA. Four nucleoside derivatives were added after hydrolysis as markers. diethylene glycol: U’, uracil-hydroxymet’hyl dbbreviations : C’, cytosine-hydroxymet,hyl diethylene glycol; d’, adenine-hydroxymethyl diethylene glycol: G’, guanine-hydrowymethyl diethylene glycol.
[facing
p. 625
DOUBLE-STRANDED
VIRAL
626
RNA
The profile of 3H radioactivit#y accompanied by ultraviolet absorption was taken as a reasonable indication that the labeling of the 3’-terminus had been almost homogeneous in all the segments, the molecular weights of which vary from 0.35 x IO6 to 2.6 x lo6 daltons (Fujii-Kawata et al., 1970). After these fractions were pooled, the efficiency of the modification was calculated using as reference [3H]uridine trialcohols prepared from uridine and the same lot of [3H]borohydride. About 30% of t’he theoretical number of 3’-termini of double-stranded RNA were labeled during the reduction step, and this is in agreement with recent findings on the reovirus RNA (Millward & Graham, 1970). 3H radioactivity observed at the bottom and near the top of the tube was not from the CP virus RNA (see section (c) below). (b) Analysis
of 3’-terminal
nucleosides
The 3’-terminal labeled RNA was hydrolyzed completely with ribonuclease T, or alkali, and the resulting four kinds of nucleoside derivatives were separated by two-dimensional paper chromatography (Plate I). The area corresponding to nucleoside tri-alcohols was extracted with water and radioactivities counted in Kinard’s scintillator. When the paper was cut into 1.5-cm square pieces and counted in toluene scintillator, little radioactivity was observed except for that at the origin. TABLE
Distributions
RNA subjected analysis
to
(1) CP virus RNA Preparation I Preparation II Preparation III (2) Phosphatasetreated CP virus RNA Preparation I Preparation II
of 3’-terminal
I
nucleosides of cytoplasmic
polyhedrosis
virus RNA
Total radioactivity of nucleoside derivatives (cts/min)
A’
C’
U’
G’
Radioactivity remaining at origin (&/mm)
RNase T, RNase T, Alkali
3526 3217 1275
2.5 0.8 0
43.4 46.6 46.9
44.4 46.6 53.1
9.8 6.0 0
24,600 2270 163
RNase T, RNase T,
293s 3498
0.4 0
45.6 49.1
45.5 48.6
9.0 2.3
16,357 1171
Hydrolysis
Nucleoside derivatives (mole %I
Conditions of hydrolysis by ribonuolease T, and terminal nucleoside determination are described in Materials and Methods. Preparation I: preparation of end-labeled RNA which was obtained by precipitation with ethanol after Sephadex G50 gel filtration. Preparation II : preparation of endlabeled RNA which was purified from preparation I by glycerol gradient sedimentation. Fractions designated in Fig. 3 were pooled and precipitated with 2.5 vol. of cold ethanol. Preparation III: preparation of end-labeled RNA which was purified by glycerol gradient sedimentation similar to preparation II. The starting viral RNA was purified by methylated albumin-kieselguhr column chromatography (Mandell & Hershey, 1960) before 3’terminal modification. Hydrolysis of this preparation was by incubation with 0.3 N-KOH, at 37OC, for 24 hr and the reaction mixture was neutralized with Dowex-50 (H+-form). Radioactive material remaining in each nucleoside derivative was determined in Kinard’s sointillator after extraction with water; radioactivity remaining at the origin was determined by soaking the paper in toluene scintillator. The counting efficiency of the latter was about a quarter of the former.
626
Y. FURUICHI
AND
K. MIURA
The relative distribution of nucleoside derivatives is summarized in Table 1. It is obvious that the major terminal nucleosides are cytosine and uridine in equal amounts, whether the RNA was treated with phosphatase or not. The sum of these pyrimidine nucleosides reached 94 to 98% of the total terminal nucleosides in the purified peparations II and III, which were purified by glycerol gradient centrifugation. Adenosine, which was common in the 3’-terminus of RNA viruses analyzed to date, was scarcely detectable in these preparations. Since guanosine was reduced markedly after purification by glycerol gradient centrifugation, it is uncertain whether 10% of the radioactivity contained in preparation I was really derived from CP viral RNA or not. However, in the case of phosphatase-treated preparations II and III, the amounts of guanosine and adenosine were negligible. Therefore, it is likely that 20 3’-termini of CP viral RNA segments consist of cytidine (50%) and uridine (50%). Essentially the same results were obtained repeatedly when another preparation of CP viral RNA was used. Further, similar results were obtained when CP virus itself was oxidized with periodate, and the RNA extracted was reduced by 3H-labeled sodium borohydride (details to be published). In order to determine whether any deamination had occurred in these experiments, 32P-labeled CP viral RNA and nucleosides were exposed to the same treatments for end-labeling and analyzed as described above. With 32P-labeled CP viral RNA, after chemical modification and digestion with ribonuclease T, , two-dimensional paper chromatography was carried out and radioactivities in the nucleotide spots were determined. The results showed that the relative distribution of radioactivity coincided with the base composition of this RNA, and no selective loss of radioactivity of the nucleotide was observed. In the case of nucleosides, such as cytosine, adenosine and guanosine, when they were subjected to paper chromatography in solvent B after chemical modification and incubation with ribonuclease T,, no ultraviolet-absorbing spots were found except for their corresponding nucleoside derivatives. These facts show that, in this set of experiments there is no specific deamination which produces uridine from cytidine, inosine from adenosine, and xanthosine from guanosine. However, there arose doubt as to whether hydrolysis by ribonuclease T, was complete, since there were large amounts of radioactivity remaining at the origin on the paper. In order to test this possibility the origin on the paper (1.5 cm x 1.5 cm) was cut out and soaked in 3 ml. of 0.01 M-Tris*HCI (pH 7.6) containing 1 mM-EDTA and kept at room temperature with occasional stirring. Half of the radioactivity was extracted from the paper after 48 hr by means of a change of equivalent volume of buffer. When the extracts were passed through a Sephadex 650 column, all radioactivity was excluded by the column. After treatment with ribonuclease T,, paper chromatography was performed in solvent A. As shown in Figure 4 all the radioactive compounds remained at the origin, and no radioactivity was detected at the positions where nucleoside tri-alcohols migrated. These results show that radioactivity remaining at the origin is not unhydrolyzed RNA. Thus, irrespective of treatment with phosphatase, preparation II, which was purified by glycerol gradient centrifugation, contains a much smaller amount of radioactive material at the origin than preparation I. Further, when alkaline hydrolysis was substituted for ribonuclease T, digestion the same results were obtained, as is shown in the case of preparation III in Table 1. Therefore, the conditions for ribonuclease digestion were complete. In order to confirm that only 3’-termini of the genome RNA are labeled with
DOUBLE-STRANDED
VIRAL
RNA
Orlgln
G’
A’,C:U’
Front
t
I
t
t
627
FIG. 4. Paper chromatogram of the radioactive compound, which was eluted from the origin part on Plate I, after repetition of ribonucleasse Ta treatment. A calibrated diagram at the top of the Figure demonstrates the mobilities of four nucleoside derivatives, which were added as markers. The solvent was solvent A.
Fraction
no
Pm. 5. Sedimentation pattern of 3H-end-1abeled 3aP-labeled virus RNA. CP virus RNA uniformly labeled with saP was purified by benzoylated DEAE-cellulose column chromatography. RNA which was &ted by O-6 to 0.7 M-N&I concentration was pooled and precipitated with 2.6 vol. of cold ethanol. The RNA was labeled at the 3’-termini with 3H as described in the text and analyzed by glycerol gradient centrifugation. Preparation was layered on toh 12-ml. linear glycerol gradient (5 to 30%) containing 0.01 M-TrisHCl (pH 7.6), 0.1 M-N&I, 0.001 M-EDTA, and centrifuged in a Spinco SW41 rotor at 33,000 rev./min for 16 hr. 3H radioaotivity ; -O--O-, s2P radioactivity. -•-a-,
628
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[3H]borohydride, 32P-labeled CP viral RNA which was purified by benzoylated DEAE-cellulose column chromatography was subjected to periodate oxidation and 3H-labeledsodiumborohydridereduction,asdescribedinMaterialsandMet,hods.Doubly labeled CP viral RNA was then analyzed by glycerol density-gradient centrifugation. AS shown in Figure 5 the RNA is almost homogeneously labeled with 3H and the radioactivity observed near the top of the tube is not accompanied by 32P radioactivity. In contrast with Figure 3, no radioact,ivity was found at the bottom of the tube. The bottom material would be removed by the benzoylated DEAE-cellulose column chromatography. The 3H radioactive compound observed near the t,op of the tube is not RNA but some other polymer, because all the radioactivities were excluded by the Sephadex G50 column. The doubly labeled CP viral RNL4 was hydrolyzed by ribonuclease T, and analyzed by paper chromatography. In Figure 6 no 3H radioactivity is observed at the positions to which nucleotides and nucleosides migrated. 3H radioactivity was detected at the origin, where unhydrolyzed RN-4 was not detected, as mentioned above, or at the position where nucleoside tri-alcohols migrate. To eliminate the possibility that there is some internal nucleoside resulting from nicks in the duplex segment, CP viral RNA terminally labeled with 3H was analyzed by 5 to 30% glycerol density-gradient centrifugation after separation of the strands by denaturation with formamide containing formaldehyde. Figure 7 shows that, all the radioactive material sediments at peaks ranging from 18 s to 30 s of single strands corresponding to the full length of every segment and no small fragment wad observed in the centrifugation. We can conclude, therefore, that the RNL4 has no random nick in the strand. These facts indicate that only the 3’-termini of the genome RNA are labeled with [3H]borohydride, and no internal nucleoside residue is labeled.
00 4-
GP
2 5
5 2mv) ‘; I c) I -P
3- --
0 0 QG G’ A U C
Ap UP CP
---I
A’ U’ c’
‘7 i ’
I-J I I I 1 I -2
&L-A 4
-
k
_ _8
_ _- ---12 16
-20
--
24
D~stonce from owqn (cm1
Pm. 6. Paper chromatogram of the ribonuclease T, digest of CP virus RNA. sH-end-labeled CP virus RNA (uniformly labeled with azP) was hydrolyzed with ribonuclease Ta and the reaction mixture was chromatographed on a strip of Whatman no. 1 paper in solvent A. The paper was cut into pieces (I.0 cm x l-6 cm) and the radioactivity was counted in a vial with Kinard’s scintillator. An ultraviolet absorption diagram above the Figure demonstrates the mobilities of four nucleotides (Up, Cp, Ap, Gp), four nucleosides (U, C, A, G) and four nucleoside trialcohols (U’, C’, A’, G’) which were added as markers. , 3H radioactivity; - - - - -, 32P radi0activit.y.
DOUBLE-STRANDED
VIRAL
RNA
629
FIG. ‘7. Strand separation of the terminal labeled double-stranded RNA. (a) The 3H-end-labeled RNA, which was purified by glycerol gradient centrifugation as in Fig. e, (fractions 14 through 17), was denatured in 95% formamide containing 1.5% formaldehyde at, 6O’C for 5 min and cooled rapidly in ice water. Denatured RNA was recovered by ethanol precipi.. tation with carrier RNA obtained from E. coli ribosome. The RNA’s were layered on a 5 to 300/, glycerol gradient containing 1.5% formaldehyde and centrifuged for 15.5 hr at 28,000 rev./min at 4°C in a Spinco SW41 rotor. For the assay of 3H radioactivity, samples of each fraction were dis.. solved in 7 ml. of Kinard’s scintillator and counted. The bottom of the tube is to the left. (b) s2P-labeled double-stranded RNA of CP virus was centrifuged under the same conditions ----, Absorption at O.D.zeonm; -a--•--, 3H radioactivity; -O-O---, a2P radio.. activity.
(c) Characterization of radioactive compounds other than cytoplasmic polyhedrosis viral RNA In order to determine the nature of the radioactive compounds which were found at the bottom and near the top of the glycerol gradient cent’rifugation, each of these in vacuum below fractions was pooled, dialyzed against water and concentrated 30°C. As both of them are excluded by a Sephadex G50 column, their molecular weights would exceed 30,000 daltons. These t.wo compounds remained at the top of t,he polyacrylamide gel on electrophoresis under the conditions in which the double. stranded RNA segments were separated. These compounds did not show ultraviolet absorption at 260nm. When they were treated with ribonuclease T, and subjected t,o two-dimensional paper chromatography as described above, all the radioactive material remained at the origin. These facts indicate that these two radioactive frac.. tions are not RNA but some other polymers. The radioactive fraction obtained from the bottom of glycerol density-gradient; centrifugation was t’reated with (a) alkali, (b) acid, (c) a-amylase and (d) trypsin. The reaction mixture was chromatographed on a Sephadex G50 column and was compared with an untreated control sample. Treatment with alkali, which can hydrolyze CP viral RNA completely, did not have any effect on the elution pattern. However, treatment with acid, the condition which hydrolyzes glycogen to glucose
630
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FURUICHI
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(Hassid & Abraham, 1957), degrades the polymer completely to small components. Further, it was found that treatment with ol-amylase and with trypsin decomposed the compound. These facts suggest that the radioactive compound resulted from a polysaccharide-protein complex. In the course of oxidation and reduction for 3H labeling, the sugar moieties in this compound would be modified and tritiated. Hamilton & Smith (1956) showed that polysaccharide can be tritiated by the same method, giving a poly-alcohol. This would explain why t’he bottom fraction in a glycerol density-gradient showed extremely high radioactivities in spite of little ultraviolet absorption. The radioactive fractions observed near the top of the glycerol density gradient showed the same characteristics as the bottom fractions. This indicates that the top component could also be a kind of polysaccharide-protein complex. 4. Discussion It has been shown that genomes of reovirus and CP virus consist of ten doublestranded RNA segments. This raises many questions about their transcription replication and the association of one set of genome and viral coat protein. Elucidation of the terminal sequence of these segments would contribute to the resolution of these problems. In this paper we have studied the relative distribution of 3’-terminal nucleoside of CP viral RNA using a tritium end-labeling technique. It was found that the amount of tritium incorporated into RNA was similar whether or not E. coli alkaline phosphatase treatment was used before labeling. This indicates that the 3’-termini of CP virus RNA are not phosphorylated, as is the case for reovirus RNA (Millward & Graham, 1970). The efIiciency of reduction with [3H]borohydride was about 30%, assuming that RNA from a CP virus particle consists of twenty 3’-termini and 1 mg units in distilled water. The explanation for low efficiency of this RNA is 20 0.D.26,-,nm of the end-labeling is in agreement with the comments of Glitz, Bradley & FraenkelConrat (1968), who applied the same techniques with single-stranded phage MS2 RNA and f2 RNA. When 3H-labeled RNA was characterized by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the results suggested that the RNA was labeled to the same extent for all segments, and no random cleavage of RNA occurred. The results in Figures 6 and 7 indicate that only 3’-terminal nucleosides of the genome RNA were labeled with [3H]b orohydride and no internal nucleoside residues was labeled. Analysis of terminal nucleosides revealed that CP viral RNA contains 50% uridine and 50% cytosine as its 3’-terminus for both phosphatase-treated and non-treated CP viral RNA. Adenosine is common to the 3’-terminus of single-stranded viral RNA such as tobacco mosaic virus RNA (Whitfeld, 1965; Steinschnedier & FraenkelConrat, 1966; Mandeles, 1967), MS2 RNA (Sugiyama, 1965; De Wachter & Fiers, 1967), f2 RNA (Lee & Gilham, 1965; Glitz et al., 1968), RI7 RNA (Dahlberg, 1968) and QB RNA (Weith & Gilham, 1967; Dahlberg, 1968) ; terminal @dine of satellite tobacco necrosis virus RNA (Wimmer & Reichmann, 1969) is an exceptional case. However, in the 3’-termini of the double-stranded CP viral RNA, adenosine was not, found at all. In the case of guanosine, some radioactivity was observed in preparation I (Table 1). However, when the labeled RNA was further purified by glycerol gradient centrifugation before hydrolysis by ribonuclease T, , most of the radioactivity in the guanosine tri-alcohol part disappeared without concomitant loss of radioactivity
DOUBLE-STRANDED
VIRAL
RNA
631
from uridine and cytidine. Therefore, it did not result from CP viral RNA. Further, it’ was confirmed that no specific deamination occurred throughout the whole course of the experiments, as described in Results. From these observations, we conclude that half of the twenty 3’-termini of the RNA segments of CP virus are uridine and the remaining half are cytidine. When RNA. was modified in situ in the virion according t.o the technique of Millward & Graham (1970), the ten distinct RNA segments were equally labeled and analysis of the 3’-terminal nucleoside gave the same results as described above, namely, 500;;, cytidine and 50% uridine (unpublished data). With regard to the distribution of uridine and cytidine of the 3’-terminus of double-stranded RNA, there are three possibilities : (1) terminal uridine and cytidine are distributed at random over the ten segments of CP virus RNA; (2) each of the five segments contains only uridine at its 3’-termini and each of the residual five segments contains only cytidine; (3) ever> segment is composed of two complementary strands in an antiparallel relationship, one strand of which contains uridine and the other strand cytidine. At present it ia: uncertain which of these three possibilities is correct. Some complexes consisting of polysaccharide and protein were observed in the preparation of RNA extracted from the purified CP virus. Since these materials were found also in the RNA preparation which was purified further by methylated albumin-kieselguhr column chromatography (unpublished data), these are likely to originat’e from a virus particle in which these compounds form a complex with RNA. We are indebted to Mr Y. Itano of Nagoya University, for his help in the preliminary st,ages of this work, especially with the preparation of guanosine tri-alcohol. This work was partly the Naito Foundation.
supported
by grants
from
the Ministry
of Education
of Japan
and
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K. W., Louw, H. & Oellermann, R. A. (1970). J. Viirol. 5, 1. Y. & Graham, A. F. (1967). J. Vi’iroE. 1, 665. Y., Prevec, L. & Graham, A. F. (1967). Proc. Nut. Acad. Sci., Wa.sh. 58, 1040. & Gilham, P. T. (1967). J. Amer. Chem. Xoc. 89, 5473. P. R. (1965). Biochim. biophys. Acta, 108, 202. E. & Reichmann, M. E. (1969). Nature, 221, 1122.