The release of viral genome RNA from virions of the cytoplasmic-polyhedrosis virus of Malacosoma disstria by acetone treatment

JOUItN.IL

OF

INVERTEl3RATB

The

PATHOLOGY

17, 433-439 (1971)

Release of Viral Genome Cytoplasmic -Polyhedrosis disstria

RNA from Virions Virus of Malacosoma

by Acetone

of the

Treatment

Y. HAYASHI AND J. C. CUNNINGHAM Insect

Pathology

Research

Institute ,I Canada Department oj Fisheries Sault Ste. Marie, Ontario, Canada Received

November

and Forestry,

30, 19’YO

Acetone treatment of the CPV of Malacosoma disstria released the viral genome RNA from nonoccluded virions, virions liberated from polyhedra by alkali treatment,, and virions still occluded in polyhedra; in the third case the RNA was retained within the polyhedra until they had been dissolved by alkali treatment. The components were separated by sucrose gradient centrifugation; typical 15 and 12 P viral RNA samples were obtained by this method. Bcetone treatment may prove useful for the extraction of double-stranded RNA from virus-infected tissues.

CPV Studies

virions have a complex structure. have been made on the CPVs of

Bombyx mori (Hosaka and Aizawa, 1964), Orgyia bucostigma, C’horistoneurafumiferana (Bird, 1965, 1966), and Malacosoma disstria (Hayashi and Bird, 1968a). It has been demon&rated that t’hey are icosahedral, about, 70 rnp in diameter, and have 12 prominent project,ions. Miura et al. (1969) working wit,h B. mori CPV report*ed t,hat the virion is composed of many small subunits within an inner shell. The major components of CPV virions are RYA and protein (Hayashi and Bird, 1968b; Xshimura and Hosaka, 1969‘). The viral genome RNA of B. mori CPV was found to have a molecular weight of about 14-18 X lo6 daltons (Nishimura and Hosaka, 1969) with a double helical structure (Hayashi and Kawase, 1964; hIiura et :%I.,196s). 0. leucostigmaCPV was also found to have a double helical structure (Hayashi, 1970a), and RNA comprised 2630 5%of the virion (Haynshi and Bird, 1968b; Xshimura and Hosakn, 1969). Richards (1970) reported that, CPV virions can be disrupt#edby ethanol treatment which 1 Contribution

No. 185.

releasesviral RXA similar to turnip yellow mosaic virus (TYMV) RNA (Markham and Smith, 1949; Dunn and Hitchborn, 1966). Acetone is a useful agent for disrupt,ing adenovirus type 5 virions; when so treated three structural units and a nucleoprot,ein core are obtained (Laver et al., 1968). In t,his paper the effect of acetone on CPV virions is evaluated, the products of degradation are analyzed, and the potent,ial use of t,he technique is discussed. MATERIMS AND BIETH~DS Virus and radioisotope labeling. The virus used in these experiments was the CPV of the forest tent caterpillar, Jlalacosoma c/isstria. The virions were SH-labeled with uridine2 as described previously (Hayashi, 1970a; Richards and Hayashi, 1970). Acetone treatment of oirions. Konocclutletl virions isolated from the cytoplasm of infected gut cells (Hayashi and Bird, 1970), and occluded virions which had been liberated by alkali trea,t.ment, from thr polyhedr:l (Hukuhara and Hashimoto, 1966; Hayaslri 2 Uridine-(i-aH (10 Ci/mmole) was ot)~~ined from New England Nuclear Corp., Bost~)~~. X~:MS:\chuset,t s.

433

434

HAYASHI

AND CUNNINGHAM

and Bird, 1970), were suspended in TK buffer (0.03 M tris-HCl, pH 7.5; 0.025 M KCl), and 5 volumes of cold acetone were then added to the suspension. These mixtures were kept for 30 min on crushed ice, then centrifuged at 10,090 g for 10 min at 2°C. The pellets were resuspended in TKbuffer, analyzed by sucrose gradient centrifugation, and examined in a Philips EM 200 electron microscope (Hayashi and Bird, 1970). A study was also made of the effect of acetone on virions occluded in polyhedra. A suspensionof purified polyhedra was divided into 3 samples. The first, a control, was treated with alkali to release the virions and then directly layered onto a linear sucrose gradient. The second was t#reated with acetone, then suspended in alkali and treated as was the first sample. The third samplewas treated with a,cetone and analyzed on a sucrose gradient without alkali treatment. RNA extraction from the virions by treatment (a) phenol and (b) acetone. (a) Nonoceluded virions were purified, then suspended in TK buffer; the suspension was divided into 3 aliquots. RNA from 2 aliquots was extracted separately by the addition of an equal volume of fresh 90% phenol, then shaken for 10 min at 4°C. The aqueous and phenol layers were separated by centrifugation at 10,000 g for 10 min, and the phenol layer was discarded. The aqueous layer of one aliquot was analyzed directly by sucrose gradient centrifugation. The RNA from the aqueous layer of the second aliquot was precipitated by adding 3 volumes of cold ethanol, washed twice with ethanol, and then dissolved in TK buffer before gradient analysis. (b) The third aliquot of the virion suspension was treated with acetone as described above, suspendedin TK buffer, and analyzed by sucrose gradient centrifugation. Preparation of ribosomesand RNA from rat liver. Rat liver ribosomes were prepared by the method of Wettstein et al. (1963), and

RNA was extracted as described by Hayashi (197Oc). Sucrose gradient centrifugation.. A Spinco Model L2-65B ultracentrifuge was used. Samples were layered over linear sucrose gradients, prepared according to Britten and RoberB (1960). A 1040% gradient in TK buffer was used for virions. They were centrifuged for 90 min at 23,000 rpm in an SW 25 rotor. For RNA analysis a 5-25% gradient was used for 20-30 hr at 21,000 rpm in an SW 40 rotor. Fractions were collected by piercing the bottom of the centrifuge tube. Each fraction was measured for optical density at 260 nm in a Beckman DU spectrophotometer, and for radioactivity in a Packard Tri-Carb scintillation spect’rometer (Hayashi, 1970b). RESULTS

AND DISCUSSION

Effect of Acetone on Virions as Observed by Electron Microscopy The virions were precipitated from the suspensionsby addit(ion of cold acetone, and kept at 4°C. The pellets were suspended in distilled water, negatively stained, and examined in the electron microscope. Compared with an unt’reated control (Fig. lA), it appeared that the virions in the acetonetreated sample released their core materials and became empty shells (Fig. 1B). However, a few virions remained intact in t,he acetone-treated preparation. A,nalysis of Acetone-Treated Virions by SucroseGradient Centrifugation To characterize the components released from virions by acetone treatment, uridine3H-labeled nonoccluded virions were treated with acetone and analyzed by sucrose gradient centrifugation. The results compared with the untreated control are shown in Fig. 2. In the cont,rol the intact virions showing uridine-3H activity were located in the cent,er of the gradient. The acetone-treated sample did not show any intact virions in the gradi-

EFFECT

OF

ACETONE

ON

WV

I::,->

FIG. 1. ISlectron micrographs of untreated viriorls and virions beated with acetone, trcg:it iwai> st:Glw~ wit,h l’/; PTA. (1A) Cont.rol sample (arrows show empty viriolls). (IB) Sample trt‘atctl \\\I h scet,oue which released the materials inside the virions (arrow itldicatcs if11 act viriun rcm:rirlitjg :!t’trlt this treatmerlt). X125,000.

ent, but a labeled, light component was found at the top of the gradient. It is possible t,hat) t,his was RNA released from the virionc. In this experiment a precipitate was seen in tlie bottom of t.he crrkrifuge t.ube and when examined in t.he electron microscope it had the same appearance as the sample shown in Fig. 1B. This result is similar to t,hat obtained by the ethanol-t,rentment of virions in l\\-hich all viral protein was precipit.ated after gradient centrifugation (Richards, 1970). The results of the experiment demonstrating the effect of acetone on virions occluded in ~polyhedra are illustrated in Pig. 3. In the first sample, polyhedra treated with alkali alone, the virions were released by the solubilizat~ion of the polyhedral protein but their struct’ure \vas unaffected. The results are similar to those shown in Fig. 2. In the sec-

ond sample, polyhedra treated tirst wit11 acetone followed by alkali, the virions \vtw completely disrupted and uridine- “H-labrlt4 materials were obtained only as :L light c’c~~nponent. 111 t.he t.liird sample, t~olyherlr:~ t,reated with acetone alone, no virious or .i+al genome RNA were det ccted in t hr gr:rditnt and a pellet of intact polyhedra 1~1s founcl at the bottom of the tube. These rw11th demonstrate that acetone can pened r:tt e through the prot,ein matrices of tlw I)olyv hedra and cause some structural damage to t.he occluded virions resulting in the freeing of viral RX4, but the RX4 cannot wc:qr from t,he polyhedra and remains occluded. The viral components can be detected OIIIJafter the alkaline dissolution of the pal\-hedral protein. Similar results have been reported by Hayashi and Bird (l!IriO). I’M-

436

HAYASHI

AND

sediment.ation values of the main peak and shoulder part (tube 23) were considered to be 15 and 12 S, respectively, by comparison with the rat liver RNA components known to be 28 and 18 S. These values are almost identical to the values obtained from the viral genome RNA of B. mori CPV (Miura et al., 1968) and of 0. leucostigma CPV (Hayashi, 1970a). It was therefore very clear that the material released from the virions by acetone treatment was viral RNA. The materials released from polyhedra by acetone and alkali treatment (Fig. 3, tubes 30-34) were separated further. After prolonged centrifugat-ion, they also clearly showed 15 and 12 S components typical of viral RNA, as seen in Fig. 5. This result supports the conclusion that these eomponents are viral genome RNA which has fragmented on extraction (Hayashi, 1970a).

250C

2000

-2 ‘2 . .a ii 15oc h .z .? t z -0 3

1ooc

5oc

C

CUNNINGHAM

10 Tube

20 Number

FIG. 2. Sedimentation profiles of nonoccluded virions and virions treat.ed with acetone. Sucrose gradient (1040%) centrifugation was performed at 23,030 rpm for 90 min in an SW 40 Ti rotor at 5°C.

Control

0

mage et al. (1970) reported that high yields of polyhedra of a nuclear polyhedrosis virus (NPV) were obtained by a combination of lactose and acetone treatment and that there was no decrease of infectivity. Results obtained in this experiment showed that CPV virions occluded within polyhedra were severely damaged by acetone treatment, but no infectivity tests were made. These results indicate that there are essential differences in virion structure between CPVs and NPVs. Characterization the Visions

of Materials

Released from

Experiments were performed to charact,erize the materials released from the virions by acetone treatment. They were compared with rat liver RNA as a marker using prolonged gradient centrifugation (Fig. 4). The

(Na$O,)

! i \I I

J

I

Acetone \

i

Ni Tube

\I

Number

FIG. 3. Sedimentation profiles of virions OCeluded within polyhedra after alkali treatment followed by acetone treatment. Centrifugal condition was in the same manner as in Fig. 2.

O-8 I

28s

Rot liver I

O-6

Tube

Tube

Number

Sedimentation profile of materials released from nonoccluded virions by acetone treatment centrifuged together with rat liver RNA as a marker. Centrifugation C5-25y0 sucrose gradient) was performed in an SW 40 Ti rotor for 20 hr at 21,000 rpm at, 5°C. FIG.

Phenol 1000

i

T : 2005.3

c

‘3-1 0

Phenol-Ethanol

T : 2810.5

t

Number

FIG. 5. Sedimentatioll profile of materials I’Pleased from polyhedra treated first with aretone and then with alkali. Centrifugafion ~:ih at 21,000 rpm for 30 hr.

4.

IO

20

30

0

10

20

Tube

30

0

10

20

30

Number

FIG. ti. The recoveries of viral RNA from nonoccluded virions by phenol-ethanol and acet,one procrdures. Centrifugal condition was as in Fig. 5. T = total cts/min recovered. Three identical sarnplrs were used.

437

43s

HAYASHI

AND

03 t 28s Acetonetreated 0.4~

2 i

Control

: p \I I

8s

”

CUNNINGHAM

of RNA was considerably higher than bJ phenol procedures. In fact, the acetone treatment can recover about double the RN,4 recovered by the phenol-ethanol precipit ation procedure, which is the commonest method of RNA extraction. Recovery of RNA from Ribosomesafter Acetone Treatment

It was of interest to invest’igate whet,her the acetone method could be used for ex,-,x traction of RNA from ot,her nucleoproteins ; such as ribosomes. The sediment,ation pat.4 tern of the isolates obtained from rat liver % ,v 0.2ribosomesby acetone treatment is shown in z Fig. 7, and is compared to t)he phenol proce0 dure where 3 components were obtained. The acet.one isolate yielded only degraded components of ribosomal RNA and all the 0.1 materials were found near t,he top of the gradient. The degradation of the RNA by acetone t.reat.ment, is clearly shown. If the extraction of RNA from ribosomes by acetone treatment is possible at least part’ of the ribosomal RNA should be represented in Tube Number the gradient pattern. Since it was not repreFIG. 7. Sedimentation profiles of rat ribosomal RNA and materials released from ribosomes after sented, it was originally assumedthat treatacetone treatment. Centrifugation was 20 hr at ment with acetone did not) extract) ribosomal 23,000 rpm in an SW 25.1 rotor. RNA. Another possibilit,y was that the RNA was freed from the ribosomesby the acetone Recovery of RNA from the V&ion after treatment and digested by ribonuclease. Acetwe Treatment Ribonuclease is resistant to acetone and The recovery of RNA from virions after ribosomal ribonuclease has been described acetone treat,ment was compared to the re- (Stavy et al., 1964; Somberg and Davis, covery by phenol and phenol-ethanol ex- 1965). If the latter explanat,ion is correct and tra&ion procedures (see Materials and the ribosomal RNA is enzyme sensitive, it Methods). The results are shown in Fig. 6. must, therefore, be single-stranded. The samples of RNA from phenol and pheThese experiments demonstrate that acenol-ethanol extractions showed similar sedi- tone t,reatment of nonoccluded and occluded mentabion profiles and recoveries in sucrose virions of a CPV results in the releaseof viral gradients, although the phenol-ethanol pro- RNA. It seemslikely that this met.hod can cedure yielded about 30% less RNA; this be employed only for the extraction of reduced yield may be due to precipitation of double-stranded RXA, which is extremely the RNA by the ethanol before overlayering resistant to ribonuclease. on the sucrose gradient. After acetone treatACKNOWLEDGMENT ment of the virions one peak of uridine-3H activity was centered at 15 S and another We wish to thank Dr. F. T. Bird for his kind of this work. We also thank Mr. W. C. peak appeared clearly at 12 S. The recovery support 5

0.3 -

EFFECT

OF

ACETO%-E

Iticharda and ?vlr. T. P. 1)onaghue for their tech!licsl assistance, and I)r. T. A. Angus and Dr. P. P. Sohi for criticizing the manuscript. REFEREXCES I%IRI), F. T. lO(i5. On the morphology and developmerlt of insect cyt oplasmic-polyhedrosis virrls particles. (‘an. J. Microbial., 11, 497-501. I311tu. F. T. 1966. The development of a cytoplasmic polyhedrosis virus in the spruce budworn,, Chorialonetrru jwnijerunu (Clemens). Con. .J. Mic~rohiol., 12, 337-339. BRITTI:N, I<. J., .LXD HOHLRTS, R. B. 1960. High resol~ltion density gradient sediment,ation analysis. Science, 131, 32-33. DuI,u.\c:I:, H. T., ~%I.LRTINEZ, A. J., AND CORKER, .J. A. 1970. Recovery of the nuclear polyhpdrosis virrls of the cabbage looper, l’richoprrlsia ni, by coprecipitation with lactose. .J. Invcrfebr. Pathol., 16, 80-83. I)VSN, I). 13., AND HITCHBOEN, J. H. 1966. The extraction of infevtiotls high molecular weight RX.4 from turnip yellow mosaic virus with ethanol. Ivirology, 30, 598-607. HAYASHI, Y. 1RiOa. Propert,ies of RNA from cyt oplasmic-polyhedrosis virus (CPV) of xhit,e-marked tussock moth, Orgyia /~ILCOStigmtc. .I. Invert&,. P&ho/., 16, 451158. H.\\-.~BHI, XT. lQ7Ob. Occluded and free virions in midgnt cells of Malec~osoma tliaslria infected with oytoplasmic polyhedrosis virus (Cl?\‘). .Z. Znvrrtebr. Puthol., 16, 442-450. H.YY.\SHI, T. lQ7Oc. IN,4 in midgut of tnssock moth, Org!/icl tatrwstigmn infected with cytol~lnsmicpol~hetir~~sis virus. Can. J. MicroGo/., 16, llOlL1107. HLKZRHI. Y., .\xI) BIRD, F. T. 1968a. The use of sucrose gradients in the isolation of cytopl:ismic-polyhcdrosjs virus particles. J. In-. mrtdr. H.~Y.\~HI.

Pathol.,

II, Y., .\%I) BIRD,

40-14.

F. T. 1968b. Properties of :t cyt oplasmic-polyhedrosis virlls from the white-marked inssock moth. .I. Znved. P&ho/., 12, 140. H-11..\~HI, Y., .%luu I~IRD, F. T. 1970. The isolaI iota t)f rytoplasmic polyhedrosis virus from the ahite-marked tllssock moth, Orgylia Zetcrws/ignrct (Smit h). <‘in. .I. dlicrobid., 16, 695ifll.

OS

CPY

cl!)

Y., A?;D E;.r\v.\s~, S. lQ6-l. Hase pairing in ribonucleic acid ext.racted from the cytoplasmic polyhedra of the silkw\-cbrrn. I’r rolog!J, 23, (i12Mll. HOSAKA, XI’., .\NI) .~IzA\\.A, li. 1964. The fi11e structure of the c~?;topl:ts”ir-polghedrl)sis virus of the silkworm, Non~h!/x mori il,irltl:t em). 1. Znscct J’ath,d., 6, 53-57. HDKUH.UU. T., .\NU H.U+HIMOTO, Y. 1Wi. Srnt. logical studies of the cytoplasmirand n,lclear-polyhcdrosis virus of the silkworm, Botnbyx tori. J. Znve/~teb~~. l’athol., 8, 234-230.

HAYASHI,

L.%VElL,

W.

AND

G.,

internal J.

~‘IGRlCIIL\,

V \I,~.:NTIXK, Hiol.,

M.~RKHAM,

I<.,

011 the sitology, RIIun.\, I<..

W.

(‘..

3W38li.

of tllrnip

I<.

kl.

yellow

39, 330-3Q. FUJII, I., S\Ic.\rir:

‘I?.,

1949.

$1 udiry

mosaic*. Fual:,

I’nr~r. %I.,

\sI>

S.

lQ68. Double-stranded ribsacid from cytoplasmic polyhrdrosis of’ the silkworm. .I. Tirol., 2, 1211 ~l?“L’.

T<.i-\V .\ s I,:,

Mm<.\.

Xi,

.~si7r) SMITH.

virils

I<1lSSl;.I,l..

1968. Isolatioll of an from adenovirus t ~1” 5

component

X0/.

nl&ic virus

11. (:.,

I<, C‘.

Ii..

FI:JII-EC.\\\-.\Y\,

I.,

1~.-.2~.\.

1t.I

.i.uI,

I<.~\v.~sb:, S. 196R. ISlectron-mic:roscol,ic I,IIservat ion of a cyt oplasmir-pol~hedrosis virlls from the silkwornl. .I. Znzwrtrbr. I’dho/.. 11, 262-265. NISHIMLXA. .4., INI) Ilos~n.~, Y. 1969. T’Xect~r~lu microscopic st tidy OII RSA of eyt,oplasrnic polyhedrosis virrls of the silkworm. I*ir&G,g!/, 38, 550-557. RIcaaitus, W. C. 1970. Disruption of :L (*!;~IIplasInic.-pol~hedrosis virus by (It hanol. ./. Znwrtebr. P&o/., 15, -ljT-15X. HICH.4RI)S, w. C., .\xn H.kr.\aMI, Y. lQ71. l’tfcc,t of snme organic solvents on the cytoplasmic:polyhedrosis virlls of the forest tpnt, c:it(lrpillar, Mulamsonw rliwtrin. J. Itlvrri’cd~r. Puthot.,

17,

42-47.