Structural Polypeptides of the Granulosis Virus of Plodia interpunctella

0022-538X/80/02-0877/10$02.00/0

Structural Polypeptides

of

the

Granulosis Virus of Plodia

interpunctellat

KATHLEEN A. TWEETEN,' LEE A. BULLA, JR.,2 ANDRICHARD A. CONSIGLI'*

Division of Biology, Section of Virology and Oncology, Kansas State University, Manhattan, Kansas66506,1 andU.S. Grain Marketing Research Laboratory, Science and Education Administration, Manhattan,

Kansas665022

Techniquesweredevelopedfor theisolationandpurificationofthreestructural components ofPlodia interpunctella granulosis virus: granulin, enveloped nu-cleocapsids, andnucleocapsids. Thepolypeptide compositionanddistribution of protein in each viral component were determined by sodium dodecyl sulfate

discontinuous

and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of15structuralproteins ranging inmolecularweight from 12,600to97,300. Five of theseproteins,havingapproximatemolecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surfaceradioiodination of the enveloped nucleocapsids. Present in purified nu-cleocapsidswereeight polypeptides.Thepredominantproteins in thisstructural

componenthadmolecularweights of 12,500 and 31,000. Whereas no evidence of

polypeptideglycosylationwasobtained,sixof the viral proteins were observed to bephosphorylated.

Granulosis and nuclear polyhedrosis viruses arestructurally complex viruses belongingtothe family Baculoviridae. They infect a number of lepidopteran insects, causing disease and larval death in infected populations. Because of their effectiveness, the baculovirusesare being consid-ered foruse asbiological insecticides forcontrol

of their insect hosts. As a result, these viruses

have acquired scientific interest, and investiga-tors have beguntoobtain

information

ontheir molecularproperties. Suchknowledgeisneeded for

developing

methods of virus identification anddetection and for

determining

theireffects atthecellular levelonnontargetspecies.

The basic structural unit of the granulosis virus (GV) and nuclear polyhedrosis virus (NPV) is a

high-molecular-weight,

covalently closedsupercoiledDNApackaged withina rod-shaped capsid. The

nucleocapsid

issurrounded bya lipidenvelope, anddepositedontheouter surface of the envelope is a thick matrix of protein(1, 10, 21,24).InNPV, several enveloped nucleocapsids are embedded within a protein crystal. This feature

distinguishes

it from

GV,

in

which the enveloped nucleocapsids are singly

occluded.

Identification of the baculoviruses based on

characteristics of their matrix

proteins

hasbeen

showntobe

inadequate.

Theseproteinsall have

molecular weightsof26,000to30,000(6, 11, 16,

22) and demonstrate

serological

cross-reactivity

t Contribution no. 79-383-j, Kansas Agricultural Experi-mentStation,Kansas StateUniversity, Manhattan,KS66506.

(8, 11, 14). On theotherhand, bothqualitative

andquantitative differences have beenobserved

intheproteincomposition of theenveloped

nu-cleocapsids and nucleocapsids from various

NPVisolates (7, 11, 22).

Comparisons of

GV,

based on polypeptide composition, cannotbe as readily made. Aside from extensive characterization ofgranulin, the majorcomponentoftheGVprotein matrix,the only structural proteins that have been exam-ined are those of the

enveloped

nucleocapsids

fromthe GVsofPieris brassicae(5), Trichoplu-sia ni, and Spodoptera frugiperda (22). Like those of

NPV,

they

consisted of 12to 18

poly-peptides

ranging

in molecular

weight

from

12,000 to 90,000. Information on the isolation

and characterization of GV

nucleocapsids

is

available

only

for the virus which infects P.

brassicae(5).

Ourlaboratory isinvestigatingthe molecular biology of the GV of the Indian meal moth Plodia

interpunctella.

To

analyze

the structural polypeptide

composition

of this

virus,

we devel-oped

biophysical

methods for its

separation

into

three structural components:

granulin,

enve-loped

nucleocapsids,

and

nucleocapsids.

These methods,

along

with the

polypeptide

composi-tion and distribution of

protein

in each viral

component, are

reported

in this paper. Other

studiesonNPV andGV havenot

provided

direct

evidence that

specific

viral

polypeptides

are

con-stituents of the viral

envelope.

In the

present

work, radioiodination which

specifically

labels

877

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virus surface proteins was used to identifyand

localize the GV envelope polypeptides.

Modifi-cations of viral polypeptides by glycosylation

andphosphorylation werealsoinvestigated.

MATERIALS AND METHODS

Production and purification of GV. GV was

produced in alaboratory colonyofP.interpunctella rearedaspreviouslydescribed(23).Earlythird instar larvae were infected peroswithGV,andtheviruswas purifiedby differentialcentrifugation,treatmentwith 1% deoxycholate, and velocity sedimentation in su-crosegradients (23,25).

Isolation of theproteinmatrix. Theprotein ma-trixwassolubilizedbyincubating purifiedGVfor10

minat37'C in0.05 Msodium carbonate-0.05 MNaCl, pH10.6.To isolate the matrixproteinsinnondegraded form, the GVwasheated before carbonatetreatment

for30min at700Ctoinactivateendogenousprotease activity (25). The dissociated viruswaslayeredon30 to70%glycerol(vol/vol,in0.01MTris-hydrochloride, pH 7.5) gradients which were centrifuged at 25,000 rpm (SW 41 rotor) for 30 min at 10°C. The matrix proteinswererecovered from the top of thegradient and concentrated inamodel12Amiconultrafiltration cell withaUM-10 membrane.

Isolation ofenvelopednucleocapsids. Purified GV wasincubated in 0.05 Msodium carbonate-0.05 MNaCl, pH 10.6, for5min at roomtemperature. The dissociated virus waslayered on 30 to 70% glycerol (vol/vol, in 0.01 MTris-hydrochloride, pH 7.5) gra-dients which werecentrifugedat 25,000 rpm(SW41

rotor)for1h at 100C.Theband ofenveloped nucleo-capsidswasrecovered from thegradients, diluted with

0.01MTris-hydrochloride,pH 7.5, and centrifugedat

25,000 rpm (SW 41 rotor) for 1 h to remove the glycerol. Theenvelopednucleocapsidswerestored in

0.01MTris-hydrochloride,pH 7.5,at4°C.

Isolation of nucleocapsids. Purified enveloped nucleocapsids wereincubatedin 1% (vol/vol, in0.01

M Tris-hydrochloride, pH 8.5) Nonidet P-40 (NP-40;

Shell Chemical Co.) for30minwith stirring at room temperature. Thenucleocapsids were separated from

thesolubilizedenvelope proteins by sedimentation on

30 to70%glycerol (vol/vol, in0.01 M Tris-hydrochlo-ride, pH 8.5) gradients centrifuged at 30,000 rpm (SW

41 rotor) for 1 h. The band of nucleocapsids was dilutedwith 0.01 MTris-hydrochloride, pH 8.5, and centrifuged at 25,000 rpm (SW 41 rotor) for 1 h to

removeglycerol.Thenucleocapsids were stored in 0.01

MTris-hydrochloride, pH 7.8, at4°C.

Radiolabeling of GV invivo. Radioactively

la-beled GVwasproduced in vivo by injection of1 d(0.1

to 0.5,uCi) ofisotope into larvae at 96 and 120 h after infection. The isotopes used included [3H]thymidine (71Ci/mmol,Schwarz/Mann),'4C-aminoacidmixture

(Schwarz/Mann), and [32P]orthophosphoric acid

(New England Nuclear). GV was purified from in-jected larvae8days afterinfection.

lodination ofenveloped nucleocapsids.

Prepa-rations ofpurifiedenveloped nucleocapsids containing

250

fig

ofproteinin100

til

of0.01 MTris-hydrochloride, pH 7.5, were mixed with 50

1l

of 0.2 M phosphate buffer, pH 7.2,0.5 mCiof['25I]iodine (ICNChemical

and Radioisotope Division), 25tu (0.5 mg) of

Enzy-mobead reagent, and 25 ll of 1% /8-glucose. After incubation atroomtemperature for30min, the En-zymobeadswereremoved bycentrifugation at 250x

g for5min. Unreactediodinewasremoved from the enveloped nucleocapsids by centrifuging the prepara-tionon a30 to70%glycerol gradient for1hat25,000 rpm (SW 41 rotor). The visible band of enveloped nucleocapsids was recovered and dialyzed for 24 h against0.01MTris-hydrochloride, pH 7.5.

Glycosylation of viral components. To label galactosyl residues,amixtureconsisting of250ug of virus (granulin, enveloped nucleocapsids, or nucleo-capsids), 12 U of neuraminidase(Calbiochem), and 25 U of galactose oxidase (Sigma ChemicalCo.) in 200 ,ul of 0.02 Mphosphatebuffer, pH 7.2, and0.1M NaCl

was incubated for 1 h at 37°C. Then, 500 ,Ci of NaB[3H]4 (New England Nuclear) in20 1L of0.01 N NaOHwasadded, and the mixturewasincubatedat roomtemperature for30min. The viralpreparations were then repurified by sedimentation on glycerol gradients. Sendai viruswaslabeled inasimilar man-ner.Controls consisted of viral preparations that

re-ceivednoenzyme treatment.The viral proteinswere

then resolved bypolyacrylamide slab gel electropho-resis andtritium-containing bandsweredetected by fluorography.

Electron microscopy of viral components. Samples ofenvelopednucleocapsids and nucleocap-sidswereplaced onFormvar-coated gridsand nega-tively stained with 1%phosphotungstic acid,pH 7.0. Grids were examined withaPhilips EM 201at60 kV. Polyacrylamide gel electrophoresis of viral polypeptides. Viral proteinsweresubjectedto elec-trophoresis on sodium dodecyl sulfate (SDS)-poly-acrylamide slab gelsby the discontinuous buffer

sys-temof Laemmli(15).Samplepreparation, electropho-resis, andstainingwere as described (25). Molecular weightsweredeterminedby the method of Weber and Osborne(26) withcytochromec(11,700), chymotryp-sinogen (27,500), ovalbumin (43,000), and bovine se-rum albumin(68,500) (Schwarz/Mann) asstandards. Destained gelswerephotographed,andthenegatives were scanned with a Photovolt photometer, model

520-A. Scanswererecorded andpeakareaswere de-termined with a Hewlett-Packard 3380A integrator. Gels were dried with an SE-540 Hoeffer Scientific Instrumentsslabgel dryer.Forautoradiography, dried

gels were placed in contact with Kodak X-Omat R

film (XR-1). Fluorography ofgelswasconducted by

the method ofBonner and Laskey (4). Films were

developed in Kodak D-19, and scans were made as described above.

RESULTS

Purification and characterization of the

protein matrix. It is well-documented that

treatment ofoccludedbaculoviruses with

alka-linecarbonateresults inthesolubilization of the

protein matrix (2, 13, 16). This technique was

used toisolate the matrixproteins from P.

in-terpunctella GV. The solubilized matrix was

separated from other viral components by

cen-trifugation on glycerol gradients. It formed a

J. VIROL.

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GRANULOSIS VIRUS STRUCTURAL POLYPEPTIDES 879

diffuse band which remained on top of the gra-dients (Fig. 1).

Polyacrylamidegelelectrophoresis ofthe iso-lated matrix showed that it consisted of a

mix-tureofpolypeptides ranging inmolecularweight

from 10,000 to 28,000 (Fig. 2B). Most of these

protein bands resulted from the activity of an alkaline protease associated withtheGV(25).If

the protease was inactivated before

solubiliza-tion ofthematrix,theproteinsrecovered on top

of the gradient consisted primarily of a

poly-peptidehavingamolecular weightof 28,000 (Fig.

2A). Also observed in the matrix preparations

were two to three minor polypeptides having

approximate molecular weights of 66,000 to

74,000 (Fig.2AandB).

Purification of enveloped nucleocapsids. Carbonate treatment was also used to release the envelopednucleocapsids from purified GV. After incubation in carbonate, the dissociated viral preparation was centrifuged on glycerol gradients. When

[3H]thymidine-labeled

virus was disrupted, a single peak of DNA-labeled virus was obtained in the gradients (Fig. 1). When

"4C-amino

acid-labeled GV wassimilarly treated, a portion of the protein label cosedi-mented with the DNA label while the majority oftheradioactivity correspondingtothe matrix

proteins remained on top ofthegradient.

Elec-4~~~~~~~~~~~~~~~~~~4

7- ~~~EN

40 1~~~

6-t

|211y

2

5-4

0~

U

4--2 4

FRACTONNUMBER

FIG. 1. Isolation ofGVenveloped nucleocapsids (EN) bysedimentation inglycerol gradients. [3H]-thymidine-labeledor14C-aminoacid-labeled GVwas

incubated in 0.05Msodiumcarbonate-0.05 MNaCl, pH 10.6, for15minatroomtemperature. The

disso-ciated GVwaslayeredonto 30to 70%oglycerol

gra-dients (in 0.01 M Tris-hydrochloride, pH 7.5) and centrifugedat25,X000rpm(SW41rotor) for1h.

A B

[image:3.514.312.409.80.263.2]

VP28- o

FIG. 2. SDS-polyacrylamide gel electrophoresis of GV matrix proteins. The protein matrix was solubi-lized andrecoveredon topof glycerol gradients as shown inFig. 1.Samples were prepared for electro-phoresisbyboiling in 2% SDS,5% 2-mercaptoethanol, and10%oglycerol. (A) Matrix proteins isolated from GV heatedbefore carbonate treatment for 30 min at 70°Ctoinactivateendogenous protease activity. (B) Matrixproteins isolatedfromprotease-active GV.

tron microscopy ofthe band of virus obtained onthesegradients showed thatit consistedofa homogeneouspreparation ofenveloped nucleo-capsids (Fig. 3A).Fewdegraded viral envelopes were observed, indicating that the carbonate

treatment and sedimentation in glycerol

gra-dients were not detrimental to the structural

integrity ofthis viralcomponent.

Purification of nucleocapsids. GV nucleo-capsidswereisolatedbytreatingenveloped nu-cleocapsids with the nonionicdetergent Nonidet

P-40. Enveloped nucleocapsids, labeled in vivo

with

['H]thymidine

or

"4C-amino

acids, were

dissociated with the detergent and then

sedi-mented on 30 to 70%

glycerol gradients.

After

centrifugation,

essentially

all of the DNA label

waspresent ina

single

band which sedimented

moreslowlyin thegradient than the

enveloped

nucleocapsids.

Approximately

43% of the amino acid label cosedimented with the viral DNA label,whereasthe remainderwaslocalizednear

the top of the gradient (Fig. 4). Material from

bothregionsof the

gradient

wasrecoveredand

examined for its

polypeptide composition

(re-sults described

below).

The band of virus was

recovered from the gradient and examined

by

electronmicroscopytoconfirm that itcontained

nucleocapsids

(Fig.

3B).

The

micrograph

dem-onstratesthat the

nucleocapsids

were

structur-allyintact and free of

contaminating

envelope

fragments.

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880 TWEETEN, BULLA, AND CONSIGLI

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FIG. 3. Electron micrographs ofpurified GVenveloped nucleocapsids and nucleocapsids. Enveloped

nucleocapsids (A)andnucleocapsids(B)wereisolatedasdescribed in thelegendstoFig.2 and4, respectively.

Samples weremounted on Formvar-coatedgrids and stained with 1%phosphotungstic acid,pH7.0. Bar represents400 nm.

12

N

10-~~~~~~~

X,,""l' .

0~~~

4-

2-5 1,5 2'0 30

FRACTIONNUMBER

FIG. 4. IsolationofGVnucleocapsids (N) by sedi-mentationofNonidet P-40-dissociatedenveloped

nu-cleocapsidsinglycerol gradients.

[3H]thymidine-la-beled (0) or '4C-amino acid-labeled (0) enveloped nucleocapsidswereincubatedin 1%Nonidet P-40in 0.01 MTris-hydrochloride, pH 8.5, for30minatroom temperature. Thepreparation was layeredon30to 70oglycerol gradients (in0.01 MTris-hydrochloride, pH 8.5)andcentrifugedat25,000rpm(SW41rotor)

for1h.

Identification of the structural proteins

ofenvelopednucleocapsidsand

nucelocap-sids.Samples ofpurifled enveloped

nucleocap-sids and nucleocapsids were dissociated with

SDS and mercaptoethanol. The proteins were

subjected to electrophoresis in discontinuous

SDS-polyacrylamide gels.Anelectropherogram

ofthe structural polypeptides oftheenveloped

nucleocapsids is shown in Fig. 5 (EN). Fifteen

proteinsranginginmolecularweightfrom12,600

to97,300wereconsistentlyidentified. Avariety

of acrylamide concentrations (10, 12.5, 15 and

20%) wasused toadequatelyseparate proteins

having similarelectrophoretic mobilities.By

us-inggradient gelsranging inacrylamide

concen-tration from 10 to 26% or 7 to 15%, threetofive

additional minor viral protein species were

re-solved. The molecular weights of these minor

structural proteins were 20,000, 36,000, 57,600,

59,800, and 86,000. The average molecular

weight of each of the enveloped nucleocapsid

proteins, determinedbycomparisonwith

molec-ularweightstandards, is presented in Table 1.

Eight polypeptides, ranging in molecular

weight from 12,500 to 64,200, were observed when nucleocapsids were dissociated and elec-trophoresedin 12.5and 15%SDSslab gels (Fig.

5, N). The average molecular weight of each

nucleocapsid polypeptide is shown in Table 1.

Two of the nucleocapsid proteins, VP49 and

VP39, had molecular weights very similar to polypeptides identified as constituents of the

viral envelope (results described below). They

were, however, demonstrated tobe unique

nu-cleocapsid polypeptides by electrophoresison 7 to 15% SDS-polyacrylamidegradient gels. This

gelsystemprovidedforseparation of enveloped

nucleocapsid proteinsVP48 and VP39 eachinto

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[image:4.514.68.261.331.526.2]

E N N

VP97--VP8

--

VP63--VP4 8-.

-VP63

VP46

\_

VP44

\=

---VP49

VP44-='

-VP42

~

~~~

-VP4

VP39

-7 -

VP36

VP31 --

4

_ - VP31

VP29

-- VP29

VP26

-VP17

-VP 16 --VP12

FIG. 5. SDS-polyacrylamide gelelectr GVenvelopednucleocapsids (EN)andni (N). Numerical designations referto th

weight (x 10-3) ofeachpolypeptide dei

comparison withmolecular weight stand

twodistinctprotein bands (resultsn

Only one of each of these polype

presentinnucleocapsids electrophor

same gel. In addition tothe eight p(

describedabove, minor proteincomp(

ing approximate molecular weights

57,000, and 68,500wereobservedinni

preparations.

Proteincomposition ofGVenv(

cleocapsids andnucleocapsids. T amount ofeach viral polypeptide in

nucleocapsids andnucleocapsidswa;

from densitometricscansofpolyacry

in which unlabeled or "C-amino a

viruswasresolved. Theresultingdist

proteinamongthe various viral

poly

shown in Table 2. VP12, VP17, V]

VP39, andVP48werethepredomina

in Coomassie-stained enveloped nue

since they represented 40,22, 20, 4,

thetotalproteinin this viralcomponm

[image:5.514.65.355.61.394.2]

tively.Theremaining polypeptidesw

TABLE 1. Molecular weights of GV enveloped nucleocapsid and nucleocapsid proteins

Envelopednucleocapsids Nucleocapsids

Poly-

Poly-peptide Mol

wta

peptide MolWtb

designa-

designa-tion tion

VP97 97,300±3,300

VP88 88,600±4,700

VP63 63,700±2,500 VP63 64,200± 3,000

VP49c 49,300±500

VP48 48,200±1,700 VP46 46,800±1,500

VP44c 44,900±1,500 VP44" 45,600±1,000

VP42 42,400±1,000 VP39-EC 39,700± 1,000

VP39-N 39,600±700 VP38 38,200± 1,100

VP36 36,700±600 VP31 31,000± 1,500 VP31 31,000±700

VP29C 29,900± 1,500 VP29C 30,100±1,300

VP26 26,300±1,300 VP17C 17,800±600 VP16 16,300±700

VP12 12,600±500 VP12 12,500±300 aValues reported are the means and standard de-viationscalculated from 10 separatedeterminations.

bValues reported are the means and standard de-viations calculated from eight separate determina-VP12 tions.

e

Phosphorylated

polypeptides.

inamountsranging from less than 1% (VP88 and

ophoresisof VP38) to 2.8% (VP42). Scans of

Coomassie-e

molecular

stained

nucleocapsid proteins

indicated that

termined

by

they

consisted

primarily

of VP12

(54%)

and

fards. VP29 and VP31 (41%). The other proteins

as-sociated withnucleocapsidswereminor

compo-iotshown). nents which represented from 2.3% (VP39) to ptides was 3.1%(VP44)of the totalnucleocapsid protein.A

esedonthe similar distribution of protein resulted from

olypeptides scans of fluorograms of gels containing

"4C-onentshav- amino acid-labeled viralpreparationsexcept for

of 27,000, VP12. Substantially more of this viral

poly-acleocapsid peptide appeared to be present in enveloped nucleocapsids and nucleocapsids, based on in-elopednu- tensityof Coomassie

staining,

thanon incorpo-'he relative ration of'4C-amino acids (Table2). The lower enveloped percent values obtained by incorporation of sestimated amino acidsmost

likely

canbeaccounted forby

lamidegels the

extremely

high arginine content of VP12

Lcid-labeled

(manuscript submitted). Polypeptides

of high tributionof argininecontentstainmoreintenselywith

Coo-Tpeptides

is massie brilliant blue than do those with lower P29, VP31, argininecontent (D.J.Stubs,M.S.thesis, Kan-ntproteins sasStateUniversity, Manhattan, Kansas,1978). -leocapsids, Identification of viralenvelope proteins.

and5%, of Radioiodination is a usefultechniquefor

exam-ent,respec- ining the distribution and organization of

pro-erepresent teinsin animal viruses(19, 27).Agentlein vitro

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882 TWEETEN, BULLA, AND CONSIGLI

TABLE 2. Proteincomposition ofGVenveloped nucleocapsidsandnucleocapsids

Envelopednucleocapsids Nucleocapsids

Totalprotein (%) Totalprotein(%)

Viralpolypeptide I Viralpolypeptide

Staineda

tionb

Stained"

Incorpora-VP97 2.7 5.9

VP89 -C -C

VP63 1.0 1.4 VP63 2.6 2.8

VP49 2.5 3.3

VP48 5.0 8.9

VP46 1.8

VP44 1.2 9.4 VP44 3.1 3.1

VP42 2.8J

VP39-E 4.1 5.7

VP39-N 2.3 2.2

VP38 -C -C

VP36 -c _^

VP31,VP29 20.7 19.1 VP31,VP29 40.9 39.2

VP26 -C -C

VP17 22.3 22.9

VP16 _C _C

VP12 40.8 27.3 VP12 54.2 44.5

aPercentage of totalproteindeterminedby calculatingtheareaunder eachCoomassiebrilliant blue-stained peak; values represent the average of six separate determinations.

bPercentage

of totalproteindetermined fromareaundereachpeakresultiilgfromautoradiographyofSDS gels of virus labeled in vivo with'4C-amino acids;valuesreportedarethemeansof three separatedeterminations.

cPeak areas less than 1% of the totalprotein.

labeling procedure employing solid-phasebound

lactoperoxidase (Enzymobeadreagent,Bio-Rad)

and['251]iodinewasusedtoidentifyGVenvelope

proteins. The large size ofthe bound enzyme

inhibits itspenetrationintothe viral membrane

so thatonlythoseproteinsexternal tothe viral

envelopearelabeled (18, 27).

To verify the specificity ofthe labeling

pro-cedureused,iodinated enveloped nucleocapsids

were incubated with the proteolytic enzyme

trypsin (2mg/ml)for 2 hat37°Csothat proteins

locatedontheoutersurface of theviral envelope

would be digested (18, 20). After trypsin

treat-ment, the enveloped nucleocapsids were reiso-latedby centrifugation in glycerol gradients. It

wasfoundthat the majority of the iodine label

wasremoved fromthevirus by theproteaseand remained on top of the gradient. Electron

mi-croscopyof the band of virusobtained in these

gradients revealed that itconsisted of particles

with intactenvelopes (results notshown)

indi-catingthat thetrypsin didnotalterthe integrity

ofthe envelopes. It was concluded from these

datathatthe lactoperoxidase labelingwas

spe-cific forproteinsexternaltothe viralenvelope.

Toidentify which viralproteinswerelabeled

bythelactoperoxidaseprocedure, and thuswere

envelope constituents, portionsof the iodinated

enveloped nucleocapsids were dissociated by

SDSandmercaptoethanoland the proteinswere

resolved by electrophoresis in 12.5% discontin-uousSDS

gels.

Densitometer

tracings

ofan

au-toradiogramof suchagel (Fig. 6A)showed that

the iodine label waslocalized atpositions

cor-responding to VP17, VP39, VP42,

VP48,

and

VP97. Ofthesepolypeptides,VP17wasthe

ma-jor component. The smallamount of label

as-sociated with

nucleocapsid proteins

VP29 and VP31

probably

resulted fromsome

envelope

dis-ruption duringpreparation of enveloped nucleo-capsids for iodination.

NP-40 treatment of the iodinated

enveloped

nucleocapsids

provided

further evidence that these five structural

polypeptides

were

envelope

proteins.Viralproteinsthatweresolubilizedby

NP-40wereprecipitatedwith 25%

trichloroace-tic acid and resolved in

polyacrylamide

gels.

Autoradiography revealed that the label mi-gratedwithelectrophoretic mobilities character-istic-ofVP17,VP39, VP42, VP48,and VP97(Fig.

6B).The viral proteinswhichwerereleasedby

NP-40 were also examined after their

electro-phoi;esis

inSDSdiscontinuousgelsand staining with Coomassiebrilliantblue. As shown inFig. 6C,theproteinsthatwerevisualizedagainwere VP17, VP39, VP42, VP48, and VP97. A small amountof VP12wasalso observed in thesegels.

Its presence mayindicate thatsome

nucleocap-sid disruption occurred during detergent treat-ment.

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Modifications of GV structural

polypep-0. tides.(i)Glycosylation.Asensitivetechnique W W,n40, _: N C for detection of carbohydrate moieties on

poly-> > > >>>>> > > peptides consists of radiolabeling with

[3H]so-diumborohydride after neuraminidase and

ga-lactose oxidase treatment (3, 9). Thisprocedure

was used to determine whether any of the struc-turalpolypeptides ofP.interpunctellaGVwere glycosylated. Even afterprolonged exposure (4

months) ofgelscontaining radiolabeled virus to

X-ray

film,

no

radioactivity

wasdetectedinany of the polypeptides associated with granulin,

enveloped nucleocapsids,

or

nucleocapsids.

Un-a

: derthe same conditionsoflabeling andexposure,

thetwostructuralglycoproteins of Sendaivirus were extensively labeled. A labeling procedure

specific

for sialic acid residues

(3)

also failedto .s, .revealanyglycosylationin GV.

.,,.

Glycosylation

ofviral

polypeptides

was also

investigated by SDS gel electrophoresis and

-Ai--

"fluorography

ofviral

components

isolatedfrom

infected larvaethat were injected with

['4C]fu-cose

(15

Ci/mmol,

New

England

Nuclear)

or

B['4C]glucosamine (30

Ci/mmol,

New

England

Nuclear). Again, noincorporation ofisotope by anyof theGV structural proteinswasobserved. (ii) Phosphorylation. Preliminary

experi-ments wereconductedtodeterminewhetherGV

uW

structural

polypeptides

were phosphorylated.

32P-labeled

viruswas

produced by injecting

1

I1

'

(25

mCi/ml)

of

[32P]orthophosphoric

acid

(neu-,a tralizedto

pH

7.4with1M

Tris-hydrochloride)

co W . 3into GV-infected larvae. Afterpurification ofthe

virus from the larvae, the granulin, enveloped

0 I' 'l

||nucleocapsids,

and

nucleocapsids

were

isolated,

dissociated withSDSandmercaptoethanol,and

electrophoresed

on

SDS-polyacrylamide

gels.

The association of radioactive

phosphate

with

viral

polypeptides

wasdetermined

by

compari-^,,,C FIG. 6. IdentificationofGVenvelopeproteins. (A)

Surface-iodinated enveloped

nucleocapsidswere dis-sociated with SDS and mercaptoethanol, and the

proteins

were resolved

by

SDS

gel

electrophoresis.

.*h

I X,

Viralpolypeptides

wereidentified by comparing den-sitometer tracings of autoradiograms of the gels

*M1 s 1

1,

(---)tothose

of

Coomassie blue-stained

gels

( ).

.*,%1

4,

(B andC)

Iodinated

orunlabeledenveloped

nucleo-capsids

were incubated in 1% Nonidet P-40for 30

min at roomtemperature. The solubilizedproteins

were isolatedas shown inFig. 4 andprecipitated

I

I ' V i U 1. with 25% trichloroacetic acid. Theprecipitateswere

washed withethanol,dissociated with SDS and mer-a

1''.t1 1'captoethanol, and resolved by SDS gel

electrophore-sis.

Envelope proteins

were identified bycomparing densitometer tracings of autoradiograms (B) or of l-e;k- i stainedgels (C) containing the solubilized proteins

->2t-\j (---) to those ofstainedgels containing enveloped

..*| nucleocapsids( ).

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884 CONSIGLI

son of densitometertracingsofautoradiograms

of each gel to those of the samegelstained with

Coomassiebrilliant blue. Themajorconstituent

of theproteinmatrix, granulin,wasobserved to

be phosphorylated (Fig. 7A). Of the proteins

present in enveloped nucleocapsids, VP17,

VP29,VP39, VP44,and VP48 contained

signifi-cant amounts of 32P-label (Fig. 7B). This

re-sultedfromincorporation of theisotope bytwo ofthe envelope proteins, VP17 and VP39. The remaining three phosphorylated species, VP29, VP44, and VP49, wereassociated with nucleo-capsidpreparations

(Fig.

7C).

DISCUSSION

The techniques described in this paper

pro-vided forthe separation of the GV ofP.

inter-punctellainto

protein

matrix,

enveloped

nucleo-capsid, nucleocapsid, and solubilized envelope

components. By isolation of each viral

compo-nentandanalysis ofitspolypeptide composition, the number andorganization ofthe

major

pro-teins in the structurally

complex

GV could be Lu

elucidated. Thepredominantconstituent of the z

proteinmatrixhadamolecular

weight

of28,000. ,

Inthisproperty,it resembled thematrixproteins ci

that have beencharacterized inother baculovi- l

ruses (6, 11, 16,22).The

high-molecular-weight

0

proteins associated withthegranulin appear to i

be unique to the protein matrix, since their < electrophoretic mobilities were different from

that of the polypeptides found in either the

enveloped nucleocapsids or nucleocapsids. McCarthy and Liu (16) have also observed a

high-molecular-weight

protein in the protein

matrix prepared from the NPV of Porthetria

dispar.The distribution oftheseminorproteins

within the protein matrix, aswell as their

bio-chemical properties andimportancetothe viral

infectionprocess, remains to bedetermined.

The number ofpolypeptides observedin the

enveloped nucleocapsidsandnucleocapsidswas dependent on the resolving power ofthe SDS gel inwhich theywereelectrophoresed. Gener-ally, 15polypeptideswerepresent inenveloped nucleocapsids and 8 were found in

nucleocap-FIG. 7. Identificationofphosphorylated GV struc-turalpolypeptides. Proteinmatrix (A), enveloped

nu-cleocapsids (B),andnucleocapsids (C) were isolated as described from GV grown in the presence of

[32P]orthophosphate.

Eachviral component was

sol-ubilizedby boilingin SDS and mercaptoethanol, and itspolypeptideswere resolved by SDS gel

electropho-resis.Phosphorylatedviralpolypeptideswere

identi-fied by comparing densitometertracings of

autora-diograms of eachgel (---) to those of the same gel

stained withCoomassiebrilliantblue ( ).

J.

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sids. The minor protein species associatedwith these viralcomponents mayrepresent cleavage

products or modified (phosphorylated or

acet-ylated) forms ofthe major viralproteins.

Two-dimensional gel electrophoresis is currently

being used to investigate these possibilities as

wellas toseparate the polypeptides of enveloped

nucleocapsids and nucleocapsids into more clearlydistinguishable proteins.

Iodination studiesshowed that at least five of the proteins found in enveloped nucleocapsids were located on the outer surface of the viral envelope. Previous studies comparingthe poly-peptide compositions of GV andNPVenveloped nucleocapsids and nucleocapsids have impli-catedcertainproteinsaspossible viral envelope components (5, 7, 11). The lactoperoxidase la-beling procedure used in this work provided a direct means of identifying these proteins as VP17,VP39, VP42, VP48, and VP97. Nonidet

P-40 treatment of the enveloped nucleocapsids

provided further evidence for localization of theseproteins in the viral envelope. These poly-peptides were readily released from the viral

structure during solubilization of the envelope

by thedetergent.

Therewere afewadditional viralpolypeptides

(VP38,VP16, andVP46) which were present in

enveloped nucleocapsids butnot in

nucleocap-sids. Although this observation suggests that

these proteins are envelope proteins, they did

notappear tobe labeledby theradioiodination

procedure. Itispossible that these proteins are located moreinternally inthe viral membrane than the other envelopeproteins, and, as a re-sult,areunexposedtothelactoperoxidase.They

mayalsobe componentsofanintermediate layer

which has been proposed by Kawamoto et al. from electron microscopic observations (12) to existbetweentheenvelopesandnucleocapsids. This intermediatelayermaybe analogousto the "membrane" (M) proteins associated with the envelopes of rhabdoviruses, orthomyxoviruses, and paramyxoviruses which are speculated to

serve as recognition sites for assembly of the

nucleocapsids into envelopes (17).

The number and relative amounts of struc-tural

polypeptides

observedinthe GVbasedon

in vivo

incorporation

of

14C-amino

acids into

viral proteins were very similar to those based

onintensityofCoomassiebrilliant bluestaining.

Theseresults indicate thatpreexistinghost

pro-teins are not incorporated to any great extent

intoGV duringits replicationandsuggest that the structural proteins are virus

specified.

The possibility isnotruled out, however, thatsome

of the proteins observed in the GV are

host-contributed polypeptides that are synthesized

after initiation of virus infection. Because the effect ofGV infection onhost protein synthesis

has notbeendetermined,the specific association

of cellular proteins with the structural

compo-nents of GV cannot be further evaluated at

present.

None ofthe GV structural polypeptides was

radiolabeled by invitrolabelingprocedures spe-cific for carbohydrateresidues on proteins. This mayindicate that:(i) none of the viral polypep-tides contains carbohydrate moieties, (ii) some of the structural polypeptides are glycosylated but with residues other than the sugars sialic acid,galactoseorN-acetyl galactosaminewhich the in vitro labeling procedureare specific for, or (iii) some of the polypeptides are glycopro-teinscontaining galactoseor N-acetyl galactos-amine residuesthat are sohighly substituted by other sugars such as fucose that they do not become labeled.Itisdifficult,atpresent, torule out any of these possibilities, particularly

be-causeviralpolypeptides synthesizedin larvae in

thepresence of

["C]fucose

or

['4C]glucosamine

may not haveincorporated labelto adetectable

specificactivity. Theseresultsare

especially

sur-prising in thecaseof theenvelope proteins. All other well-characterized proteins found onthe

outersurface of animal cell membranesor

ani-mal virusenvelopes havebeen found to be gly-coproteins (9).

The 80 x

106-dalton

genomeofthe GV of P.

interpunctella (24) is

genetically complex

enoughto codefor

approximately

150proteins having an average molecularweight of 42,000. To synthesize the total number of structural polypeptides identified so far in this

virus,

it would benecessary to utilize

only

20 to 25%of theDNA molecule. Thisindicatesthat theGV

genome probably also contains information to

code for nonstructural proteins having

regula-tory or

enzymatic

activities. Whether

virus-coded enzymes or

regulatory

proteins

such as early

proteins

are involvedin the viral

replica-tionprocess hasnotbeen

investigated.

Comparison

ofthe

polypeptide composition

of the GV ofP.

interpunctella

tothatof the GVs

of T.ni, S.

frugiperda,

andP. brassicaereveals

several

differences,

basedon

electrophoretic

mo-bilities, in the structural

proteins

of these

vi-ruses. Thesedifferences appeartobemore

pro-foundfor the

proteins

of the

enveloped

nucleo-capsids than for those of the

nucleocapsids,

in-dicatingthata

major

divergence

between these

GVsmay be due to their

envelope

proteins.

If

the envelope

proteins

are

responsible

for the

tissue and host

specificity

exhibited

by

the

GV,

changes in their

composition

may account for

intrinsic differences observed in the

biological

33,

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http://jvi.asm.org/

activity of these viruses. From these

observa-tions, the importance of determiningthe number

and structural position of theviralpolypeptides

forelucidating their biologicalfunctionbecomes

obvious. This information isalso necessary for

thedevelopment ofimmunologicalreagents

pre-ciseenoughtoprovidesatisfactoryidentification

ofbaculoviruses as well as to analyze specific

steps in the infectionprocess ofthese complex viruses.

ACKNOWLEDGMENTS

This workwassupported byPublic Health Servicegrant ES02036fromtheNational Institute ofAllergyand Infection-Environment Safety. K.A.T. was supported bya research

associateshipfrom the U.S. GrainMarketingResearch Labo-ratory,U.S. DepartmentofAgriculture,Science and Educa-tion AdministraEduca-tion and from the KansasAgricultural Exper-iment Station.

We thankKimberly Osborne, Diane Potts,andViolaHill for theirexcellent technical assistance.

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