Polyhedrin gene of Bombyx mori nuclear polyhedrosis virus.

Copyright © 1985, American Society forMicrobiology

Polyhedrin Gene of Bombyx mori Nuclear Polyhedrosis Virus

KOSTAS IATROU,* KENICHI ITO,AND HALINAWITKIEWICZ

Department ofMedical Biochemistry, Faculty of Medicine, The University of Calgary, Calgary, Alberta T2N4NJ, Canada

Received 18October 1984/Accepted 14January 1985

Aportion of thegenomeof thenuclearpolyhedrosisvirusofBombyx morihas beencloned.Thispartof the viralgenomecontainsthegeneencodingthe viral occlusionbody protein, polyhedrin. The polyhedringenehas beensequencedinitsentirety togetherwithsomeof its 5' and3'flankingsequences.Theprimarystructureof polyhedrin predicted from the nucleotidesequenceof thegene wasfoundtobe somewhatdifferent fromtheone reported previously for the authentic protein (E. A. Kozlov, T. L. Levitina, N. M. Gusak, and S. B. Serebryani, Bioorg. Khim., 7:1008-1015, 1981; S. B. Serebryani, T. L. Levitina, M. L. Kautsman, Y. L. Radavski, N. M. Gusak, M. N. Ovander, N. V. Sucharenko, and E. A. Kozlov, J. Invertebr. Pathol., 30:442-443, 1977). Comparisonof theprimarystructuresofthepolyhedrinof the nuclearpolyhedrosisvirus of B.moriwith that ofAutographacalifornicasuggeststhatconsiderable selectivepressurehas been exercised attheproteinlevelduring evolution. Nucleotidesequencecomparisonsof the twostructuralgenesrevealthat the coding sequences have diverged significantly through the accumulation of silent and replacement substitutions. In contrast, a remarkable degree ofsequence conservationwas foundto existinthe domains corresponding tothe 5' and3' noncoding regionsof thepolyhedrin mRNAs.

Nuclear polyhedrosis viruses(NPVs) representagroupof insect viruses (family Baculoviridae, genus Baculovirus) whosevirions areembeddedinto polyhedron-shaped

occlu-sion bodies or polyhedra in the nuclei of host cells (for extensive reviews see references4, 13,and 19). Thegenetic information of baculoviruses is stored in the form of a relatively large molecule of covalently closed double-stranded DNA ranging in size from 50 x

106

to 100 x 106

daltons.

NPVshave been recognized formany years as the

infec-tious agents that cause acute diseases in a wide range of

insects, including the silkmoth, Bombyx mori (4), and the first tissue culture propagation of the NPV of B. mori (BmNPV) was reported as early as 1935 (47). Occlusion bodieswereshowntobemade almostexclusively ofasingle protein, polyhedrin, which can be solubilized by treatment

ofthepolyhedra withweakalkalinesolutions(3). In B. mori,

about350nucleocapsids enveloped singly orinmultiplesof two tofiveare occluded in each occlusionbody.

AlthoughNPVs mayinfectinsectsother than their normal

hosts, their host

range

is usually restricted to different

species or (at most) families within an order. Only very

rarelycanthehost range extend to differentinsectordersor to different arthropod classes (9). There hasonly been one case in which infection of a vertebrate cell culture was caused byan NPV (21), but thatcase has not been studied

anyfurther. In contrast, repeated attemptsbyother

investi-gators to propagate NPVs in a wide variety of vertebrate cells were unsuccessful (18, 25, 46). Because of their re-stricted host range and their effectiveness ininfecting their hosts, baculoviruses have beenconsidered as potential

bio-logical pest control agents(33, 45). This has in turn stimu-lated the molecular characterization ofsome NPVs, partic-ularly theNPV ofAutographa californica (AcNPV) (13).

Anumber of mutations have beenfound in AcNPV which result in the formation of abnormal, few, or no occlusion

bodies in the nuclei of infected cells (7, 12, 27, 53). In

addition, it has been established that extensive serial

pas-*Corresponding author.

sageof NPVs in cell cultures results inadramatic decrease in virus occlusion (14, 22, 31). In suchcases the production of nonoccluded virususually remains unaffected, and non-occluded virus becomes the predominant viral form in the infected cells.Onthebasis of theseobservations it has been concluded that occlusion body formation and, presumably,

polyhedrin synthesisarenotrequired forviralpropagationin

laboratorytissue cultures.

With the demonstration that polyhedrin is a protein en-codedbyaviral gene (50) and with the advent of recombi-nant DNA technology, it became apparent that the poly-hedrin gene might represent a site of the viral genome amenable togenetic manipulation, suchasthe introduction offoreigngenes intothe DNA of the virus. Experiments in which the structure of the polyhedrin gene from spontane-ous, chemically induced, or in vitro-constructed mutants wasexaminedshowedmutational changes inthe gene orits

immediatevicinity(8,11, 40),thussupportingthehypothesis that the polyhedringene representsa convenient target for

geneticengineering.Finally, polyhedrinissynthesizedin the host cells late in infectionin large amountsandthis in turn

reflects acorresponding abundance in the amount of accu-mulated polyhedrin mRNA (1, 42), probably achieved

throughhigh ratesofmRNA synthesis. This suggestedthat

heterologous genes might be appropriately introduced into the genomes of baculoviruses and expressed effectively

under the control ofthe polyhedrin promoter. The use of

AcNPVas avectorfor theexpressionof

foreign

genesunder

polyhedrin promoter control was

successfully

attempted recently and resulted in the production of human ,B-interferon and Escherichia coli 3-galactosidase in large

amounts in insect tissue culture cells(34, 41).

Unfortunately, themolecular characterization of BmNPV has lagged far behind that of AcNPV.

Although

some

controversialreportsonthenatureof the viral genome have

appeared in the literature (reviewed in reference 19), no accurategeneticmapor measurementsofthesize of BmNPV DNA have beenreported duetothelack of

published

work basedon currentcloning methodology. Weareinterested in the mechanisms regulating chorion gene expression in the

436

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BmNPV POLYHEDRIN GENE 437

silkmoth B. mori(forarecent review see reference 16) and

feel that BmNPV may be used effectively as a vehicle for introducing authentic or mutagenized silkmoth genes into

thetissuesinwhich they arenormally expressed inorder to

study theirexpression invivo. To testmodels of differential

regulation

of choriongeneexpression (24),wewould liketo recombine well-characterizedchoriongenesintothe genome ofBmNPV, using the vicinity of the polyhedrin gene as a target site, and to infect follicular cells in vivo with the

recombinant virus. As afirststepin this directionwereport here the localization, cloning, and complete nucleotide se-quence ofthe

polyhedrin

gene ofBmNPV and its vicinity.

The primary structure of polyhedrin predicted from the

nucleotide sequence of the gene is not identical to that

derivedfrom the protein itself (26, 39). Comparison of the polyhedrin genes of BmNPV and AcNPV indicates that

although a large degree of point mutations were fixed in someparts of the genesduring evolution, other domains of

the genes and theproteins themselves have been subjected

toconsiderable selective pressure.

MATERIALS ANDMETHODS

Cells and virus. B. moritissue culturecells(Bm-5 [17])and

BmNPV passedonce in Bm-5 cellswerekindly supplied by

J. L.

Vaughn,

Insect

Pathology Laboratory,

U.S.

Depart-mentofAgriculture, Beltsville,Md.Cellsweremaintainedat

28°C

in

complete

IPL-41 growth medium (52) supplemented

with 0.24,uM

ZnSO4

and 16 ,uMAIK(SO4)2 in the absenceof antibiotics. The cells were subcultured weeklyat aseeding

density

of0.2 x 106 cells per ml (usuallya 1:5 dilution ofa 1-week-oldculture). Infection ofthecells with BmNPVwas

carriedout

by

removing

themediumfrom2 x

107

cells and replacing it with 5 ml ofa nonoccluded virus inoculum of tissue culture

origin containing

1 x

107

to 5 x

107

PFUper ml. After 1 h the viralinoculum wasremoved and replaced with25mlof fresh medium

containing

gentamycin (50

,ug/ml).

Cellswerecollected

by low-speed centrifugation

3to4

days

later whenmore than98% ofthemexhibiteda

large

number ofviralocclusion bodiesintheir nuclei. Cellular

pellets

were

used as thesource of occluded

virus,

whereasthe superna-tants were used as the source of nonoccluded virus and as

inoculafor further infections.

A. californica polyhedrin gene probe. A cloned HindIII

fragment

ofthe polyhedrin gene ofAcNPV (HindIII-V) in

pBR322 (clone pM5 HindV) waskindly supplied by Eric B.

Carstens, Department

of

Microbiology

and

Immunology,

Queen's

University, Kingston, Ontario,

Canada.Thiscloned

fragment

is 937 base pairs long and contains the coding

region

ofthe gene downstream from amino acid number 83

(nucleotide

251ofthe

polyhedrin

gene sequencepublishedin

reference23)and almosttheentire 3' noncodingpartofthe gene

(E.

B.

Carstens, unpublished data).

Preparationof occlusionbodies.Atthe endoftheinfection

period

cellswere

pelleted by

centrifugation

andwashed once

with phosphate-buffered saline (10). Cellular pellets were

solubilized with 0.4% sodium

dodecyl

sulfate-10 mM Tris-HCI(pH 7.8) by gentle rocking for2 h(approximately5mlof

solution per4 x 107 cells),and the cellularsuspension was

layeredona30-ml cushion of

65%

(wt/vol)sucrosein 10mM Tris (pH

7.8)-10

mM EDTA. Aftercentrifugationat110,000

x g (24,000 rpm) in the SW27 rotor of a Beckman

ultracen-trifuge

for4h at 15°C, the supernatant was removedpartly

by aspiration

and finally by decanting, and the pelleted occlusion bodies were suspended in a small volume of

distilledwaterwith theaidofaPasteurpipette. Finally,the occlusion

body suspension

wasmade 0.25Mwithrespectto

NaCl andcentrifugedat 17,000 x g (12,000 rpm)in the SS-34 rotorofaSorvall centrifuge. Thepelleted occlusion bodies were washed twice with 0.25 M NaCl as described above, and the finalpellets were stored at -20 to -70°C until used for the extraction of viral DNA.

Viral DNAisolation.Occlusion bodies were solubilizedin 0.1 MNa2CO3-10 mMEDTA-0.1M NaCl (pH 10.8)

(occlu-sion bodies from2.5 x

107

cells per ml ofbuffer)withgentle

swirlingatroomtemperature overaperiodof 2 h. At the end of theprocess the volume of the solution was increased by 50%by the addition of distilledwater, and the solution was

finally made 1% with respect to sodium dodecyl sulfate. A small amountof insoluble matrix was removed by

centrifu-gation,and the supernatant was extractedexhaustivelywith

phenolandchloroform-isoamylalcohol(24:1, vol/vol). After

the extractions, the aqueous phase was concentrated to a volume equal to that ofthe Na2CO3 solution used at the

beginning of the process, made 0.25 M with respect to

ammoniumacetate, and spun at30,000x g(17,000 rpm)in a Sorvall centrifuge. The supernatant was precipitated with 2.5 volumes of ethanol. After being washed with 70% ethanol, the DNApelletsweredissolved in 5 mM Tris-HCl

(pH 7.8)-0.1 mM EDTA and stored at4 or

-40°C.

The Na2CO3-insoluble matrix was also partially

solubil-ized inamixture ofequal volumes of0.1 M

Na2CO3-10

mM EDTA-0.1 M NaCl (pH 10.8) and 7 M urea-0.135 M

NaCl-10 mMTris-(pH

7.5)-i

mM EDTA-2% sodium

dod-ecyl sulfate (occlusion bodies from 5 x 107 cells per ml of

buffer) for3 to4h atroomtemperature with

swirling.

After

extractions andprecipitationsasdescribedabove, the DNA was pelleted bycentrifugation and storedin thecold.

The distributions of DNA in the fraction solubilized in

carbonatesolutionalone and in the

partially

insoluble matrix were 66% and33%, respectively. The yield ofDNA was in the rangeof1.2 ,ug per106cells.

Othermethods. Restriction enzyme

digestions

were

per-formed with the buffers recommended

by

the

suppliers.

After

electrophoresis

on agarose

gels,

the resolved

frag-ments were immobilized on a membrane support

(Gene-Screen;

New

England

Nuclear

Corp., Boston, Mass.)

as

previously described (44)except thattwo15-mintreatments

of the

gels

with 0.25 N HCl were included before alkaline

denaturation to

depurinate

the DNA into sizes of

approxi-mately

1 to 2 kilobases

(kb) (51).

Nick translations and

Southern

hybridizations

were as described

previously

(24). Probeswerenicktranslatedto

specific

activities of2 x

107

to 4 x

107

cpm Cerenkov per pLg. Hybridizations were

per-formedat

70°C

for 14to16hwith400,000cpmCerenkov of

each

probe

per mlof

hybridization

solution.DNA

fragments

were isolated from gel slices by electroelution (20), but no

bovine serumalbuminwas used.

Cloning

of various restric-tion fragments ofthe viral genome was done as described previously (24) by

using equimolar

mixtures of

linearized

and dephosphorylated

plasmid

pUC9 (48) and

purified

re-striction

fragments

isolated

by

electroelution. After the

transfection of competent Escherichia coli cells (a strain

derivative ofE. coli K-12 RRI

having

the genotype leupro thistrA hsdr- m- lacZ AM15 F'

lacIQ

pror [38]), transfor-mants were selected on ampicillin-containing plates.

Ampi-cillin-resistant colonies were further plated on plates

con-taining

5-bromo-4-chloro-3-indolyl-,-D-galactoside

and

isopropyl-p-D-thiogalactoside

(30), and colorless colonies

werepickedfrom thoseplates forfurther biochemical

anal-ysis.

The clones of the EcoRI

fragment

of BmNPV DNA inserted in

pUC9

in two orientations have been

designated

Bmp/pR5

and

Bmp/pR8,

whereas thoseofthePstI

fragment

VOL. 54, 1985

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438 IATROU ET AL. J.VIROL.

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FIG. 1. Southern hybridizations ofBmNPVDNArestrictiondigests. BmNPV DNA (0.15 to 0.22 ,ug perdigest)wasdigested with avariety of restriction enzymes, and the digests were resolved on 0.7% agarose gels in the presence of ethidium bromide (0.5 ,ug/ml). After electrophoresis the gels were photographed (middle portion of each panel), and the digests were subsequently Southern transferred to membrane sheets, hybridizedtonick-translated clonepM5HindVcontainingpartofthe AcNPVpolyhedringene,andautoradiographed (left portion of each panel). After autoradiographyandwithoutanyprior melting of thehybridizedAcNPVpolyhedringene sequences,thesame filterswereprobed with total nick-translatedBmNPV DNA andautoradiographed (rightportion of each panel). The orderofthedigestswas

asfollows: (upperpanel)1, AvaI;2, BalI;3,BamHI;4,Clal;5,EcoRI;6,EcoRV; 7,HindlIl;8,NdeI;and(lowerpanel) 1, NruI;2, PstI; 3,PvuI;4,PvuII;5,SalI;6,SphI. For sizemarkers (M), a mixture ofHindlll-digestedandEcoRI-digestedA DNA wasusedoneach ethidium bromide-stained gel.Forsize markersonthe narrowstrips oftheethidium bromide-stainedgels,amixture ofHindIl-HindIll doubledigest of X-DNAandHaeIII-digestedpMB9 DNA wasused.The numbers at therightside of eachpanel indicate thelengthinkilobases ofsome

ofthesizemarkers described above. Asterisksonthe bandsoftheethidium bromide-stainedgels indicatethefragmentshybridizingtothe AcNPV polyhedringeneprobe.

have been designated Bmp/pP3 and Bmp/pP14. 32P end

labeling

ofplasmid DNA preparations with T4 polynucleot-ide kinase after linearization and dephosphorylation has

beendescribed previously (24).Restriction enzymemapping wascarriedoutbythepartial digestionmethod with

single-end-labeledfragments of cloned viralDNA(43). Nucleotide sequencingwasdonebythechemical method of Maxam and

Gilbert (29), and chemical reactions were analyzed on

85-cm-long 6% polyacrylamide gels. 32p labeling at the 3' termini of restriction fragments was performed by repair

synthesis of3' recessed endsbyusingtheKlenow fragment

of E. coli DNA polymerase in a 20-,l reaction mixture

containing50 mMTris-HCI(pH 7.5), 5mM MgCl2, 10 mM

,B-mercaptoethanol, 70,uMeachofdATP, dGTP, andTTP,

gm am

40

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BmNPV POLYHEDRIN GENE 439

1 kb

PstI R I Sal I Sal I Bam H I SalI

r

-Pst I R I

1 5'

S i

Sal I

I Coding

'11

XbaI

qtl11 AATAAA

...

..-Nde I Hindm Sal I Bam H I

o 0.2 kb

FIG. 2. Cloned portions ofBmNPV. The9.6-kbEcoRl and10-kbPstlfragments ofBmNPVcloned inplasmidpUC9arerepresented by the top line. Some of the enzymes relevant to the sequences of the polyhedrin gene are also presented. In the lower line an expanded version ofthe SalI-BamHI fragment containing the polyhedrin gene is presented togetherwith thecleavage sites usedfor generating end-labeled fragments for nucleotide sequence determination in the polyhedrin gene region. The designations 5'U and 3'U symbolize the 5' and 3' untranslated regionsofthe polyhedrin mRNA, and the end of the mRNA has been tentatively placed 30nucleotidesdownstreamfromthefirst AATAAApolyadenylation signal(seealsothe text).

100,uCi ofa-[32P]dCTP (ca. 3,000Ci/mmol),10UofKlenow

fragment,and 5 to 20pug ofthe DNA digest. Sites usedfor5' or3' end labelingare listedinorderfromleft torightin the

lower part of Fig. 2, and their locations are identified by referencetothe numbered DNA sequence shown inFig. 3 as

follows: Sallat -192,XbaIat 147,NdeI at595, and HindIll

at 1177. In all cases sequencing was performed in both directions.

RESULTS

To identify the part of the genome of BmNPV that contains the polyhedrin gene, viral DNA was isolated and

restricted with a large number of restriction enzymes with hexanucleotide recognition sites. After electrophoresis of the restrictedDNA,the resolvedfragmentsweretransferred

to amembrane filterandsubsequently hybridizedto aDNA

clonecontainingalargeportion ofthecodingsequencesand some 3' noncoding sequences of the polyhedrin gene of AcNPV (HindIII-V; see above for details). Typical results

fromsuch hybridizations are shownin Fig. 1. Through the

hybridizations ofthe BmNPV digests to the AcNPVprobe

(left hybridization panels ofFig. 1), the sizes of the

restric-tion fragments that included the BmNPV polyhedrin gene and the flanking sequences that are homologous to the

hybridization probe weredetermined. Thus, several

restric-tionenzymes were identified thatproduced single hybridiz-ing fragments whoselengthswerewithin the size range that may be cloned relatively easily in E. coli and also allowed

the prediction to be made that the entire polyhedrin gene may be contained withinthem.

Todetermineunequivocallytheposition of the polyhedrin

gene-containing fragmentswhen bandswere closely spaced and to visualize the smaller restriction fragments of the BmNPV genome that might have escaped detection in the ethidium bromide-stainedgels, the same immobilized digests weresubsequently hybridized to radioactive BmNPV DNA. Through such hybridizations (right hybridization panels of

Fig. 1), allrestrictionfragments of a size equal to or greater than 500 base pairs were detected. Based on this

informa-tion, we wereableto deducealength of125 ± 4kbforthe genome of BmNPV.

The EcoRI and PstI fragments of BmNPV DNA

hybrid-izing to theAcNPV polyhedringene probe (approximately

9.6 and 10kb,respectively; Fig. 1)werecloned andmapped

in detail. Partofthismapping information isshown inFig.2. ThecombinedEcoRIand PstI clonedfragmentswerefound to spanatotallengthof 11.8 kb of the viralgenome.On the basis ofthe complete restriction mapping information de-rived from these clones as well as from additional Southern

hybridizations (datanot shown), we were abletodetermine the approximate location of the polyhedrin gene in the middleofthe 11.8 kbof the cloned viral DNA andtoinitiate

its sequencing.

Thecompletenucleotidesequenceof the polyhedringene

withsomeof its5'and 3'flankingsequencesis showninFig.

3. Upon inspection ofthe 2,060-nucleotide-long sequenced

part ofthe cloned DNA, an open readingframewas identi-fied inoneofthe DNA strands thatresultedin thetranslation ofa 244-amino acid-long polypeptide that, with few excep-tions (seebelow),wasidentifiedasthepolyhedrin ofBmNPV based on comparisons with the published sequence ofthe authentic protein (26, 39)and with the predictedaminoacid

sequence of the AcNPV polyhedrin (23). The exact locali-zation ofthepolyhedringeneanditstranscriptional

orienta-tion in the cloned DNA,asdeduced fromthedetermination oftheprimarystructureofthe gene,are shown in the lower part of Fig. 2 along with the restriction sites used as the

major starting points forthenucleotide sequenceanalysis.

ThesequencedDNAwasalsoscannedforthe presenceof

other open reading frames.Two moreputativeopenreading

frameswere detected at thebeginningand at the endofthe sequenced DNA. The first one, at the beginning of the sequence, is read in the sameorientation asthe polyhedrin

gene, comprises 82 amino acids, and terminates at nucleo-tide -260. The second one, atthe end of the sequence, is read in the opposite orientation (from the complementary

strand).This openframe translates into 239 amino acids and terminatesatnucleotide 768 of the sequence shown inFig.3. The consequences of the possible presence ofone or two VOL. 54, 1985

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440 IATROU ET AL.

-l:. -55z -542 -S32 -522 -512-5i

CCGCCCACTA TTAATGAAATTAAAAATACC AATTTTAAAAAACGCAGCAAAAGAAACATT TGTATGAAAG

-492 -442 -472 -462 -452 -442 -472

AATGCGTAGAAG&AAAAAAT AATGTCATCG ACATGCTGAA CAGCAAGATC AATATGCCTCCGTGCATACA

-422 -412 -402 -392 -302 -772 -76:

AAAAATATTl GGCGATTTGA AAAAGAACAA TGCAGCGCGG CGGTATGTACAGGAAGAGGTTTATACTAAA

-352 -3,42 -@3 _-22 -312 -302 -292

CTGTTACATT GCAAACGTGGTTTCGTGTACCAAATGTGAAAACCGATGTTTAATCAAGGCTCTGACACAT

-208 -272 -26: -252 -242 -232 -222

TTTTACAATT ACGACTCCAAGTGTGTGGGT GAAGTCATGC ATCTTTTAATCAAATCCCAAGATGTGSATA

-212 -202 -192 -192 -172 -162 -152

AACCACCAAA CTGCCAAAAA ATGAAAACTGTCGACAAGCTCTGTTCGTTTGCTGGCAACT GCAAAGGTCT

-142 -132 -122 -112 -12 -92 -42

CAATCCTATCTGTAATTATTGAATAATAAA ACAATTATAAATGTCAAATTTGTTTTTTAT TAACGATACA

-72 -62 -52 -42 -32 -22 -1I

AATGGAAATA ATAACCATCT CGCAAATAAA TAAGTATTTTACTGTTTTCGTAACAGTTTT GTAATAAAAA

AACCTATAAA T

15 30 45 63

ATG CCGAATTAT TCATACAACCCC ACCATC GGG CGTACTTAC GTGTAC GAC AATAAATAT MetPro Asn Tyr SerTyrAs% ProThr lIe Gly ArgThrTyrVal Tyr AspAsnLys Tyr

75 90 105 1

TACAAAAAC TTG GGCGGT CTC ATCAAAAAC GCC AAG CGCAAG AAGCACCTAATC GAACAT

TyrLys A-nLeo GlyGlyLouIleLys Asn Al1 LysArg Lys Lys His LouII1GluHis

135 150 165

GAA 66A GA6 GAG AAGCAATGG GATCTTCTAGACAACTAC AT6 GTT GCC GAA GATCCC TTT Glu Lys GluGlu LysGinTrp Asp Leu Leu AspAsn TyrMet Val Al& GluAspProPhe

195 210 225 240

TTA GGACCG GGC AAAAAC CAA AAACTTACCCTTTTT AAAGAG 6TT CGC AAT0TG AAACCC

Leo GlyProGlyLys Asn Oln LysLouThrLouPh- LysGluVal ArgAsn Val LysPro

255 270 295 300

GATACCATGAAGTTAATCGTCAACTGG AGC GGC AAA GAGTTTCTGCOT G66 ACT TGG ACC Asp Th7r Ht Lye LouII Val AsnTrp Ser GlyLye GluPh-LouArgGluThrTrpThr

315 330 345 360

CGTTTT GTTGAG GAC AGCTTCCCC ATT GTAAACGACCAAGAG GTG ATGGAC GTG TAC CTC ArgPh- Vol Glu Asp S6r Phe ProIleVal AsnAspOlnOlu ValMetAspVal TyrLeu

375 390 405 420

GTC GCC AACCTCAAACCCACACGCCCCAACAGG TGCTAC AAGTTCCTC GCTCAACACGCT

Vol Al6 A-n LouLysProThr ArgPro Asn Aro Cy- Tyr Lys Ph- L-o AlaG61 HisAl&

435 450 465 480

CTTAGG T7GGACGAA GACTAC GTGCCCCAC GAAGTAATC AGA ATT ATG GAGCCATCCTAC

LouArgTrpAsp Glo Asp TyrVal ProHisGluVal leArg IleMetGI.Pro6-r Tyr

495 510 525 540

GT6 GGC ATG AACAAC GAATACAGA ATT AGT CTG GCT AAAAAG GGC GGC GGC TGCCCAATC

Val GlymetAsnAsnGlu Tyr Arg Ile Sor Lou A61 Lye LysGly Gly Gly CysPro

11-555 570 5ss 600

ATG AACATCCACAGC GAG TAC ACCAAC TCGTTCGAGTCG TTT GTG AAC CGCGTCATATGG MetAen lie HisSerGlu Tyr ThrAsn SerPh- Glu SOr Pho Val AsnArg Val Ile Trp

615 630 645 660

GAG AACTTC TACAAACCCATCGTTTACATCGGCACASAC TCTGCC GAA GAA GAG GAAATC

Glu Asn Ph- Tyr LysPro lieVal Tyr IleSlyThr Asp S-rAl&GluGlu Glu Glu lie

675 690 705 720

CTAATTGAG GTTTCTCTCGTTTTC AAA ATA AAGGAG TTT GCACCAGAC GCGCCT CTG TTC Lou11-GluVal SOr LouVal Ph- LysIle Lys Glu PhC Al6 ProAspAl&ProLeou

Ph-75

ACT GGT CCG GCA TAT TAA Thr GlyPro Al&Tyr Tor

749 758 768 779 798 798e 9o

AACACTATACATT0TTATTA GTACATTTATTAAGCGTTAGATTCTOTACGTTGTTGATTT ACAGACAATT

1e 92 93 94 95 96 97

GTTGTACGTA TTTTAATAAC TCATTAAATT TATAATCTTT AGGGTGGTAT GTTAGAGCGA AAATCAAATG

9ee e99 909 919 929 939 949

ATTTTCAGCGTCTTTGTATC TG7 7TTTAAATATTAAATCC TCAATAGATT TGTAAAATAGGTTTCGATTG

959 968 978 999 999 1009 0l1e

GTTTCAAACA AGGGTTGTTTTTOCAAACCGATGOCTGGACTATCTAATG7 ATTTTCGCTC AACACCACAC

1028 1439 1048 1059 1069 1078 1098 GACTTGCCAAATCTTGTAGC AGCAATCTAGCTTTGTCGATATTCGTTTGT GTTTT6 TTTT GTAATAAAGA

1099 l1o1 ill 1129 1139 1149 1159

TTCGACOTCG TTCAAAATAT TATGCGCTTT T7TATTTTTTTCATCACTOT CGTTA0 TGTA CAATTGACTC

116e 1179 l1es 1199 1209 1219 1229

GAC0TAAACACGTTAAATAAAGCTTGGACATATTTAACAT CGGGC0CGTTAG6CGCATTATT7CC0CC0T

1239 1249 12s3 1268 1279 1299 1299

CGTCCCAACCCTCOTCOTTAGAAGTTGCTT CCGAAOACGA TTTTGCCATA OCCACACGAC GCCTATTAAT

1309 1319 1329 1339 1349 1359 1369

TGTGTCGACTAACACOTCCOCGATCAAATT TTTAGTTGTT GA0CTTTTCG 6AATTATTTCTGATTGCGGA

1379 1389 1399 1409 1418 1429 1438

CGTTTTTG0 CGG0TTTCAATCTAACTGTG CCCGATTTTA ATTCAGACAA TAC7TTAGAA AGC6ATGGTG

1449 1458 1408 1479 1499

CAGGCG0 TG7 T7ACATTTCAACC60C06ATCTACTATG TGGCTGTAATG

FIG. 3. Nucleotide sequence of the polyhedrin gene and

sur-rounding region. The first nucleotideof the initiatorATG codonhas

beendesignatednumber 1. Forassignmentsofcap site, polyadenyla-tion signals,and otherconsensussequencesin thepromoterregion,

seethe text.

structuralgenes in the immediatevicinity ofthepolyhedrin gene are discussed below.

DISCUSSION

Aprerequisitetothe successful genetic manipulationofa particularsite inagenomeis the detailed characterizationof the target site.Wearepredicting that,inamannersimilar to that showntooccurin AcNPV(8, 11, 27, 34, 40, 41), direct

or indirect mutational alteration, inactivation, and even removal ofthe polyhedrin gene from the BmNPV genome should bear no genetic consequences in terms ofviability and virulence of the nonoccluded form of the virus in

laboratoryanimals(silkmoths)and tissueculture cells.

There-fore, as the first step toward introducing foreign gene

se-quencesinto thegenomeofBmNPV,we identified, isolated

by molecular cloning, and characterized by sequence

anal-ysis thegene encodingthe viralprotein polyhedrin.

Genome sizeof BmNPV andpolyhedringenecloning.Inthe course of ourpreliminarycharacterization of thegenomeof BmNPV by restriction analysis and hybridizationaimed at the identification of restriction fragments containing the

polyhedringene, we hadtheopportunity tofirmly establish the size of theviral DNA. Over the past 30years, various

values have been published on the size of the circular

double-stranded BmNPVgenome, rangingfrom 3 to 180kb

(see reference 19 for a review). Through our restriction

digestion analysis (Fig. 1 and other data not shown), we calculatedalengthof 125 ±4 kb for the viralgenomea size very similar to that established for the genome ofAcNPV (32, 49). No restriction pattern polymorphisms (restriction

fragments appearing in submolar quantities) were detected when BmNPV DNA that had been serially passaged five

-5 IC 1S5

20-ProAsn TyrS-r Tyr Asn Pro Thr IleGly Arg ThrTyr Val Tyr AspAsn Lys TyrTyr

25 S.) ZX5 40

Lys Asn LeuGlyGly Leu Ile Lys Asn Ala LysArgLys LysHis Leou Ie GuHis Glu

Glu His

45 50 55 60o

|LysGluGluLysGln Trp Asp Lou Lou AspAsn TyrMWetVal Ala GluAspProPho Lou

65 70 75

GlyPro Gly LysAsnGin Lys LeuThr LeuPhe LysGluVal ArgAsnVal LysProAsp

85 90 95 100

Thr MetLysLeouIle Val AsnTrp SerGlyLysGlu Fhe LeouArg GluThrTrp ThrArg

105 -110 115 120

PheVal GluAspSer PhePro IleVal AsnAsp GinGluValMetAsp Val TyrLeuVal

125 1l0 135 140

AlaAsn Leu LysProThr ArgPro Asn ArgCysTyrLysPheLeuAlaGinHis AlaLeu

145 lS0 l5S 160

Arg TrpAspGluAsp TyrVal Pro HisG1u Val II Arg Ie*M1et Glu ProSer TyrVal

GinAsn

165 170 175 1_0

GlyMet AsnAen Glu Tyr Arg IleSer LouAla LysLysGlyGly Gly Cys Pro IieMet

185 190 195 200

Asn IlieHis Ser GluTyr.ThrAsn Scr PheGluSer Phe Val AsnArg Val IleTrpPlu

205 210 215 220

AsnPhe TyrLys Pro IleVal Tyr IleGly Thr AspISGr AlaGlu GluGIlo Gluo

Ala Seri . . IGInL

225 230 235 240

IleGluVal

~~~~~.

Ser LeuVal PheLysIle Lys GluPhe AlaProAsp AlaProLeuPheThr

. . ...*.**.**.*.**. ..

G1lyProA1l& Tyr1

FIG. 4. Comparisonof two BmNPVpolyhedrinsequences.The

polyhedrinsequence predictedfromthegene sequence(upperline)

is compared to that previously reported for the purified protein

(lowerline;[26,39]).Dots in the lowerline indicatethe same amino

acidsas in thetopone and blocksof identicalsequencesareboxed.

Noticethechangeinreadingframeoccurringat aminoacid114and

itsrestoration at residue145.

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BmNPV POLYHEDRIN GENE 441

Bm Ac Bm Ac

Bm

Ac Bm Ac Bm Ac Bm Ac Bm Ac Bm Ac Bm Ac Bm Ac

rF Asn Tyr Ser Tyr Asn Pro Thr Ile Gly Arg Thr Tyr Val Tyr Asp Asn Lys Tyr Tyr

I* Asp _-5 Arg. . . ._,)7,5. . .

_40:

Lys Asn Leu G1y G1y Leu Ile Lys Asn Ala Lys Arg Lys Lys His Leu I 1e Giu His G1u

Ala Val .. . . Phe Ala

45 5 0 6

LysGluT Glu Lys Gln Trp[sp| Leu Leu Asp Asn Ty Met Vai Ala Glu Asp Pro Phe Leu

Ile| Ala Thr Leu Pro 0 Leu e ;

65 7

6uW--G1ly Pro G1ly Lys Asn G1ln Lys Leu Thr Leu Phe Lys G1lu ValI Arg Asn Val Lys Pro Asp

0 . .

~~~

. . . Ile . .

85 90 95 1(C)

Thr MietLys Leu|IleVal sn r Ser Gly Lys Glu Phe Leu Arg Glu Thr Trp Thr Arg

* * * * Val Gly Lys Tyr[r* * *

105 1T1F 1151i5

e1ValIGiu Asp Ser Phe Pro le Vai Asn Asp Gin Giu Val Met Asp Val Tyr Leu Val

Met Phe

I-5 1 'M0 17i 140

Ala n Leu Lys Pro Thr Arg Pro Asn Arg Cys Tyr Lys Phe Leu Aia Gln His Ala Leu

Val I 18etM Arg . . . .

145 15iC 155 160e

rg Trp Asp Glu Asp Tyr Val Pro His Glu VaIle Arg Ile Met Giu Pro Ser Tyr Val

* Cys . Pro . Asp . Val .Trp

-. 165 170; 175 1E30

bMet Asn Asn Glu Tyr Arg Ile Ser Leu Ala Lye Lys Gly Gly Gly Cys Pro Ile Met

* Serj Gi. l T T A S Phe G . [ . . . .

sn Ile|His Ser Glu Tyr Thr Asn Ser Phe Glu |Ser |he jVal Asn Arg Val Ile Trp Glu

*ILeul * Glnl *Ile Asp

Bm Asn Phe Tyr Lys Pro Ile Val Tyr Ile Gly Thr Asp Ser Ala Giu Giu Glu Glu Ile Leu

Ac * * * * * * * * * * * * * * . . . 0

225 r=*>;230 2735 244:

Bm Ile Glu Vai Ser Leu Val Phe Lye Ile Lys Glu Phe Ala Pro Asp Ala Pro Leu Phe Thr

Ac Leu * * .Val . * . * . . . .

Bm Gly Pro Ala Tyr

Ac *

FIG. 5. Comparisonof thepolyhedrinsequencesofBmNPVand AcNPV. The BmNPV

polyhedrin

sequence

predicted

from thesequence of thegeneis comparedwith thatpredictedfromthe

corresponding

geneofAcNPV(23). Dotsin thesequenceof AcNPV indicate thesame amino acids asthosein the BmNPV sequence,andblocksofidentical sequencesareboxed.

times through Bm-5 cells and restricted with enzymes that

produce reasonably small numbers ofwell-separated DNA

fragments wasanalyzed onagarosegels.

After hybridizations of Southern transferred restriction

digests of BmNPV DNA to the probe (937 base pairs) containing part of the AcNPV polyhedrin gene, single re-strictionfragmentswerefound tohybridizeto theprobeina number of different digests (Fig. 1). These fragments pre-sumablycontainthecorrespondinggeneof BmNPV. Two of

them, a 9.6-kb EcoRI and a 10-kb PstI fragment (Fig. 2), werecloned,and theapproximate locations ofthesequences hybridizing to the AcNPV probe were determined. After nucleotide sequence determination (Fig. 3), the hybridizing portion of BmNPV DNA was identified as the polyhedrin

gene. This identification was based on the amino acid sequence ofthe polypeptide that resultedfrom the concep-tual translation of the corresponding RNA sequence after

comparisons with the published sequence of the authentic

BmNPV polyhedrin (26, 39) and with that derived from the

polyhedrin gene sequences of AcNPV (23).

Nucleotide sequences of the polyhedrin gene. The

se-quencesdeterminedfor thepolyhedringene of BmNPV and

its vicinity (Fig. 3) include 571 nucleotides offlanking and

mRNA noncoding sequences to the 5' end of the ATG

initiator codon (nucleotide 1 inFig. 3),acodingsequenceof

738 nucleotides(includinginitiationand terminationcodons), and 751 nucleotides of mRNA noncoding and flanking re-gions downstream from the 3' end of the TAA terminator

(nucleotide 738 in Fig. 3). Since no information is yet availableonthe BmNPVpolyhedrin mRNA sequences, the cap addition site has been inferred at position -57 by homologytothecapsitereported recentlyforthepolyhedrin

gene of AcNPV (23, 42). DNA sequences similar to the

consensus TATAand CCAAT boxes that have been shown to

represent

important

elementsof

eucaryotic

gene

promot-ers

(6)

werefound in the 5'

flanking region

ofthe BmNPV

polyhedrin

gene 28 and 63 nucleotides

upstream

from the

putative

cap

site,

respectively (Fig.

3,

positions

-85 and

-120).

As with the AcNPV

polyhedrin

gene

(23),

the

ob-served TATAbox deviates from the established canonical

sequence

(TACAAA

for

TATAAA).

Deviations of this

type,

however,

are not

unusual,

particularly

in genes of viral

origin

(2).

Anextra set ofTATAand CAATsequences was

alsoobservedat

positions

-116and

-151,

respectively (Fig.

3).

Of course, the contributions ofany ofthe

signals

men-tioned above to

polyhedrin

gene

promoter

function are

speculative

and may

only

be deduced

through

transcrip-tionalstudies.

The

coding

portion

ofthe

polyhedrin

gene of BmNPV is

not

interrupted

by intervening

sequences. This is also true

with the

polyhedrin

genesofAcNPV

(23, 42)

and ofthe NPV

of

Orgyia

pseudotsugata

(OpNPV [36]),

whose N-terminal

polyhedrin

sequencewasfound tobe verysimilartothatof

BmNPV

polyhedrin

(37).

In termsofcodonusage,theTAC

codon for

tyrosine

was found to be used at a

frequency

of

13/16,

whereas the AAC codonfor

asparagine

occurredata

frequency

of 15/18

(the

frequencies

of the

corresponding

codons in the AcNPV

polyhedrin

gene are 12/15 and

12/14,

respectively

[23]).

Except

for these two cases, no other

strong codon usage

preferences

were noted.

The

polyhedrin

mRNAs of AcNPV and

OpNPV

have

been shown to be

polyadenylated

(36, 50),

and most

likely

the same is true for the

polyhedrin

mRNA of BmNPV. Because the

published

sequenceofthe AcNPV

polyhedrin

genedoesnotextendtothe

point

ofthe mRNA

polyadenyl-ateaddition

site,

this sitecannotbe inferredonthegenefor

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[image:6.612.124.496.71.346.2]

-18. -172 -162 -152 -142 -132 -122 Sm TCGACAAGsI C1,TTCTTT G rE&C GrE&AGGTCT CAATCCTATC TGIAATIATI IT4AAp

Ac iiGTCTGCGAGG A AGCA TTGTAATGAGACGCACAAAC TAAXAAC

-11 -102 -92 -82 -72

Bm ACAATTA A ATGT AATT TGTTTTTTAT TAAATc5CA --

-Ac

AWT664A

ATGTC*A;CA ATATATAGTT A

-67 -57 -47 -37 -27 -17 -7

Sm AAATAATAAC CATCACGCAA ATAAATAAGT ATTTTACTGT TT'TCGTAACA GTTTTGTAAT AAAAAAACCT

Ac AAATGATAAC CATCTCGCAA ATAAATAAGT ATTTTACTGT TTTCGTAACA GTTTTGTAAT AAAAAAACCT

Bm ATAAAT.

Ac ATAAATA

15 30 45 60

Bm ATG CCG AAT TAT TCA TAC AO CCC ACC ATC GGGCGT ACITAC GTG TAC GAC AAI AAATAI

Ac ATG CCG SAT TAT TCA TAC CGT CCC ACC ATC6G6 CGT ACCTAC GTGTAC GAC AACAAGTAC

75 90 105 120

Bm TAC AAA AAC TTG GGC GGT CTC ATC AAA AACGCSAAG CGC AAG AAG CAC TA ASICGAA CAT

Ac TAC AAA AAT TTASSTG CCGTT ATC AAGAAC GCT AAG CGC AAGAAG CAC TTC GCC GAA CAT

135 150 165 180

Bm GAA AAA GAGGAGAAG CAA TGG GAT CII CTA GAC AAC TACeTG GTGCCTGAAG GAT CCC TTI

Ac GAG ACGAA GAG GCTACC CTCGACCCC CTA GAC AAC TAC CTA GTG GCT GAG GAT CCT TTC

195 210 225 24i

Sm ITA GGA CCG GGCAAA AAC CAA AAA CTT ACC CTTTTI AAA GAG GTT CGC AAT GTGAAA CCC

Ac CTGGSACCCGGC AAS AAC CAA AAA CTCACTCTCTTC AAGGAA ATCCGT AAT GTt AAA CCC

255 270 285 300

Bm GAT ACC ATGAAG TTA ATC GTCAA, TGGA,G GG, AAA GAS TTI CI ,GST GAA ACT TGG ACC

Ac SAC6AC8 ATGAAGCT;tTC GTt GGbTGGAAA GGA AAA GAG TTCtA A GG GAA ACT TGG ACC

31S 330 345 360C

Bm CGTTTT STT GAG GAC AGC TTC CCC ATT GTA AAC GAC CAA GAG GTG ATG GACGTG TAC CTC

Ac CGC TTC ATG GAA GAC AGC TTC CCC ATT GTT AAC SAC CAA GAA GTG ATG GAT GTt TTCCTT

375 390 405 42C)

Bm GTC GCC AAC CTC AAACCC ACA CGC CCC AAC AGGTGC TAC AAG TTC CT, GCT CAA CAC GCT

Ac GTT G6CAAC ATGS6TCCC ACT AGA CCC AAC CGT TGTTAC AAA TTC CTG GCCCAA CAC GCT

435 450 465 480

Bm CTTAGGTGGSAC SAA GAC TAC GTGCCCCAC GAO GT ATC AGAAT! ATGGAGCCA TCC TA

Ac CTG CGT TGCGAC CCC GAC TAT GTACCTCAT GAC GTG ATT AGG ATC GTCGAGCCt TCA TGG

49S 510 525 540

Bm GTG GGC ATGAAC AAC GAA TAC AGA AT! AGS CTG GCTAAA AAG GGC GGC GGC TGC CCA AT;

Ac GTG GGC AGCAAC AAC GAG TAC CGCATCAGC CTG GCTAAGAAG GGC GGC GGC TGC CCA ATA

555 570 585 600

Bm ATG AAC ATCCAC AGCGAG TAC ACC AAC TCGTTC GAGICG TT GTG A,AA CGS GTCATA TGG

Ac ATGAAC CTT CAC TCT GAGTACACC AAC TCGTTC GAA CAGTTCATC A CG GTC ATCTGG

61S 630 645 66L0

Bm GAG AAC TTC TAC AAACCC ATC GTT TAC ATC GGC ACAGAC TCT GC GAA GAAGAG GAA AT,

Ac GAGAAC TTC TAC AAGCCC ATC GTT TAC ATC GGt ACC GACTCT GCTGAA GAG GAG GAA AT?

675 690 705 720

Bm CTA ATT GAG GTT TCI CTC GTTTTC AAA ATA AAGGAGTTT GCA CCA GAC GCG CCT CTGTTC

Ac CTC CTT GAA GTT TCCCTGGTGTTC AAA GTA AAG GAGTTT GCA CCA GAC GCA CCTCTGTTC

735 Bm ACT GGT CCG GCA TAT TAA

Ac ACTGGT CCG GCGTAT TAA

748 758 768 778 788 798 B80

Bm AACACTATAC ATTGTTATTA STACATTTAT TAAGCGTTAG ATTCTGTACGTTGTTGATTTACAGACAATT

Ac AACAC6ATAC ATTGTTATTA GTACATTTAT TAAGCGCTAGATTCTGTGCG TTGTTGATTTACAGACAATT

18B 828 838 848 858 868 878

Bm GTTGTACGTA TTTTAATAAC TCATTAAATT TATAATCTTT AGGGTGGTAT GTTAGAGCGA AAATCAAATG Ac GTTGTACGTA TTTTAATAAt TCATTAAATT TATAATCTTT AGGGTGGTAT GTTAGAGCGA AAATCAAATG

s88 898 908 918 928 938

Bm ATTTTCAGCGTCTTTQTATC TGAATTTAAA TATTAAATCC TCAATAGATT TGTAAAATAG GTTTCGA Ac ATTTTCAGCG TCTTTATATC TGAATTTAAATATTAAATCC TCAATAGATT TGTAAAATAGGTTTCGA

FIG. 6.

Sequence comparison

betweenthe BmNPVand AcNPV

polyhedrin

genesandtheir

surrounding

sequences.(A)The 5'flanking sequencesof thetwogeneswhich have diversified

considerably

are

compared

aftergapswereintroducedtomaximize

homologies.

Blocks oftwoor moreidentical nucleotidesareboxed,anddotsindicate nucleotide differences.(B)The

comparison begins

atnucleotide -76of the sequenceofthe BmNPV gene andcontinuestothe lastknown nucleotideofthe AcNPV

polyhedrin

gene sequence(23).

Except

forablank introduced after nucleotide-1ofthe BmNPV

polyhedrin

gene sequencetoaccommodateanextraAresiduepresentin thegeneof AcNPV

polyhedrin,

no sequencerearrangementswerenecessaryfor maximum

homology.

Nucleotide differencesareindicated

by

dots.

BmNPV polyhedrin by homology. However, the sequence AATAAA, showntobe presentin the 3'noncodingregion of

almostallpolyadenylatedeucaryoticmRNAs approximately 25to30nucleotidesupstreamfrom the site of polyadenylate addition (35), occursatposition 1081ofourgene(Fig. 3). It should be noted thatasecond AATAAA sequence ispresent

approximately 90nucleotides downstream from the firstone

(position 1174).Anunambiguousanswer astothe location of thepolyadenylationsite mayonlybederivedbySinuclease

protection experiments (5). Protection experiments ofthis kindto determine the length ofAcNPV polyhedrin mRNA have indicated a cytoplasmic mRNAsize of approximately

1,200 nucleotides, excluding the polyadenylate tail (42). Assuminga similar mRNAsizefor BmNPVpolyhedrin, that value wouldplace theend of the BmNPV mRNA at around 60 nucleotides downstream from the first polyadenylation signal.

Amino acid sequence ofpolyhedrin. When the amino acid sequencepredictedfrom the geneprimary structure (Fig. 3) was compared to that of the authentic protein (26, 39), a small number ofdiscrepancies were noted whichprevented

the absolutematchingof thetwosequences.The differences are shown in Fig. 4 and are summarized as follows (the numbers inFig. 4 and those mentioned below correspond to

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BmNPV POLYHEDRIN GENE 443

oursequence and areexclusive ofthe initiating methionine residue): (i) amino acid substitutions in dipeptides at posi-tions 39-40 (His-Glu for Glu-His) and 213-214 (Ser-Ala for

Ala-Ser); (ii) an amino acid substitution at position 218 (Glu for Gln); (iii) the presence of a single valine residue at

position 114instead oftwo,resulting in ashift oftheamino acidreading frame by one residue; (iv)the presence ofan extraamino acid residueat position 144(Glu) missing from

thepublished polyhedrinsequence (26, 39),whichresults in therestoration ofthecolinearamino acid reading frame;and (v) amino acid substitutions at positions 142 (Trpfor Gln)

and 143 (Aspfor Asn) within theregion in whichthereading frameshift has occurred.

Ofthe observed differences, those described in (i) above are positional. Ofthe remaining amino acid substitutions, those at positions 143 and 218 may be explained by the

accumulation of single point-mutational events. The amino

acid substitution at position 142, however, requires the

accumulation ofatleasttwo point mutations on the

partic-ular codon. More complex mechanisms should, ofcourse,

be used toexplain the deletion and insertion ofthe codons fortheamino acid residuesat positions 114and 144. These

differences areobviously morethan whatonewouldexpect from simple variants of the same virus. Since we have

confirmed our nucleotide sequence analysis and since the

sequenced moiety of the cloned

fragment

was the only portion of the 3mNPV genome that hybridized to the AcNPVpolyhedringeneprobe,wefeel thatareexamination of the polyhedrinprotein sequencing data may be

appropri-ate.

Polyhedrins of BmNPV and AcNPV. The sequence of

BmNPV polyhedrin predicted by the primary structure of

the gene was also compared with that predicted from the

correspondingsequences ofAcNPV

(Fig.

5).No rearrange-mentswere requiredtoalignthetwoamino acidsequences,

which areclearly highly homologous. The degree of

diver-gence between the two sequences is 13.9% (34 amino acid substitutions inatotalof244

residues).

The collectiveresult

ofthe amino acid substitutionsappears tobe

neutral,

since

the net change in charge effected by them amounts to one and the overall degree of

hydrophobicity

remains

un-changed. In

addition,

computerized predictions

ofthe

sec-ondary structures of BmNPVand AcNPV

polyhedrins (15,

28) reveal that although some regional differences may occur, the overall

distributions

of a-helical and

p-pleated

sheet structures within the two molecules were identical (datanot shown). Inthis respectitis also worthmentioning

that BmNPV polyhedrin has 11 arginine

residues,

all of which arealso present in the polyhedrin ofAcNPV, which

has atotal of13 arginine residues. Ofthecodonsfor the 11

arginine residues sharedbythe twosequences, 5 have been altered by double point mutations resulting in codons again specifying arginine residues. Thus, itappears that thechanges

in thepolyhedrin molecule during evolution occurred under considerable selectivepressure. This isfurther corroborated by the results obtained when the N-terminal amino acid

sequences of the

polyhedrins

from two types of

OpNPV

werecompared with those of the polyhedrin of BmNPV (37). Asequenceidentityof

88.9%

wasfoundinthecomparisons involving the first 36 residues of a multicapsid OpNPV,

whereas the sequence homology for 34 residues of a

uni-capsid OpNPVwas85.3% (thecorrespondingvaluesfor the

comparisonbetween BmNPV and AcNPVpolyhedrinswere 88.2and 86.8%for the first 34 and 36residues, respectively

[Fig.

5]).

Sequence homologybetweenthepolyhedringenesofBmNPV

and AcNPV. The nucleotide sequences

determined

for the

polyhedrin

gene ofBmNPV were

compared

with those of the

polyhedrin

geneof AcNPV. This

comparison

indicated

thatmajor portionsofthetwosequences may becompletely correlated

(Fig.

6).

Forthe protein-encoding

regions

ofthe genes (shown to have

diverged by

13.9% at the amino acid level

[Fig. 5]),

a

22.2%

divergence

was determined

(164

nucleotide substitu-tions in a total of

738).

Of the observed

changes,

110

(two-thirds) represented

silent substitutions

resulting

inthe appearance ofsynonymous

codons,

whereas 54

(one-third)

were replacement substitutions

leading

to amino acid

changes.

From these results it is apparent that a

large

number of silent substitutionshavebeenfixed inthe

coding

portion

of the genes

during

evolution and that the two

nucleotide sequences have

diverged

somewhat more than

the

corresponding

amino

acid

sequences. This

indicates,

in

turn,

that the

protein

itselfhas been undermore

stringent

selectivepressure than the

corresponding

DNA sequence.

Incontrast tothe

divergence

found in the

coding regions

ofthetwo

polyhedrin

genes, thenucleotidesequencesof the

5'

noncoding regions

andthose ofasmall

portion

of the5'

flanking

regions

show a remarkable conservation. In

fact,

exceptforan extra

nucleotide

that is presentin the

58-nu-cleotide-long

5'

noncoding

region

ofthe AcNPV gene

just

before the ATGinitiation codon

(Fig. 6),

thetwo

noncoding

sequences are identical. Even this difference may not be

significant,

since in another isolate of AcNPV the extra

nucleotide was not observed

(E.

B.

Carstens, unpublished

data).

This remarkable

length

and nucleotidesequence

con-servationin the 5'

noncoding regions strongly

suggests that

capping

ofthetwomRNAsmayoccur atthe same

position

(-57 in the sequences shown in

Fig. 6).

The sequence

homology

continues fartherupstreamfor another20 nucleo-tides

(19

identical residuesoutof

20)

and stopsatnucleotide

-76

just

beforethe

beginning of

the

presumed

TATAboxes. A

similarly

striking homology

wasalsoobserved in the 3'

noncoding regions

ofthegenes.

Only

5nucleotide

substitu-tions

wereobserved in the 207 nucleotidesofthe sequences that were

compared

(2.4%

sequence

divergence).

Unfortu-nately,

because more sequence information on the AcNPV

polyhedrin

genewasnot

available,

wecould notextendthe

comparison

to cover the entire 3'

noncoding region

and

flanking

gene sequences.

Taking

into

consideration,

how-ever, that mutations

probably

occurred

uniformly

through-outthetwogenomes

(including

the

polyhedrin genes),

it is obvious

that,

in contrast to the

point

mutations

that have beenfixed inthe

coding

regions, only

averysmall numberof substitutions were allowed to be fixed in the 5' and 3'

noncoding regions

and inthe immediate5'

flanking

regions

of the genes

during

evolution. This of course should be

indicative of the

degree

of selective pressure which the

different

portions

of the genes are

under

because of the

functions

they

haveto

accomplish.

No

significant homology

canbe

recognized

atfirst

glance

in the 5'

flanking

gene sequences upstreamfromnucleotide

-76

(Fig.

6,

upper

panel).

With the aid of a dot matrix

program,wescanned thetwosequences for the presence of

homologies

thatmayoccurwithout respectto

position.

Asa result of this search we were able to construct different versionsof sequence

alignments.

Oneofthese

alignments

is

presented

in

Fig.

6

(upper

panel).

Although

the overall sequence

identity

is

only

38.8%,

several blocksofcommon sequences may bediscerned

(boxes

in

Fig. 6). Except

for the

large

gap of ninenucleotidesintroducedatthe 3' endof the BmNPV sequence,

only

three

single

nucleotide gaps have

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beenintroduced inthe sequence of AcNPVtoallow forthe alignment. We would like to emphasize not the nucleotide sequencesassuch but thefact that thedistancesamong all of theobservedhomology blocks are thesame(±1nucleotide) in both flanking sequences. At this point we can only speculateonthesignificance of these blocks.Transcriptional

experiments would be needed to determine whether they participate in the regulation of polyhedrin gene activity. Despite our uncertainties, sequences of this type should be

considered, particularly when molecular engineering in the

vicinity of thepolyhedringene isto beundertaken. Is the polyhedrin gene immediately flanked by other viral genes? Since we may need to insert a fragment offoreign

genetic material such as B. mori genomic DNA in the

vicinity ofthepolyhedringenewhile preservingtheabilityof the virus to function properly (including the formation of

polyhedra),itisimportantnotonlytoknow the exact limits of the polyhedrin gene but also what lies next to it. In this respect we have noticed the two openreadingframesat the twoendsof thesequenced portionof theclonedDNA. More extensive studies are needed to determine whether the observed open reading frames are parts ofadjacent struc-turalgenes. Such studies are now in progress.

ACKNOWLEDGMENTS

We thankJ. L. Vaughn and R. Stone ofthe Insect Pathology Laboratoryfor thegifts ofBm-5cellsandinitial inocula ofBmNPV as wellasfor theiradvice for the establishment ofthecellsin our laboratory, E.B. Carstens for supplying us with the cloned HindIII-Vfragment of the AcNPV genome and forcommunicating to ushis sequences of the AcNPVpolyhedrin gene before publica-tion;G. Chaconas of theCancer ResearchLaboratory, University of WesternOntario,formakingavailableto usthecomputer program

onprotein secondary structurepredictions, J.C. States of the De-partmentofMedicalBiochemistry, UniversityofCalgary,for hisgift ofpUC9 and host cells, D. McKay of the Department of Medical Biochemistry, University of Calgary, for providinguswithsomeof the computer software used for the analysis ofour nucleic acid sequences, B. Pinder and R. Haselden for photography, Anne Vipondfor technicalassistance, and Susan Carlson for secretarial assistance.

This work was supported by grants from the Cancer Grants Program of the Alberta Heritage Savings Trust Fund (Alberta CancerBoard)andthe Medical Research Council of Canada (toK. Iatrou)and byapostdoctoralfellowshipfromthe AlbertaHeritage Foundation for Medical Research (to H.W.).

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