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Properties of RNA from cytoplasmic-polyhedrosis virus of the white-marked tussock moth, Orgyia leucostigma
JOWNAL
OF
INVEIITEBI~ATE
PATHOLOGY
Properties of RNA White-marked
16,
451458
from Cytoplasmic-Polyhedrosis Virus Tussock Moth, Orgyia Zeucostigma Y.
Department
( 1970)
of Fisheries Canada,
and Sault
HAYASHI
Forestry, Insect Puthology Research Ste. Marie, Ontario, Canadur
Received
of the
April
Institute.
10, 1970
When the genome RNA is extracted from the cytoplasmic-polyhedrosis virus of the white-marked tussock moth, Orgyia Zeucostigma, by the phenol method, it has two components with sedimentation rates of about 15 and 12 S. Their base composition includes adenine and uracil as well as guanine and cytosine. The grraninc + cytosine content was 41.2%. Melting temperature of the RNA was about 80°C in a solution containing 0.0015 M NaCl-0.00015 M sodium citrate. Most of the RNA was found to be resistant to ribonuclease digestion; some of it was partially sensitive and was presumed to be single-stranded. After enzyme treatment, the 15 and 12 S components of the genome sedimented at about 9 S. From these findings it is proposed that viral genome RNA from cytoplasmic-polyhedrosis virus is double-stranded.
Cytoplasmic-polyhedrosis viruses ( CPV) are widely distributed among lepidopterous insects. Serological evidence suggests that they are not the same (Krywienczyk et al., 1969). CPV from the silkworm, Bombyx nzori (Hosaka and Aizawa, 1964) and the forest tent caterpillar, Malacosomu disstriu (Hayashi and Bird, 1968a) have an icosahedral capsid about 70 rnp in diameter which has 12 prominent projections. It was previously reported (Hayashi and Kawase, 1964) that the RNA of CPV from B. mori displays an equimolar base composition of adenine to uracil and guanine to cytosine suggesting that the RNA is doublestranded. Recently Miura et al. (1968) have confirmed the double-stranded nature of the RNA of CPV of B. mori. They have shown that the RNA, in addition to having a complementary base composition, has a sharp melting profile, resistance to ribonuand a characteristic clcase (RNase), 1 Contribution
No.
156
from
this
double-stranded X-ray diffraction picture. The CPV virion obtained from the whitemarked tussock moth, Orgyia leucostigma (Bird, 1965; Hayashi and Bird, 196813) was found to be similar to B. mori CPV in size, surface structure (Nishimura and Hosaka, 1969), and sedimentation behavior (Miura et al., 1968). This virion also contained about 28% RNA (Hayashi and Bird, 1968b). These similarities suggest that the RNA of CPV of 0. Zeucostigmu might also be a double-stranded polynucleotide. Evidence is presented in this paper to show that the CPV-RNA is double-stranded and is similar to RNA from reovirus (Gomatos and Tamm, 1963a; Langridge and Gomatos, 1963), wound tumor virus (Gomatos and Tamm, 1963b), rice dwarf virus (Miurn et al., 1966), and bluetongue virus (Verwoerd, 1969).
MATERIALS
AND
Virus. The CPV was larvae of 0. leucostigma.
Institute. 451
METHODS
multiplied in the Unless otherwise
,452
HAYASHI
stated, the free virions from midgut epithelial cells were used in all the experiments (Bird, 1965; Hayashi and Bird, 1970). Mode of infection and radioisotope labeling. The larvae were reared on a meridic diet (McMorran, 1965) and, when they reached the 4th instar, were transferred to diets coated on the surface with a virus suspension containing lo7 polyhedra in 1 ml of water. Twenty-four hours after virus inoculation, the diets were replaced with radioactive ones containing 5 yCi uridine-3H/cm3 diet. The larvae were removed from the radioactive diet after 10 at which time the midguts were days, chalky-white due to the occurrence of the virus. The midguts of 10,000 insects were dissected out, washed twice, and stored in distilled water at 4°C until required. During this period the cells lysed. Purification of virus. The lysed midguts were homogenized in a Teflon-glass homogenizer. The homogenate was filtered through 4 layers of cheesecloth, and centrifuged at 10,000 g for 10 min at 2°C. The free virions remained in the supernatant whereas the polyhedra sedimented. The purification of the virus was carried out as described previously (Hayashi and Bird, 1968a, 1970). The virus isolated by sucrose gradient centrifugation was further purified by rebanding in a sucrose gradient (Fig. 1). This virus preparation was estimated to be 95% pure by electron microscopy. Extraction of RNA. The virions were suspended in TK buffer (0.03 M Tris-HCl, pH 7.5, 0.025 M KCl) and sodium dodecyl sulfate (SDS) was added to make a final concentration of 0.5%. After 2 min, RNA was extracted by the addition of an equal volume of 90% fresh phenol to the virus suspension and shaking for 10 min at 4°C. The aqueous and phenol layers were separated by centrifugation at 10,000 g for 10 mm and the phenol layer discarded. The purified by aqueous phase was further
20 Tube
no.
FIG. 1. Sedimentation profile of purified CPV virions. Virus preparation was layered over sucrose gradient ( 1040% ) and centrifuged in the SW 25.1 rotor of a Spinco model L-2 ultracentrifuge for 90 min at 23,000 rpm, -O-m-, optical density at 260 rnp; -O--O--, cpm. repeating the process with fresh phenol. The aqueous layer containing the RNA was carefully collected and the RNA was converted to its sodium salt by adding 2 drops of 1 M NaCl. It was then dehydrated by adding 3 volumes of cold 95% ethanol, which gave it a fibrous appearance similar to DNA. The fibrous RNA was washed several times with cold 70% ethanol and stored at -20°C until required. Cellular RNA from the midguts of tussock moth and rat liver was extracted by the method of Applebaum et al. (1966) using bentonite instead of polyvinyl sulfate (Petermann and Pavlovec, 1963). Sedimentation analysis and radioactivity assay. All RNA analyses were carried out by zonal centrifugation in a S-25% linear sucrose gradient by the method of Britten and Roberts (1960). RNA was dissolved in TK buffer, layered on the gradient, and
PROPERTIES
OF
centrifuged in an SW 25.1 or SW 40 Ti rotor in a Spinco Model L-2 or L-2-65B preparative ultracentrifuge at 5°C (see legends of Fig. 1, 2, 3, and 6 for details). The optical densities of the fractions that were collected from the bottom of the tube after centrifugation were measured at 260 mu in a Beckman DU spectrophotometer. To each fraction was added 200 ng of yeast RNA as a carrier, followed by the addition of an equal volume of cold 10% trichloroacetic acid (TCA) to precipitate the RNA. The precipitates were collected on glass fiber filters and washed several times with cold 5% TCA, placed in scintillation vials, and dried under an infrared lamp. Ten milliliters of toluene scintillation fluid (LiquiHuor, 4 g 2, 5diphenyloxazole, and .50 mg 2. 2-p-phenylenebis (5-phenyloxazole) per liter of toluene) was added to each vial and the radioactivity, counted in a Packard Tri-Carb scintillation spectrometer. Digestion of RNA z&h RNase. The RNA purified bv the sucrose gradient method was reprecipitatcd with ethanol at -20°C for 3 days. The precipitate was dissolved in
4.3
CT\‘-RX.%
SSC (0.15 ~1 NaCI-6.015 M sodium citrate) (Bellamy et al., 1967) and digested with different amounts of RNase for 30 mm at 30°C. The reaction mixtures were prchcipitated with TCA and the radioacti\.it\. was determined. The comets rcpresentcd the radioactivity of the viral R?rTA that VYIS not digested by the RNase. Thermal denaturation of RNA. RNA ( 50 ug/ml) was dissolved in 0.01 SSC (MuIra et al., 1966) and the melting curve was ob tained using the heated cell assemblv of ;I Gilford recording spectrophotometcr. Chemicals. Uridine-6-‘:H ( 10 Ci/mmole j and RNase (ribonuclease A) were obtairrcd from New England Nuclear Corp., Boston, Massachusetts, and Worthington Biochelnical Corp., Freehold, New Jrrsc!., resp(x;tivcb.. REWLTS
Sedimentation
Profiles of Viral RNA
Sedimentation profile of RNA extracted from the virion was obtained b\. sucrose gradient centrifugation, and the results arc shown in Fig. 2. IVhen 400 ug RNA \v;ts
Tube
no.
FIG. 2. Sedimentation profiles of RNA extracted from virions. (a) 400 pg RNA crose gradient (5-25% ) and centrifugation was performed in the SW 25.1 rotor for (b) 150 pg RNA after dialysis was centrifuged for 22 hr at 24,000 rpm.
was analyzed by su19 hr at 23,000 rpm.
454
HAYASHI
Tube
FIG. 3. Sedimentation liver RNA (a) or host at 23,000 rpm; O-O,
cell
profiles RNA optical
no.
of viral RNA labeled with uridine-sH centrifuged together (b). Centrifugation was performed in the SW 40 Ti rotor density; 0 - - - 0, cpm.
loaded on the gradient, only one component was observed in the gradient with broad banding (Fig. 2a). A dilute RNA sample ( 150 ug), however, showed a heterogeneous component with a shoulder at tube 18 (Fig. 2b) suggesting the existence of different sizes of viral RNA. The sedimentation coefficients of the main peak and the shoulder part were determined to be 15 S and 12 S, respectively, using RNA from rat liver (Fig. 3a) and host midgut (Fig. 3b) as markers. These values are virtually the same as those obtained from the CPV of B. mori (Miura et al., 1968). The appearance of the second component when RNA was diluted is also similar to that of the RNA of the CPV of B. mori (Hayashi et al., 1965; Miura et al., 1968). The separation of these components from the RNA of the CPV by prolonged centrifugation is possible and is currently under study. The absorption spectrum of the RNA preparation showed a characteristic peak at 259 mu with a 2591232 ratio of 2.2 (Fig. 4) similar to that of RNA from B. mori CPV (Hayashi and Kawase, 1965).
with rat for 19 hr
Base Composition The base composition of the RNA from free virions was determined by the method of Markham and Smith (1951) and is
Wave
length
FIG. 4. Absorption spectrum from CPV-virion. The sample TK buffer.
(mr)
of RNA extracted was dissolved in
PROPERTIES
TABLE
IF
1
I
Base
Guanine Adenine Cytosine I’racil
21.7 28.4 19.5 30.4
A/U
0.93 -
C/C
pu/pyc
-
-
1.11
1.00
-
-
I
I
occluded
BASE COMPOSITION OF V-RNAa Molar ratio”
455
CPV-RNA
I
,
virus-RNA
130 I
G+C (% )
I
,-
I’ !/
/
41.2 -
a Expressed as moles per 100 moles of total base. b Based on 5 analyses on 3 preparations. c pu, purine; py, pyrimidine.
shown in Table 1. It is apparent from the table that the molar percent of guanine closely approximates that of cytosine, and the ratio of adenine and uracil is approximately the same. This indicates that the RNA of the CPV has base pairing similar to that reported from double-stranded RNA viruses as mentioned above. The molar percent of guanine and cytosine together is 41.2% and is comparable to the RNA of the CPV from B. mori (Hayashi and Kawase, 1964; Miura et al., 1968).
Melting
Behavior
The thermal denaturation of RNA in 0.01 SSC from the occluded virion, free virions, and ribosomes is shown in Fig. 5. The RNA from occluded as well as free virions exhibit sharp melting profiles with a Tm value of about 80°C. The hyperchromic increase of RNA from the free virions was about 26%, but that obtained from the occluded virions was about 34%. On the other hand, the single-stranded rRNA (ribosomal RNA) under similar conditions melted over a wide range. Thus, the typical difference between singlestranded and double-stranded RNA became apparent. The sharp melting profile from the viral RNA corresponded precisely with the melting point profile obtained from the RNA of the CPV of B. mori (Mima et al., 1968) under the same conditions.
FIG. 5. Thermal denaturation of viral RNA extracted from free and occluded virion, and midgut ribosomal RNA.
Resistance to Digestion It
has been
shown
by RNase in the
case of the
reovirus that the genome RNA was doublestranded, based on RNase resistance among other properties (Gomatos and Tamm, 1963a). If the RNA of the CPV is resistant to RNase, then a secondary structure for the RNA can be inferred. With such a goal in mind, RNase resistance of the RNA of the CP\’ was studied. RNA of the CPV
labeled with uridine-“H was incubated with various amounts of RNase. Table 2 shows the radioactivity of the acid-insoluble material after RNase treatment, indicating that it is resistant to the enqme digestion. About 20% of tritium activity was found in the acid-soluble fraction when 5 pg/ml of RNase was used, suggesting that the RNA is either single-stranded in part or the base pairing is loose in some regions. The rate of RNA digestion did not increase significnnth,
even
Altkoueh
at a lcvcl
of 150 lr,g RN&. is rt’-
double-stranded RNA
456
HAYASHI
EFFECT
OF RNASE
Concentration ( wc/mI 1 0 1 5 10 30 75 150
TABLE 2 CONCENTRATION
ON THE V-RNAQ
Radioactivity acid-insoluble material ( cpnl)
OF HEAT
of Recovery (So)
Temperature (‘-Cl
100 80.9 81.6 84.6 78.7 74.7 70.2
1058.3 856.3 863.5 895.0 833.2 790.6 742.8
a Viral RNA was dissolved cubated at 30°C for 30 min.
EFFECT
in
1.0 SSC
and
in-
&ant to RNase, the sedimentation behavior is strongly influenced by the enzyme treatment (Fenwick et al., 1964). Figure 6 shows the sedimentation profile of viral RNA after RNase treatment. The sedimentation rate of the material forming the peak which is resistant to RNase was reduced to about 9 S. This presumably represents a single component with perfect base-pairing by hydrogen bonding resembling the rigid structure of DNA. On the other hand, the broad band at 15-12 S before RNase treatment may contain the
Tube
FK. 6. Sedimentation profiles (200 ug RNA) were centrifuged optical density; 0 --0 -0,
of viral RNA in the sucrose 0, cpm.
( Zontrol 23 40 50 60 70 80 90 100
TABLE 3 ON THE RNASE OF V-RNAa
Radioactivity acid-insoluble material (wm) 3618.4 3650.0 3038.0 3030.4 2909.3 3073.1 3035.9 2892.6 2370.7
STABILITY
of Recovery (%) 100.0 100.9 84.0 83.7 80.4 84.9 83.9 79.9 65.5
a V-RNA was dissolved in 1.0 SSC, incubated at different temperatures for 15 min and cooled iu ice. These RNA solutions were treated with 10 pg of RNase/ml for 30 min at 30°C.
enzyme-susceptible components as mentioned above. RNase-resistance of heat-denatured RNA in 1.0 SSC was examined, and the results are shown in Table 3. The acid-insoluble materials (viral RNA) decreased about 16% at 40°C after RNase treatment. No further digestion occurred until the temperature was increased to 90°C. There was
no
after RNase treatment. gradient at 24,000 rpm
After RNase treatment, samples for 22 hr in the SW 25.1 rotor.
PROPERTIES
rapid digestion between 90 and 100°C. This is probably due to the denatured RNA becoming partly single-stranded permitting the RNase to digest the singlestranded portion. Therefore, the melting profile of the denatured RNA was quite different from that of the RNA in 0.01 SSC (Fig. 5). The results obtained in this experiment support the hypothesis that the RNA of the CPV from 0. Zeucostigma is double-stranded similar to that obtained from the CPV of B. mori (Hayashi and Kawase 1964; Miura et al., 1968). DISCUSSION
It is clear that the RNA extracted from the virions of CPV is double-stranded because of its characteristic base composition, sharp melting profile, and resistance to RNase. However, CPV-genome RNA was released from the virions as a mixture of RNA fragments consisting of at least 2 components (Figs. 2, 3). The distribution of size of these fragments is highly reproducible in the gradient analysis even when 6-month-old virions are used. This finding, however, is not consistent with the electron microscopical observation reported by Nishimura and Hosaka ( 1969). According to their results, some of the RNA units from the CPV-virion of silkworm were 6.8 p long with a molecular weight between 14 and 18 X 10” daltons. Smaller fragments of the RNA, presumed to be subunits, were of two types with length of 1.12 and 0.36 11. These subunits have molecular weights of approximately 3 X loo and 1 X 10” (Miura et al., 1968). The 6.8 p contour length of RNA is probably the unbroken RNA genome from the virion, while the smaller fragments are probably produced by breakage at weak points during extraction. These subunits of viral gcnome appear as 15 and 12 S components in the sedimentation nrofile in this exneriI l.
OF
Cl’\‘-RNA
-157
ment (Figs. 2, 3). The sedimentation rates of the RNAs extracted from the virions of tussock moth, 0. Zeeucostigma, are very sinlilar in s value and molecular weight to the RNA subunits from the CPV of silkworm, B. mori (Miura et al., 1968). The fragmentation of the double-stranded viral RNA molecule, during extraction, is ill a
The author wishes to thank Dr. J. M. Cameron and Dr. F. T. Bird for their kind support of this work, Dr. A. Retnakaran for reading the manuscript, and Mr. W. C. Richards, Mr. R. Luft, Mr. D. G. Grisdale, and Mrs. R. Burke for their technical assistance. REFERENCES APPLEBAUM, S. W., EBSTEIN, R. P., ASU WYATT, G. R. 1966. Dissociation of ribosomal ribonucleic acid from silkworm pupae by heat and dimethylsulfoxide: Evidence for specific cleavage points. J. Mol. BioE., 21, 29-41. BELLAMY, A. R., SHAPIRO, L., AUGUST, J. T.. AKD JOKLIK, W. K. 1967. Studies on reovirlls RNA. I. Characterization of reovirlls gmome RNA. 1. Mol. Biol., 29, 1-17. BIRI), F. T. 1965. On the morphology and development of insect cytoplasmic-polyhedrosis virus particles. Can. J. Microhiol.. 11, 497501. BRITTEN, R. J., AND ROBERTS, R. B. 1960. IIigh s~dimcntatitm resolution density gradient analysis. Science, 131, 32-:S3.
458
HAYASHI
FEN~ICK, M. L., ERIKSON, R. L., AND FRANKLIN, R. M. 1964. Replication of the RNA of bacteriophage R 17. Science, 146, 527530. 1964. GOMATOS, P. J., AND STOECKENSUS, W. Electron microscope studies on reovirus RNA. PTOC. Nat. Acad. Sci. U. S., 52, 1449-1455. GOMATOS, P. J., AND TAMM, I. 1963a. The secondary structure of reovirus RNA. Proc. Nat. Acad. Sci. U. S., 49, 707-714. GOMATOS, P. J., AND TAMM, I. 1963b. Animal and plant viruses with double-helical RNA. Proc. Nat. Acad. Sci. U. S., 50, 878-885. HAYASHI, Y., AND BIRD, F. T. 1968a. The use of sucrose gradients in the isolation of cytoplasmic-polyhedrosis virus particles. 1. Inoertebr. Pathol., 11, 4044. HAYASHI, Y., AND BIRD, F. T. 1968b. Properties of a cytoplasmic-polyhedrosis virus from the white-marked tussock moth. J. Inuertebr. Pathol., 12, 140. HAYASHI, Y., AND BIRD, F. T. 1970. The isolation of cytoplasmic polyhedrosis virus from the white-marked tussock moth, Orgyia Zeucostigma (Smith). Can. J. MicrobioZ., 16, 69% 701. HAYASHI, Y., AND KAWASE, S. 1964. Base pairing in ribonucleic acid extracted from the cytoplasmic polyhedra of the silkworm. ViroZogy, 23, 611-614. HAYASHI, Y., AND KAWASE, S. 1965. Studies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombyx mori L. I. Specific RNA extracted from cytoplasmic polyhedra. 3. Se&x&. Sci. lap., 34, 83-89. HAYASHI, Y., HAYASHI, Y., AND KAWASE, S. 1965. Studies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombyn: mori L. (III) Comparison between IPBand HPB-RNA. 3. Se&x&. Sci. fup., 34, 167-170. HOSAgA, Y., AND ACZAWA, K. 1964. The fine structure of cytoplasmic-polyhedrosis virus of the S~UCWO~, Bombyx mori (Linnaeus). ]. Insect Pathol., 6, 53-57. KLEINSCHMIDT, A. K., DUNNEBACKE, T. H., SPENDLOVE, R. S., SCHAFFER, F. L., AND WHITcOMe, R. F. 1964. Electron micro-
scopy of RNA from reovirus and wound tumor virus. J. Mol. Biol., 10, 282-288. KRYWIENCZYK, J., I&YASHI, Y., AND BIRD, F. T. 1969. Serological investigation of insect viruses. I. Comparison of three highly purified cytoplasmic-polyhedrosis viruses. J. Inuertebr. Puthol., 13, 114-119. LANGRIDGE, R., AND GOMATOS, P. J. 1963. The structure of RNA. Science, 141, 694-698. MARKHAM, R., AND SMITH, J. D. 1951. Chromatographic studies of nucleic acids. IV. The nucleic acid of the turnip yellow mosaic virus, including a note on the nucleic acid of the tomato bushy stunt virus. Biochem. J., 49, 401-406. MCMORRAN, A. 1965. A synthetic diet for the spruce budworm. Choristoneura fumiferana ( Clem. ) ( Lepidoptera: Tortricidae). Can. Entomologist, 97, 58-62. MIURA, K., KIMUR.4, I., AND SUZUKI, N. 1966. Double-stranded ribonucleic acid from rice dwarf virus. Virology, 28, 571-579. MIURA, K., FUJII, I., SAKAKI, T., FUKE, M., AND KAWASE, S. 1968. Double-stranded ribonucleic acid from cytoplasmic polyhedrosis virus of the silkworm. J. ViToI., 2, 1211-1222. NISHIMURA, A., AND HOSAKA, Y. 1969. Electron microscopic study on RNA of cytoplasmic polyhedrosis virus of the silkworm. Virology, 38, 550-557. PETERMANN, M. L., AND PAVLOVEC, A. 1963. Ribonucleoprotein from a rat tumor, the Jensen sarcoma. III. Ribosomes purified without deoxycholate but with bentonite as ribonuclease inhibition. J. Biol. Chem., 238, 318323. SHATKIN, A. J., SIPE, J. D., AND LOH, P. 1968. Separation of ten reovirus genome segments by polyacrylamide gel electrophoresis. J. Viral., 2, 986-991. VERWOERD, D. W. 1969. Purification and characterization of bluetongue virus. Virology, 38, 203-212. WATANABE, Y., AND GRAHAM, A. F. 1967. Structural units of reovirus ribonucleic acid and their possible functional significance. J. Viral., 1, 665-677.
OF
INVEIITEBI~ATE
PATHOLOGY
Properties of RNA White-marked
16,
451458
from Cytoplasmic-Polyhedrosis Virus Tussock Moth, Orgyia Zeucostigma Y.
Department
( 1970)
of Fisheries Canada,
and Sault
HAYASHI
Forestry, Insect Puthology Research Ste. Marie, Ontario, Canadur
Received
of the
April
Institute.
10, 1970
When the genome RNA is extracted from the cytoplasmic-polyhedrosis virus of the white-marked tussock moth, Orgyia Zeucostigma, by the phenol method, it has two components with sedimentation rates of about 15 and 12 S. Their base composition includes adenine and uracil as well as guanine and cytosine. The grraninc + cytosine content was 41.2%. Melting temperature of the RNA was about 80°C in a solution containing 0.0015 M NaCl-0.00015 M sodium citrate. Most of the RNA was found to be resistant to ribonuclease digestion; some of it was partially sensitive and was presumed to be single-stranded. After enzyme treatment, the 15 and 12 S components of the genome sedimented at about 9 S. From these findings it is proposed that viral genome RNA from cytoplasmic-polyhedrosis virus is double-stranded.
Cytoplasmic-polyhedrosis viruses ( CPV) are widely distributed among lepidopterous insects. Serological evidence suggests that they are not the same (Krywienczyk et al., 1969). CPV from the silkworm, Bombyx nzori (Hosaka and Aizawa, 1964) and the forest tent caterpillar, Malacosomu disstriu (Hayashi and Bird, 1968a) have an icosahedral capsid about 70 rnp in diameter which has 12 prominent projections. It was previously reported (Hayashi and Kawase, 1964) that the RNA of CPV from B. mori displays an equimolar base composition of adenine to uracil and guanine to cytosine suggesting that the RNA is doublestranded. Recently Miura et al. (1968) have confirmed the double-stranded nature of the RNA of CPV of B. mori. They have shown that the RNA, in addition to having a complementary base composition, has a sharp melting profile, resistance to ribonuand a characteristic clcase (RNase), 1 Contribution
No.
156
from
this
double-stranded X-ray diffraction picture. The CPV virion obtained from the whitemarked tussock moth, Orgyia leucostigma (Bird, 1965; Hayashi and Bird, 196813) was found to be similar to B. mori CPV in size, surface structure (Nishimura and Hosaka, 1969), and sedimentation behavior (Miura et al., 1968). This virion also contained about 28% RNA (Hayashi and Bird, 1968b). These similarities suggest that the RNA of CPV of 0. Zeucostigmu might also be a double-stranded polynucleotide. Evidence is presented in this paper to show that the CPV-RNA is double-stranded and is similar to RNA from reovirus (Gomatos and Tamm, 1963a; Langridge and Gomatos, 1963), wound tumor virus (Gomatos and Tamm, 1963b), rice dwarf virus (Miurn et al., 1966), and bluetongue virus (Verwoerd, 1969).
MATERIALS
AND
Virus. The CPV was larvae of 0. leucostigma.
Institute. 451
METHODS
multiplied in the Unless otherwise
,452
HAYASHI
stated, the free virions from midgut epithelial cells were used in all the experiments (Bird, 1965; Hayashi and Bird, 1970). Mode of infection and radioisotope labeling. The larvae were reared on a meridic diet (McMorran, 1965) and, when they reached the 4th instar, were transferred to diets coated on the surface with a virus suspension containing lo7 polyhedra in 1 ml of water. Twenty-four hours after virus inoculation, the diets were replaced with radioactive ones containing 5 yCi uridine-3H/cm3 diet. The larvae were removed from the radioactive diet after 10 at which time the midguts were days, chalky-white due to the occurrence of the virus. The midguts of 10,000 insects were dissected out, washed twice, and stored in distilled water at 4°C until required. During this period the cells lysed. Purification of virus. The lysed midguts were homogenized in a Teflon-glass homogenizer. The homogenate was filtered through 4 layers of cheesecloth, and centrifuged at 10,000 g for 10 min at 2°C. The free virions remained in the supernatant whereas the polyhedra sedimented. The purification of the virus was carried out as described previously (Hayashi and Bird, 1968a, 1970). The virus isolated by sucrose gradient centrifugation was further purified by rebanding in a sucrose gradient (Fig. 1). This virus preparation was estimated to be 95% pure by electron microscopy. Extraction of RNA. The virions were suspended in TK buffer (0.03 M Tris-HCl, pH 7.5, 0.025 M KCl) and sodium dodecyl sulfate (SDS) was added to make a final concentration of 0.5%. After 2 min, RNA was extracted by the addition of an equal volume of 90% fresh phenol to the virus suspension and shaking for 10 min at 4°C. The aqueous and phenol layers were separated by centrifugation at 10,000 g for 10 mm and the phenol layer discarded. The purified by aqueous phase was further
20 Tube
no.
FIG. 1. Sedimentation profile of purified CPV virions. Virus preparation was layered over sucrose gradient ( 1040% ) and centrifuged in the SW 25.1 rotor of a Spinco model L-2 ultracentrifuge for 90 min at 23,000 rpm, -O-m-, optical density at 260 rnp; -O--O--, cpm. repeating the process with fresh phenol. The aqueous layer containing the RNA was carefully collected and the RNA was converted to its sodium salt by adding 2 drops of 1 M NaCl. It was then dehydrated by adding 3 volumes of cold 95% ethanol, which gave it a fibrous appearance similar to DNA. The fibrous RNA was washed several times with cold 70% ethanol and stored at -20°C until required. Cellular RNA from the midguts of tussock moth and rat liver was extracted by the method of Applebaum et al. (1966) using bentonite instead of polyvinyl sulfate (Petermann and Pavlovec, 1963). Sedimentation analysis and radioactivity assay. All RNA analyses were carried out by zonal centrifugation in a S-25% linear sucrose gradient by the method of Britten and Roberts (1960). RNA was dissolved in TK buffer, layered on the gradient, and
PROPERTIES
OF
centrifuged in an SW 25.1 or SW 40 Ti rotor in a Spinco Model L-2 or L-2-65B preparative ultracentrifuge at 5°C (see legends of Fig. 1, 2, 3, and 6 for details). The optical densities of the fractions that were collected from the bottom of the tube after centrifugation were measured at 260 mu in a Beckman DU spectrophotometer. To each fraction was added 200 ng of yeast RNA as a carrier, followed by the addition of an equal volume of cold 10% trichloroacetic acid (TCA) to precipitate the RNA. The precipitates were collected on glass fiber filters and washed several times with cold 5% TCA, placed in scintillation vials, and dried under an infrared lamp. Ten milliliters of toluene scintillation fluid (LiquiHuor, 4 g 2, 5diphenyloxazole, and .50 mg 2. 2-p-phenylenebis (5-phenyloxazole) per liter of toluene) was added to each vial and the radioactivity, counted in a Packard Tri-Carb scintillation spectrometer. Digestion of RNA z&h RNase. The RNA purified bv the sucrose gradient method was reprecipitatcd with ethanol at -20°C for 3 days. The precipitate was dissolved in
4.3
CT\‘-RX.%
SSC (0.15 ~1 NaCI-6.015 M sodium citrate) (Bellamy et al., 1967) and digested with different amounts of RNase for 30 mm at 30°C. The reaction mixtures were prchcipitated with TCA and the radioacti\.it\. was determined. The comets rcpresentcd the radioactivity of the viral R?rTA that VYIS not digested by the RNase. Thermal denaturation of RNA. RNA ( 50 ug/ml) was dissolved in 0.01 SSC (MuIra et al., 1966) and the melting curve was ob tained using the heated cell assemblv of ;I Gilford recording spectrophotometcr. Chemicals. Uridine-6-‘:H ( 10 Ci/mmole j and RNase (ribonuclease A) were obtairrcd from New England Nuclear Corp., Boston, Massachusetts, and Worthington Biochelnical Corp., Freehold, New Jrrsc!., resp(x;tivcb.. REWLTS
Sedimentation
Profiles of Viral RNA
Sedimentation profile of RNA extracted from the virion was obtained b\. sucrose gradient centrifugation, and the results arc shown in Fig. 2. IVhen 400 ug RNA \v;ts
Tube
no.
FIG. 2. Sedimentation profiles of RNA extracted from virions. (a) 400 pg RNA crose gradient (5-25% ) and centrifugation was performed in the SW 25.1 rotor for (b) 150 pg RNA after dialysis was centrifuged for 22 hr at 24,000 rpm.
was analyzed by su19 hr at 23,000 rpm.
454
HAYASHI
Tube
FIG. 3. Sedimentation liver RNA (a) or host at 23,000 rpm; O-O,
cell
profiles RNA optical
no.
of viral RNA labeled with uridine-sH centrifuged together (b). Centrifugation was performed in the SW 40 Ti rotor density; 0 - - - 0, cpm.
loaded on the gradient, only one component was observed in the gradient with broad banding (Fig. 2a). A dilute RNA sample ( 150 ug), however, showed a heterogeneous component with a shoulder at tube 18 (Fig. 2b) suggesting the existence of different sizes of viral RNA. The sedimentation coefficients of the main peak and the shoulder part were determined to be 15 S and 12 S, respectively, using RNA from rat liver (Fig. 3a) and host midgut (Fig. 3b) as markers. These values are virtually the same as those obtained from the CPV of B. mori (Miura et al., 1968). The appearance of the second component when RNA was diluted is also similar to that of the RNA of the CPV of B. mori (Hayashi et al., 1965; Miura et al., 1968). The separation of these components from the RNA of the CPV by prolonged centrifugation is possible and is currently under study. The absorption spectrum of the RNA preparation showed a characteristic peak at 259 mu with a 2591232 ratio of 2.2 (Fig. 4) similar to that of RNA from B. mori CPV (Hayashi and Kawase, 1965).
with rat for 19 hr
Base Composition The base composition of the RNA from free virions was determined by the method of Markham and Smith (1951) and is
Wave
length
FIG. 4. Absorption spectrum from CPV-virion. The sample TK buffer.
(mr)
of RNA extracted was dissolved in
PROPERTIES
TABLE
IF
1
I
Base
Guanine Adenine Cytosine I’racil
21.7 28.4 19.5 30.4
A/U
0.93 -
C/C
pu/pyc
-
-
1.11
1.00
-
-
I
I
occluded
BASE COMPOSITION OF V-RNAa Molar ratio”
455
CPV-RNA
I
,
virus-RNA
130 I
G+C (% )
I
,-
I’ !/
/
41.2 -
a Expressed as moles per 100 moles of total base. b Based on 5 analyses on 3 preparations. c pu, purine; py, pyrimidine.
shown in Table 1. It is apparent from the table that the molar percent of guanine closely approximates that of cytosine, and the ratio of adenine and uracil is approximately the same. This indicates that the RNA of the CPV has base pairing similar to that reported from double-stranded RNA viruses as mentioned above. The molar percent of guanine and cytosine together is 41.2% and is comparable to the RNA of the CPV from B. mori (Hayashi and Kawase, 1964; Miura et al., 1968).
Melting
Behavior
The thermal denaturation of RNA in 0.01 SSC from the occluded virion, free virions, and ribosomes is shown in Fig. 5. The RNA from occluded as well as free virions exhibit sharp melting profiles with a Tm value of about 80°C. The hyperchromic increase of RNA from the free virions was about 26%, but that obtained from the occluded virions was about 34%. On the other hand, the single-stranded rRNA (ribosomal RNA) under similar conditions melted over a wide range. Thus, the typical difference between singlestranded and double-stranded RNA became apparent. The sharp melting profile from the viral RNA corresponded precisely with the melting point profile obtained from the RNA of the CPV of B. mori (Mima et al., 1968) under the same conditions.
FIG. 5. Thermal denaturation of viral RNA extracted from free and occluded virion, and midgut ribosomal RNA.
Resistance to Digestion It
has been
shown
by RNase in the
case of the
reovirus that the genome RNA was doublestranded, based on RNase resistance among other properties (Gomatos and Tamm, 1963a). If the RNA of the CPV is resistant to RNase, then a secondary structure for the RNA can be inferred. With such a goal in mind, RNase resistance of the RNA of the CP\’ was studied. RNA of the CPV
labeled with uridine-“H was incubated with various amounts of RNase. Table 2 shows the radioactivity of the acid-insoluble material after RNase treatment, indicating that it is resistant to the enqme digestion. About 20% of tritium activity was found in the acid-soluble fraction when 5 pg/ml of RNase was used, suggesting that the RNA is either single-stranded in part or the base pairing is loose in some regions. The rate of RNA digestion did not increase significnnth,
even
Altkoueh
at a lcvcl
of 150 lr,g RN&. is rt’-
double-stranded RNA
456
HAYASHI
EFFECT
OF RNASE
Concentration ( wc/mI 1 0 1 5 10 30 75 150
TABLE 2 CONCENTRATION
ON THE V-RNAQ
Radioactivity acid-insoluble material ( cpnl)
OF HEAT
of Recovery (So)
Temperature (‘-Cl
100 80.9 81.6 84.6 78.7 74.7 70.2
1058.3 856.3 863.5 895.0 833.2 790.6 742.8
a Viral RNA was dissolved cubated at 30°C for 30 min.
EFFECT
in
1.0 SSC
and
in-
&ant to RNase, the sedimentation behavior is strongly influenced by the enzyme treatment (Fenwick et al., 1964). Figure 6 shows the sedimentation profile of viral RNA after RNase treatment. The sedimentation rate of the material forming the peak which is resistant to RNase was reduced to about 9 S. This presumably represents a single component with perfect base-pairing by hydrogen bonding resembling the rigid structure of DNA. On the other hand, the broad band at 15-12 S before RNase treatment may contain the
Tube
FK. 6. Sedimentation profiles (200 ug RNA) were centrifuged optical density; 0 --0 -0,
of viral RNA in the sucrose 0, cpm.
( Zontrol 23 40 50 60 70 80 90 100
TABLE 3 ON THE RNASE OF V-RNAa
Radioactivity acid-insoluble material (wm) 3618.4 3650.0 3038.0 3030.4 2909.3 3073.1 3035.9 2892.6 2370.7
STABILITY
of Recovery (%) 100.0 100.9 84.0 83.7 80.4 84.9 83.9 79.9 65.5
a V-RNA was dissolved in 1.0 SSC, incubated at different temperatures for 15 min and cooled iu ice. These RNA solutions were treated with 10 pg of RNase/ml for 30 min at 30°C.
enzyme-susceptible components as mentioned above. RNase-resistance of heat-denatured RNA in 1.0 SSC was examined, and the results are shown in Table 3. The acid-insoluble materials (viral RNA) decreased about 16% at 40°C after RNase treatment. No further digestion occurred until the temperature was increased to 90°C. There was
no
after RNase treatment. gradient at 24,000 rpm
After RNase treatment, samples for 22 hr in the SW 25.1 rotor.
PROPERTIES
rapid digestion between 90 and 100°C. This is probably due to the denatured RNA becoming partly single-stranded permitting the RNase to digest the singlestranded portion. Therefore, the melting profile of the denatured RNA was quite different from that of the RNA in 0.01 SSC (Fig. 5). The results obtained in this experiment support the hypothesis that the RNA of the CPV from 0. Zeucostigma is double-stranded similar to that obtained from the CPV of B. mori (Hayashi and Kawase 1964; Miura et al., 1968). DISCUSSION
It is clear that the RNA extracted from the virions of CPV is double-stranded because of its characteristic base composition, sharp melting profile, and resistance to RNase. However, CPV-genome RNA was released from the virions as a mixture of RNA fragments consisting of at least 2 components (Figs. 2, 3). The distribution of size of these fragments is highly reproducible in the gradient analysis even when 6-month-old virions are used. This finding, however, is not consistent with the electron microscopical observation reported by Nishimura and Hosaka ( 1969). According to their results, some of the RNA units from the CPV-virion of silkworm were 6.8 p long with a molecular weight between 14 and 18 X 10” daltons. Smaller fragments of the RNA, presumed to be subunits, were of two types with length of 1.12 and 0.36 11. These subunits have molecular weights of approximately 3 X loo and 1 X 10” (Miura et al., 1968). The 6.8 p contour length of RNA is probably the unbroken RNA genome from the virion, while the smaller fragments are probably produced by breakage at weak points during extraction. These subunits of viral gcnome appear as 15 and 12 S components in the sedimentation nrofile in this exneriI l.
OF
Cl’\‘-RNA
-157
ment (Figs. 2, 3). The sedimentation rates of the RNAs extracted from the virions of tussock moth, 0. Zeeucostigma, are very sinlilar in s value and molecular weight to the RNA subunits from the CPV of silkworm, B. mori (Miura et al., 1968). The fragmentation of the double-stranded viral RNA molecule, during extraction, is ill a
The author wishes to thank Dr. J. M. Cameron and Dr. F. T. Bird for their kind support of this work, Dr. A. Retnakaran for reading the manuscript, and Mr. W. C. Richards, Mr. R. Luft, Mr. D. G. Grisdale, and Mrs. R. Burke for their technical assistance. REFERENCES APPLEBAUM, S. W., EBSTEIN, R. P., ASU WYATT, G. R. 1966. Dissociation of ribosomal ribonucleic acid from silkworm pupae by heat and dimethylsulfoxide: Evidence for specific cleavage points. J. Mol. BioE., 21, 29-41. BELLAMY, A. R., SHAPIRO, L., AUGUST, J. T.. AKD JOKLIK, W. K. 1967. Studies on reovirlls RNA. I. Characterization of reovirlls gmome RNA. 1. Mol. Biol., 29, 1-17. BIRI), F. T. 1965. On the morphology and development of insect cytoplasmic-polyhedrosis virus particles. Can. J. Microhiol.. 11, 497501. BRITTEN, R. J., AND ROBERTS, R. B. 1960. IIigh s~dimcntatitm resolution density gradient analysis. Science, 131, 32-:S3.
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FEN~ICK, M. L., ERIKSON, R. L., AND FRANKLIN, R. M. 1964. Replication of the RNA of bacteriophage R 17. Science, 146, 527530. 1964. GOMATOS, P. J., AND STOECKENSUS, W. Electron microscope studies on reovirus RNA. PTOC. Nat. Acad. Sci. U. S., 52, 1449-1455. GOMATOS, P. J., AND TAMM, I. 1963a. The secondary structure of reovirus RNA. Proc. Nat. Acad. Sci. U. S., 49, 707-714. GOMATOS, P. J., AND TAMM, I. 1963b. Animal and plant viruses with double-helical RNA. Proc. Nat. Acad. Sci. U. S., 50, 878-885. HAYASHI, Y., AND BIRD, F. T. 1968a. The use of sucrose gradients in the isolation of cytoplasmic-polyhedrosis virus particles. 1. Inoertebr. Pathol., 11, 4044. HAYASHI, Y., AND BIRD, F. T. 1968b. Properties of a cytoplasmic-polyhedrosis virus from the white-marked tussock moth. J. Inuertebr. Pathol., 12, 140. HAYASHI, Y., AND BIRD, F. T. 1970. The isolation of cytoplasmic polyhedrosis virus from the white-marked tussock moth, Orgyia Zeucostigma (Smith). Can. J. MicrobioZ., 16, 69% 701. HAYASHI, Y., AND KAWASE, S. 1964. Base pairing in ribonucleic acid extracted from the cytoplasmic polyhedra of the silkworm. ViroZogy, 23, 611-614. HAYASHI, Y., AND KAWASE, S. 1965. Studies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombyx mori L. I. Specific RNA extracted from cytoplasmic polyhedra. 3. Se&x&. Sci. lap., 34, 83-89. HAYASHI, Y., HAYASHI, Y., AND KAWASE, S. 1965. Studies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombyn: mori L. (III) Comparison between IPBand HPB-RNA. 3. Se&x&. Sci. fup., 34, 167-170. HOSAgA, Y., AND ACZAWA, K. 1964. The fine structure of cytoplasmic-polyhedrosis virus of the S~UCWO~, Bombyx mori (Linnaeus). ]. Insect Pathol., 6, 53-57. KLEINSCHMIDT, A. K., DUNNEBACKE, T. H., SPENDLOVE, R. S., SCHAFFER, F. L., AND WHITcOMe, R. F. 1964. Electron micro-
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