Separation and Structure of Components of Nuclear Polyhedrosis Virus of the Silkworm

Copyright( 1971 American SocietyforMicrobiology Prinited inU.S.A.

Separation and Structure

of

Components

of

Nuclear

Polyhedrosis Virus of the Silkworm

TOSHIYUKI KHOSAKA, MICHIO HIMENO, AND KONOSHIN ONODERA

Laboratory ofBiochemistry, Department of Agricultural Chemistry, Kyoto Untiversity, Kyoto, Japan Received for publication 21 September 1970

Morphology of structural components of nuclear polyhedrosis virus (NPV) particles of the silkworm (BombyxmoriLinne) wasstudiedbyelectronmicroscope using negative staining. NPVparticles isolated from polyhedra could be separated into five structuralcomponentsby centrifugation in sucrosedensity gradients. The

lowest band (band I) was found to consist of thick rod-shaped particles (330 by

80nm) with knobby surfaces and with occasional protrusion at one end. The

second band from the bottom (band II)wasshowntoconsistmainlyof slender

rod-shaped particles (360 by 60 nm), in which internalstructureswerevisibleas adense mass. Regular striations werealso seen on the surface of theseparticles. By treat-ment with mercaptoethanol, these particles weredrastically damaged,and in some cases the internal substances werepartially released, producing empty inner

mem-branes ofvarious degrees of disintegration.In bands III andIV,both empty

spheri-cal and empty rod-shaped membranes were present. Band III was rich in empty

spherical membranes which wereshownto be theouter membranes of thick

rod-shaped particles. The empty rod-shaped membranes, the inner membranes, were

mainly located in band IV and havecrossstriationsonthe surface. It is remarkable

that the uppermost band (band V) consisted purely of small spherical particles,

somewhat heterogeneous in size and shape (around20 to 25nm in diameter),

in-dicating the particles to be the degradation product of the virus particles. Similar

particlescouldalsobeobservedwithin the empty inner membranes.

Although the structure of nuclear polyhedrosis

virus (NPV) ofinsectshas been investigated by a number of workers, and was found toconsist of

complex rod-shaped particles, the fine structure of theparticles still remains obscure. Earlier

ob-servationsontheshadowedpreparations ofvirus particles isolated from polyhedra by treatment with weakalkali(1,2,3) haveledtotheworking modelthat severalspherical subunits with a

diam-eterof about40 nm wereenveloped witha setof

membranes (outer and inner membranes) to

form rod-shaped particles (4). On the contrary, Krieg (8) observed that the rod-shaped particles haveahelicalstructuresimilartothatof tobacco mosaic virus and concluded thatthesubunits do

not represent viral developmental stages but

artifactsproducedby alkaline degradationof rod-shaped particles. Such structural components

were also found innegatively stained virus prep-arations but were interpreted as discs or groups ofdiscs of the innermembranes (5).

Examination ofultrathin sections ofpolyhedra

(3, 9,10), however, gave no evidence for support-ing the existence of a subunit structure in rod-shaped particles. More recently, Kozlov and

Alexeenko (7) observed the central helical core in virusrods treated withchloroformorureaand

suggested that the structure of the inner core is

similartothreadlikebacteriophage

fd.

In aprevious report (6), it wasshownby thin sectioning of tissue infected with NPV of the silk-wormthat,attheearlier stages of the maturation process, theflexuous rod particleslooselypacked with inner membrane first appeared in the cell

nucleus and have laterallystriatedinternal

struc-tures. These particles were, then, envelopedwith

the outer membrane to form a compact rigid

structurebeforeembeddinginpolyhedralprotein.

Inthe presentstudy, the finestructure of virus

particles isolatedfrompolyhedrawasinvestigated by separating viral structural components by means ofsucrose density gradient centrifugation and byexamination of each component by

elec-tronmicroscopy.

MATERIALS AND METHODS

Preparation of polyhedra. The sources for

prepa-ration of polyhedra were the hemolymph, collected

shortly

before

instar larvae of the silkworm. The polyhedra were

267

on November 11, 2019 by guest

http://jvi.asm.org/

collected from the hemolymph by settling out for a

few days in the cold. Thesediment containing

poly-hedra was placed onandcentrifuged through a 50%c;

sucroselayerat8,000 X gfor15 min at 2 C. As a

re-sult, the polyhedra were recovered as a pellet at the

bottom of the centrifuge tube, whereas the majority

of the impurities remained over the sucrose layer.

After washing several times by cycles ofsuspending

in distilled water and centrifugation (3,000 X g,

15 min, 2 C), the polyhedra were further treated

with massive doses of lipase and trypsin for 2 hr at

room temperature to remove any residual impurities.

Finally, thepolyhedra wereagainwashedextensively

with distilled waterand stored as dry powder in the

cold.

Isolation of virus. Virus particles were isolated

from polyhedra by treatment with Na2CO3 solution

of various concentrations (from 0.005M to 0.05M)

containing 0.05M NaCl for 1 to 3 hr at 5 C. After

removal of impurities by low-speed centrifugation,

centrifuge. Thepelleted virus particlesweresuspended

thesuspension wascentrifugedforI hrat 105,000 X g

at 5 C in the RP 30 rotor ofaHitachi 55P-2

ultra-in -0.067 M phosphate buffer (PB), pH 7.8, and

purified by another cycle of low- and high-speed

centrifugation.

Sucrose density gradient centrifugation. The

puri-fiedviruswassuspended inasmallamountof -0.067

M PB (pH 7.8), and Tween 80 was added to give a

final concentration of 0.05% The suspension was

layered onto 30 to 50%X, gradient of sucrose in the

same buffer, andcentrifuged for 2 hr at 25,000rev/

min, 5C, in aswinging bucket rotor. After the run,

fractions were collected through a needle by

punc-turing the bottom of the tube, and measured as

absorbance at260nm.

Electron microscopy. The structural components

thusseparated were removed from sucrose either by

high-speed centrifugation or by filtration through a

shortcolumn (I by 20 cm) ofSeplhadex G-25,

equi-librated with -0.067M PB, pH 7.2. The pellets

ob-tained in the former case were resuspended in a

small volume of the same buffer. Each drop ofthe

samples was mixed ondental wax with one drop of

2%' phosphotungstic acid adjusted to pH 6.8, and

the specimens were examined immediately in a

JEM 7Aelectron microscope.

RESULTS

Asdescribed byBergold (4), NPV of insects is

easily purified, practicallyfree of host materials,

because polyhedra can readily be obtained in a

very pureform forthefollowingreasons.(i) They

are highly characteristic macromolecules several

microns indiameter withhighdensity (2), and (ii)

although proteinaceousin nature, theyare

resist-ant totheusualproteolytic enzymes. In addition

polyhedra consist only of virus particles besides

polyhedralproteinmatrix(3, 10).

Examination ofisolated virus particles in the

electron microscope revealed the presence of

several structural components. To separate these

E 0

cs 0.1 5 Band

II

uX0.10 m \ BandIVI

c / \ Band I>/

1i

0.05

0. 0

10 20 30 40

Tube Number

FIG. 1. SUcrose delnsity gradienit profile of} NPV

stru(ctlurlal componzenits of B. mori. Virutsparticles were

isolated from polyhedra by treatment with 0.01 f

Na2C&O soluitionl conztaininiig 0.05m NaCl for I h1rat

SC. A 0.3-ml anmounlitofvirues suspenisionlin 0.067,ii

PB (pH 7.8) vith 0.05(%, Tween 80 waslayeredo0ito

4.4 mloJ30 to

50%(i,

25,000 rev,lmiiifor 2 hr- at 5C in a Hitachi SW 40

Rotor.Fractioniswere collectedasdescribedin the text.

SedimnentationiisfromrighttoleJt.

components, sucrosedensity gradient

centrifuga-tion wasfoundtobeuseful. Undertheconditions

used, virus particles, isolated by dissolving

poly-hedra with 0.01 M Na2CO3 solution containing

0.05 MNaCIfor1 hr,could beseparatedintofive

bandsin 30 to 50% sucrosedensity gradients by

centrifugation in the SW 40 rotor at25,000 rev,

minfor2hr at 5C(Fig. 1).

ItisshowninFig.2that thelowest band(band

I) is foundtoconsist ofthick rodparticles,

meas-uring330by 80nminaveragesize, which havean irregular knobby surface structure and occasion-ally a terminal protrusion at one end. When

en-larged, however, further details could notbe ob-tained, since the staining merely outlined the particles.

BandII (Fig. 3a) consistsmainly of slender rod particles,360 nmlong and60 nmwide.Asseenin

Fig. 3b, this particle appears to have transverse

regular bandingonthe surface, in whicha dense

mass ofinternal structure can also be seen. Of

theseparticles,some wereobservedtobedistorted

inshapeand some werepartially degraded.When

treated with 1 (j' mercaptoethanol, the slender

rodsweredrasticallydamaged,resultinginpartial release oftheinternalsubstance in someparticles

and producing empty rod membranes with

vari-ousdegrees of disintegration(Fig. 4).

BandsIIIand IV werefairly close together so

that the two bands could not be easily distin-guished. For furtherseparation, thesetwobands

were combined and recentrifuged in 20 to 40%c

sucrosegradientat25,000rev/minfor2 hr at 5C.

on November 11, 2019 by guest

http://jvi.asm.org/

MORPHOLOGY OF NUCLEAR POLYHEDROSIS VIRUS

FIG. 2. Virusparticles, negativelystainedwith 2%cphosphotunigstic acid,from thelowestband(ban1d1) in the'

gradient.Kniobby surfaced rod-shaped particles, 330by 80tim inaverage size, areseen. A termitalprotrusion is

observedat oneendofsomeparticles. X 50,000.

a

FIG. 3. (a) Virusparticlesfoundinband 11. Theseslenderrod-shaped particles,about 360 nim longand 60 tinm

wide, arepartially penetrated by the stain andare somewhat elongated longitudinally anddistorted in shape.

X160,000. (b) Slender rod-shapedparticles. On thesurface, regularbanding is visible. Note that an internfal

substatice canalsobeseen inside the particle. X 160,000.

269

on November 11, 2019 by guest

http://jvi.asm.org/

Afterthe run,however, only single a diffuse band

appeared in the gradient. Whenfractionated and

measured by opticaldensityat280nm, twowidely diffused, overlapping bands could be resolved, indicating a considerable size variation of these components. Examination of these bands in the electron microscope revealed that both empty sphericalandemptyrod-shaped membranes were present.

Band III is rich in empty spherical and frag-mented membranes (Fig. 5). Even at higher magnification, further details could not be

re-solved in these membranes. During examination

of band I, such membranes could frequently be

seenfree from the thick rodparticles to give the

appearance of slender rod-shaped particles (Fig. 6). Theemptysphericalmembranes are thus

con-sidered to be the outer membrane of the thick

FIG. 4. Slenider rod-shaped particles treatedWith 1%O

mercaptoethaniol.

degradationz

apparenit.

Olne

particleis releasin7gitsintternialsuibstance, anidaiiotherontehas partiallyreleased. X 200,000.

FIG. 5.

Menmbranleolis

fragme,itary

membranies

X 50,000.

on November 11, 2019 by guest

http://jvi.asm.org/

rods. Similar findings have been reported in the

isolated virus preparations from some species of NPV polyhedra (2, 7). The empty rod-shaped membranes were mainly located in band IV. On

the surface, regular striations are clearly visible (Fig. 7) as can be seen with the slender rod-shaped particles (Fig. 3b). It is noted that the same ob-servation has been made by Kozlov and Alexeenko(7) with NPV of the silkworm. It seems obvious that these membranes are the inner mem-braneof the thick rod-shaped particles.

The uppermostband (band V) appeared atthe

sucrose-suspensioninterfaceas asharp band and was found to consist purely of small spherical particles with a diameter of about 20 to 25 nm

(Fig. 8). It must be noted that the particles are somewhat heterogeneous in size and shape.When

analyzed in a10to 40c% sucrose density gradient bycentrifugation for3 hrat25,000rev min, these particles behaved as a broad diffuse band, indi-cating size heterogeneity. It is also noteworthy that withinthe emptyinner membranes found in

band IV, similarparticlesarefrequentlyobserved

(Fig.

9).

DISCUSSION

NPV of the silkworm was shown to be

de-graded in a progressive fashion from thick rod

particlestosubviralstructuralcomponents by the

action ofalkali.

The thick rod particles observed in the lowest

band (Fig. 2) are apparently identical to those

found inultrathin section ofpolyhedra (3, 9, 10)

andwereelucidatedto consist of an internal

sub-stance enveloped with two layers of

membrane,

outer and inner membranes (Fig. 3a, 3b, 5, and

7). The outer membrane may serve to retain the

compact rigid structure of virus rods, since the

slenderrods, which had lostthe outer

membrane,

wereelongated longitudinallyand frequently were

distorted in shape during the process ofelectron

microscopy (Fig. 3a). This is supported by the

results obtained by thin sectioning of the tissue infected with NPV of the silkworm (6).

It is evident that when slender rods degrade,

furtherbreakages may occur preferentially at the end of the particles to release internal substances (Fig. 4) because the structure of the empty inner

~~~~~~~~~~~,.

lip/A

FIG. 6. Outermembranies arefreeing from thick rod-shaped particles as menitedmembranles. Thissamplewastakeni frombalndI. X 50,000.

empty sphericalmembrcanies or

fraig-FIG. 7. Emptyrod,iinnier memnbranesfrombauid IV.Reglalarstriationsareclearly seeuionithe suirfcace. X 150,000.

on November 11, 2019 by guest

http://jvi.asm.org/

membrane was well preserved exceptatthe ends

ofthe membrane (Fig. 7). Although the internal

substance which appearedas a dense mass (Fig. 4) mayrepresent ahighly organized structureand

is possibly helical in configuration, as suggested

previously (5, 7),no direct evidence couldbe ob-tained.

The facts that the small spherical particles

which seemed heterogeneous in size and shape (Fig. 8) were consistently found and shown to

::k.

:

F'"4

I

a,

P..

i.

FIG. 8. Smallsphericalparticlesfrom theuppermost band(band V). It is remarkable that these particlesar.

somewhatheterogeneouis insizeandshape. X 186,000.

FIG. 9. Membraneouis materialsfoundinband IV. Itmust be notedthat within the emptyinnermembranes

particles similar to thoseshownIinFig. 8 are observed. X 192,000.

n

.4,_{,.i '.}

v...t,f:.'

11

AM

Av.

on November 11, 2019 by guest

http://jvi.asm.org/

behaveas abroadlydiffused band insucrose

gra-dients indicatethat the component doesnot

rep-resent any structural unit but is a degradation

product of virus particles. Similar particles are

frequently observedwithin theemptyinner

mem-braneafter variousdegreesofdisintegration. This

strongly suggests that the component (Fig. 8) might be thedegradation product of the internal

substance.

The small spherical viral subunits observed earlier in the shadowed preparation of virus particles (1,8) maybeinterpretedas

correspond-ingto those shown in Fig. 8. It is probablethat

metalshadowingcaused misunderstanding ofthe

componentas aviral subunit.

Theinfectivity ofeach fractionwas examined,

and it has been found that bands I andIIshow the

activity ofNPV. The detailed accounts will be

reportedelsewhere.

LITERATURE CITED

1. Bergold, G. H. 1953. Themultiplication of insect viruses,

p. 276-283.In P. Fildes andW. E. vanHeyningen (ed.),

Thenatureofvirus multiplication, 2nd Symposium Soc. Gen. Microbiol. Cambridge University Press, London and New York.

2. Bergold, G. H. 1958. Viruses ofinsect, p. 60-142. In C. Hallauer and K. F. Meyer (ed.), Handbuch der Virus-forschung, vol. 4. Springer, Vienna.

3. Bergold, G. H. 1963. Finestructureofsomeinsectviruse3.

J.InsectPathol.5:111-128.

4. Bergold, G. H. 1963. The natureof nuclear-polyhedrosis. viruses,p.413-456.InE. A.Steinhaus (ed.), Insect pathol-ogy, an advanced treatise, vol. 1. Academic Press Inic.,

NewYork.

5. Harrap, K. A., and B. E.Juniper. 1966.The internal struc-tureofan insect virus. Virology 29:175-178.

6. Himeno, M., S. Yasuda, T. Khosaka, and K. Onodera. 1968. The finestructureofanuclear-polyhedrosisvirus of

thesilkworm. J. Invertebr. Pathol. 11:516-519.

7. Kozlov, E. A., and I.P. Alexeenko. 1967.

Electron-micro-scope investigation of the structure of the nuclear-poly-hedrosis virus of the silkworm, Bombyxmori. J.Invertebr.

Pathol. 9:413-419.

8. Krieg, A. 1961. Uber den Aufbau und Vermehrungsmoglich-keitenvonstabchenformigen Insekten-Viren.II. Z.

Natur-forsch.16b:1 15-117.

9. Morgan, C., G. H. Bergold, D. H. Moore, and H. M.Rose.

1955. The macromolecular paracrystalline latticeof insect

viralpolyhedral bodies demonstrated in ultrathinsections examined intheelectronmicroscope. J. Biophys. Biochem. Cytol.1:187-190.

10. Morgan, C., G. H. Bergold, and H. M. Rose. 1956. Use of serial sections to delineate the structure of Porthetris dispar virus in the electron microscope. J. Biophys. Bio-chem.Cytol. 2:23-28.

273

on November 11, 2019 by guest

http://jvi.asm.org/