Inhibition of Bombyx mori nuclear polyhedrosis virus (NPV) replication by the putative DNA helicase gene of Autographa californica NPV.

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0022-538X193/106239-07$02.00/0

Inhibition of

Bombyx

moni

Nuclear

Polyhedrosis Virus

(NPV)

Replication by the Putative

DNA

Helicase Gene of

Autographa

califomica

NPV

SHIZUO GEORGE KAMITAANDSUSUMU MAEDA*

Department of Entomology, University of California, Davis, California 95616-8584

Received 29 March1993/Accepted 12July 1993

Coinfection ofBombyx mori nuclear

polybhedrosis

virus(BmNPV) withAutographa californicaNPV(AcNPV)

in theBmNPV-permissiveBmNcellline resulted in thecomplete inhibition ofBmNPVreplication. Coinfected

BmN cells exhibited an atypical cytopathic effect (CPE) and synthesis of viral and host proteins was

dramatically

attenuated by 5 h postinfection (p.i.) and nearly

completely

blocked by 24 h p.i. Viral

transcription, however, appearedto occur

normally

during both

early

(5-h-p.i.) and late (24-h-p.i.) stages of

infection. Superinfection of BmN cells with AcNPVat 5 and 12hpost-BmNPV infection resulted inlimited

inhibition of BmNPVreplication. BmN cells singly infected with AcNPV also showed similarCPE, premature

inhibitionof viral and host protein synthesis, and

apparently

normal viraltranscription. BmNPV replication

occurred

normally

following coinfection ofBmNPVand eh2-AcNPV,anAcNPVmutantidenticaltoAcNPV

except fora572-bp regioninitsputativeDNAhelicase geneoriginatingfrom BmNPV(S. Maeda, S. G. Kamita,

andA. Kondo, J. Virol.67:6234-6238, 1993). Furthermore,atypical CPEandpremature attenuationof host

andviralprotein synthesiswere notobserved. These results indicated that the inhibition of BmNPVreplication

was causedeither

directly

or

indirectly

at the translational level by the putative AcNPV DNAhelicasegene.

Nuclear polyhedrosis viruses (NPVs), a genus of the

familyBaculoviridae, arecharacterized by circular

double-stranded DNA genomes. Recombinant NPVs are efficient

vectors for the high-level expression of foreign genes for

basic research and medicalapplications (15,16, 25). Recent

advances in genetic engineering have resulted in

recombi-nantNPVsthat holdpromiseaspotent yetsafealternatives

to traditional chemical insecticides (5, 24, 31, 33). NPVs

haveonly beenisolated from arthropods (mainly insects) and

areconsidered to have narrowhost ranges, i.e., they

repli-cate in only a few species of the same or similar families.

Previous host range studies have aimed to identify a

com-monregion in the genomes of baculoviruses with low DNA

homology but whichcan infect the samehost. The general

mechanism(s) of baculovirus hostspecificity, however,

re-mains undetermined.

Bombyx

mon NPV(BmNPV) and Autographa califomnica NPV(AcNPV) arehighly homologousatthegenomiclevel

(23),yettheyexhibitnonoverlappinghostspecificities;e.g.,

BmNPV replicates in BmN cells, but not in SF-21 cells,

whereas AcNPVreplicates in SF-21, but not in BmN. We

have demonstrated that BmNPVreplicationcanbeinduced

in SF-21 cells coinfected with AcNPV (12). This

phenome-non was shown to be the result of (i) a helper function of

AcNPVtowards BmNPV replication and (ii) the appearance

of AcNPV-BmNPVhybridswith expanded host ranges. By

backcross infection of one of these hybrid NPVs with

AcNPV,wegenerated eh-AcNPV, abaculovirus capable of

replicating in both BmN and SF-21 cells and possessing

DNA restriction endonuclease patterns nearly identical to

those of AcNPV.By cotransfection andnucleotide

sequenc-inganalysis,a572-bpregion was hypothesized to be

respon-sible for the hostrange-expanded phenotype of eh-AcNPV.

This572-bpregionof eh-AcNPVwasidenticaltothe

corre-*

Corresponding

author.

sponding 572-bp region of BmNPV and was localized within the putative DNA helicase gene. Cotransfection of the 572-bp BmNPV fragment with AcNPV genomic DNA

gen-erated the hostrange-expanded virus eh2-AcNPV,

confirm-ing that DNA helicase playsarole inhost range expansion

(20).

In this study, we found that BmNPV replication was

completely inhibited in BmN cells coinfected with BmNPV

and AcNPV. The coinfected BmN cells also exhibited a

unique cytopathic effect (CPE), and synthesis of both host

and viral proteins was prematurely attenuated. Viral

tran-scription, however, appearedto occurnormally during both

the early and late stages of infection. BmN cells coinfected

with eh-AcNPV and BmNPV oreh2-AcNPV and BmNPV

didnotexhibit any abnormal CPEorprematureattenuation

of viral or host protein synthesis, and viral replication

occurred normally. These analyses suggested that the

puta-tive DNA helicase gene of AcNPV(23)caneitherdirectlyor

indirectly cause the inhibition of BmNPV replication in

BmNcells.

MATERIALSANDMETHODS

Cell lines and viruses. BmN(BmN-4)and SF-9 cellswere

maintained in TC-100 medium supplemented with 10% fetal

bovineserumand inEx-Cell 400(JRH Biosciences,Lenexa,

Kans.) supplementedwith2.5% fetal bovine serum,

respec-tively, as described previously (17, 23). The BmNPV T3

isolate (21) and the AcNPV mutants, eh-AcNPV and

eh2-AcNPV (20), were propagated on BmN cells. The

AcNPVOT2 isolate(23)was propagatedonSF-9 cells.

Viral infection andplaque assay.BmN or SF-9cellswere

infected with BmNPV, AcNPV, eh-AcNPV, and/or

eh2-AcNPVat amultiplicityof infection (MOI)of 10(exceptas

noted) for each virusasdescribedpreviously (12,17). In all

experiments, time zero was defined as the point at which

freshmediumwas added following the 1-hviral

adsorption

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200 ,u ofmethionine-freeculturemediumat 1,5, 12,24, 36,

48, and 72 h p.i. as described previously (11). Cell pellets

were dissolved in 100 pl of sample buffer, and 6

RI

was

subjectedtosodiumdodecyl sulfate-polyacrylamide gel

elec-trophoresis (SDS-PAGE) as described by Laemmli (13) on

10% acrylamide gels (0.4% bis-acrylamide). Gels were

treated with an enhancer (Resolution; EMCorp, Chestnut

Hill, Mass.), dried under avacuum, and exposed to X-ray film (Eastman Kodak Co., Rochester, N.Y.). Low-range SDS-PAGE molecular weight standards

(14.4

to 97.4 kDa)

werefromBioRad Laboratories

(Richmond,

Calif.).

Isolation of

poly(A)+

RNA. Poly(A)+ RNAwas isolated

from approximately 9 x 107 BmN cells which were either

mock infectedorinfected withvirus(es)asappropriate.Cells

were harvested at either 5 or 24 h p.i., washed once in

ice-cold phosphate-buffered saline, and oligo(dT) cellulose

wasusedto extractpoly(A)+ RNA asdescribedpreviously (19). The concentration of the poly(A)+ RNA was

deter-minedbymeasuring

A260-DNAprobes. Twoprobescontainingsequencesfromearly

and latebaculovirus geneswerelabeled with

[a-32P]dATP,

using a random primed DNA

labeling

kit (United States

Biochemical, Cleveland, Ohio). The probe OT2EH was

derived from the AcNPV 0T2 EcoRI H

fragment

which

corresponds to the EcoRI H fragment

(79.8

to 86.4 map

units)in the conventional AcNPV map(foranexample, see

reference 28). This fragment contains the open reading

frames (ORFs) gp64, ORF1, ORF2, PE34, ORF4, ORF5,

and 94K,whichareconsidered

immediate-early

and/orlate

baculovirus genes (4, 27, 34) (see Fig. 4A). In this region,

BmNPV and AcNPV have over 95% nucleotide sequence

identity excluding the 94,000-Da gene (94K gene) (18). A gene corresponding to the AcNPV 94K gene is

lacking

in

BmNPV(11).

Theprobe BE284BE was derived from the 1.3-kbp

frag-mentofthe BmNPV transfervectorpBE284

(17)

whichwas

digestedwith BamHI and EcoRI. BE284BE contains the 3'

nontranslatedregion of thepolyhedringene and1,340bpof

the 3' end of theORF1629codingregion(2.4to3.4 map units

in the BmNPV genome [22]) (18, 30) (see Fig. 4B). The

polyhedrin geneandORF1629 areconsidered very late and late baculovirus genes, respectively (30). Nucleotide

se-quences in this region are greater than 97% identical for

BmNPV andAcNPV(10, 18, 30).

Northern(RNA) blothybridization.Poly(A)+ RNAs (3 ,ug

per lane) were electrophoresed (1.5 h) in a formaldehyde denaturing gel (1% agarose) in morpholinopropanesulfonic

acid(MOPS) (0.02 M) buffer. Molecularweightwas

deter-mined by comparison with coelectrophoresed molecular

weightmarkers(3 ,ug of 0.24-to9.5-kbRNAladder; GIBCO

1-> 7

6 5

0 24 48 72

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HOURSPOSTINFECTION

FIG. 1. Growth curves of BmNPV(0)followingcoinfectionof BmNcells with BmNPVand AcNPV(O) (A), superinfection of BmNPV-infected BmN cells with AcNPV at 5(0)or12(-)hp.i. (A), andcoinfection of BmN cells with BmNPV andeh2-AcNPV (0,eh2-AcNPV;El, BmNPVplus eh2-AcNPV)(B).

BRL, Inc., Gaithersburg, Md.). Following electrophoresis,

RNAs were denatured, transferred, and fixed to a nylon

membrane (Zeta-Probe; Bio-Rad Laboratories)bya

modifi-cation of themethod describedpreviously(19).

The DNAprobesweredenatured inboilingwater(10min)

and hybridized to the mRNAs at 42°C as described

previ-ously (23). Filters used forhybridizationwerewashed twice

in 2x SSC (lx SSC is 0.15 M NaClplus 0.015 M sodium

citrate)-0.1% SDS and once in 0.1x SSC-0.1% SDS and

thensubjectedtoautoradiographyusing X-rayfilm(Eastman

KodakCo.). The filterswerereprobed after the firstprobe

wasstripped from the mRNAsbyboilingin 10 mMTris(pH

7.5)-0.1% SDS for 5 minfollowedbyincubation at80°Cfor 1 h.

RESULTS

Inhibition of BmNPV replication by coinfection with

Ac-NPV.OnBmNcells, BmNPV showedatypical viral growth

curvewhich reached aplateau maximum of about 2 x 108

PFU/ml at 72 h p.i. (Fig. 1). AcNPV doesnot replicate in

BmNcellsasreportedpreviouslyby Maedaetal.(23).When

BmN cells were coinfected with BmNPV and AcNPV,

BmNPV replication was completely inhibited (5,000-fold

from 2 x 108 PFU/ml to a residual level of about 4 x 104

PFU/ml) (Fig. 1A). BmNPV replication was also inhibited

1,000-fold and2,500-fold whencoinfected with AcNPVat2

and4MOI,respectively (datanotshown). Superinfectionof

BmNPV-infected(5MOI)BmNcells with AcNPV(20MOI)

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FIG. 2. The morphology ofBmN cells at 72 hp.i.followingBmNPV and AcNPVcoinfection(A), BmNPVinfection(B),AcNPV infection (C), andmock infection(D).Magnification, x400.

at 5 or12 h post BmNPV infection resulted in 16-fold and

2-foldinhibition, respectively (Fig. 1A).

BmN cell morphology and growth. The morphology and

growth characteristics (data not shown) of mock-infected

BmNcells was typical ofhealthy cultures during a log phase

ofgrowth (Fig. 2D). BmN cellgrowth was not detected in

anyof thevirally infected BmN cells. At 72 h p.i.,

BmNPV-infected BmN cells exhibited CPE typical of wild

type-infected cultures, including rough cell surface, detachment

from the culture surface due to rounding, nuclear expansion,

andformation ofpolyhedra(Fig. 2B).In contrast,coinfected

BmN cells did not exhibit rounding, remained strongly

attachedtothe culture surface during all stages of infection,

and did not produce polyhedra; however, they did exhibit

nuclear expansion (Fig. 2A). These characteristics were

initiallyobserved around 12 hp.i.and were maximized by 48

hp.i. The morphology of AcNPV-infected BmN cells (Fig.

2C)was similar to thatofBmNPV and AcNPVcoinfected

cells. BmN cells superinfectedwith AcNPV (at 5 and 12 h

post BmNPV infection) did not exhibit cell morphology

specificfor AcNPV infection.

Protein synthesis. In order to examine the mechanisms behind the AcNPV-induced inhibition of BmNPV

replica-tion,proteinsynthesiswasinitiallyanalyzed by SDS-PAGE.

Mock-infected BmN cells showed auniform increase in all

protein synthesis correspondingtonormal cellproliferation

(data not shown). BmNPV-infected BmN cells showed a

pattern ofprotein synthesis typical of baculovirus-infected

cells(Fig.3A),asreportedpreviously (32).Between5 and 24

h p.i., the synthesis of two or three putatively virally

encodedorvirally induced proteinswas detected. By24 h

p.i., the expression of host- and virus (e.g., the

38-kDa)-specific proteins was retarded or terminated. Between 24

and 72 h p.i., nearly all host- and virus-specific protein

synthesis was terminated or highly retarded except for a

highly expressed 30-kDa protein(indicated bythe arrowhead

inFig. 3A),whichwasconsideredtobeBmNPV-expressed

polyhedrinonthe basis of its size and time ofexpression.

In BmNPV-andAcNPV-coinfected BmNcells, synthesis

of both host- and virus-specific proteins was prematurely

anddramaticallyinhibited(Fig.3B).By 5 hp.i.,totalprotein synthesis droppedtoabout20%of thelevels foundfollowing

BmNPVsingleinfection, andby24 hp.i.,protein synthesis

was nearlycompletely inhibited. Between 36 and 72 h p.i.,

three putatively virally encoded bands (approximately 30,

38, and 42kDa)were slightlydetectable. The 30-kDa band

(indicated bythearrowheadinFig. 3B)wasconsideredtobe

polyhedrin expressed byBmNPV or AcNPV.

To examine whether the premature attenuation ofprotein

synthesis was induced by the interaction of BmNPV and

AcNPVor wassolelythe result of AcNPVinfection, protein

synthesis of AcNPV-infected BmN cells was analyzed. A

dramatic and premature drop in protein synthesis nearly

identicalto thatfollowing BmNPVandAcNPVcoinfection

was observed in BmN cells singly infected with AcNPV

(Fig. 3C). At 12 h p.i., at least one virus-specific band

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21.5-1 5 12 24 3648 72

VW -.

97-66-i

45-31-_ 21

.5-FIG. 3. SDS-PAGE of pulse-labeled proteins from BmN cells thatwereinfectedwith BmNPV(A),BmNPVand AcNPV(B),or AcNPV(C).Proteinswerepulse-labeledwith[35S]methionineat1,

5, 12, 24, 36, 48,or72 hp.i.Theaffowheadsindicate themigration

of the putative polyhedrins. The approximate sizes of marker proteinsareindicated totheleftinkilodaltons.

A _Bm

_Bm

_cmB

eh-Aceh-Ac Ac m 5 24 524 524 524 5 24 5 24

I. I.

coinfected SF-9 cells was only about 1/10 that found in

AcNPV-infected SF-9 cells at 72 h p.i. Furthermore, the

normalattenuationof hostproteinsynthesisdid notseem to

occur as quickly in eh-AcNPV-infected SF-9 cells as in

AcNPV-infected cells (datanotshown). These results

indi-cated that the dramatic inhibition of host and viralprotein synthesis was induced by the AcNPV DNA helicase in

conjunctionwith ahost factor(s), e.g., interaction ofDNA

helicasewithhostproteinsor nucleicacids.

Northern blot

analysis.

Northern blot analysis was

per-formedtoexaminewhether the inhibition ofprotein

synthe-sis inducedbyAcNPVinfectionwasduetothe lack of viral

transcripts.Poly(A)+RNAscollectedat5and24hp.i.from

mock- and virus-infected BmN cells wereanalyzedwith the

probes BE284BE and OT2EH, which were derived from

BmNPVandAcNPV,respectively (Fig. 4).Ingeneral,viral

transcription of both earlyand late (5- and

24-h-p.i.)

genes

occurred normally in AcNPV-infected and BmNPV- and

AcNPV-coinfected BmNcells.

OT2EH (gp64, ORF1, ORF2, PE34, ORF4, ORF5, and

94K) did not hybridize with any transcripts in the

mock--_9.5 7.5

-4.4 4.2_

-2.4 2.2 _

-1.4 1.3

I--0.24

Bm eh-Ac Bm Bm Ac m

ehSAc S4 2Ac

5 24 524 524 524 5 24 5 24

- 9.5

7.5

-4.4

-2.4

-1.4

-0.24

gp64

79.8 ORF--- ORPE34EzRl

EcoRI ORF2jOHF4ORFs probe (OT2EH)

94K Xbal

_ 1.9 I .-. ORF1629

86.8mapunits l - PoFyherin 3.4 mapunits

EcoRi -- BamHl

mp_ 1

probe (BE284BE)

FIG. 4. Northern blot analysis of mRNAcollected from BmN cells at 5 and 24 h following infectionwith BmNPV and eh-AcNPV (Bm/eh-Ac),eh-AcNPV(eh-Ac),BmNPVand AcNPV(Bm/Ac),BmNPV(Bm), AcNPV(Ac),orfrommock-infectedcells(m)probed with OT2EH(A)orBE284BE(B).Sizestandardsareindicatedtothe right inkilonucleotides. Major hybridized transcripts are indicated to the

leftbythearrowheads.Thecompositionsof theprobesareindicatedbelowthehybridizations.BE284BEcontainsthe 3'nontranslatedregion

of the BmNPVpolyhedringeneand1,340 bpof thecoding regionofORF1629. OT2EHcontainssequencesencoding gp64,ORF1, ORF2, PE34, ORF4,ORF5,and 94K.

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infected BmN cultures at either 5 or 24 h p.i. (Fig. 4A). At 5 h p.i., OT2EH hybridized to a 2,000-nucleotide (nt)

tran-script in BmNPV-infected, AcNPV-infected, and

BmNPV-andAcNPV-coinfected BmN cells. A 2,800-nt transcript was

also veryweakly hybridized at 5 h p.i. in AcNPV-infected

and in BmNPV- andAcNPV-coinfected BmN cells. At 24 h

p.i., OT2EH hybridized to 2,000-, 3,600-, and 4,300-nt

transcripts in BmNPV-infected, AcNPV-infected, and

BmNPV- and AcNPV-coinfected BmN cells. A 2,800-nt

transcript was also recognized by OT2EH in AcNPV-in-fected BmN cells but not in BmNPV- and

AcNPV-coin-fected cells. The 2,800- and 2,000-nt transcripts were most

likely derivedfrom the 94K (4) and gp64 (34) genes,

respec-tively,onthebasis oftheir size, expression at the early stage ofinfection, and relative abundance. The 2,800-nt transcript

mayalso result from the activation of other (non-early gene)

promoterscoveringPE34, ORF4, or ORF5 (27). The

3,600-and 4,300-nt transcripts were similar to those described

previously (27).

Followingthe initial hybridizations, OT2EH was stripped from the filter and the mRNAs were hybridized with

BE284BE (Fig. 4B). BE284BE (the 3' noncoding region of

polyhedrin

and coding region of ORF1629) recognized a

2,200-nttranscript from BmNPV-infected, AcNPV-infected,

and BmNPV- andAcNPV-coinfected cultures at 5 and 24 h

p.i.,

albeit weakly at 5 h p.i. BE284BE also recognized a

1,300-nt transcript in BmNPV-infected and in BmNPV- and

AcNPV-coinfected cultures. The 1,300- and 2200-nt

tran-scriptsmostlikely originated from ORF1629 and polyhedrin,

respectively,onthe basis of size and abundance during a late

stageofinfection. BE284BE alsohybridized weakly at 5 and

24 hp.i.to a2,200-nttranscript in mock-infected BmN cells. This hybridization, which was also found in virus-infected

BmN cells at 5 h p.i., was possibly due to nonspecific

bindingwithahost transcript. Probes specific toORF1629 of

AcNPV E2 show similar hybridization to host transcripts

isolated from uninfected larvae under stringent conditions

(29).

Effects ofeh2-AcNPV and eh-AcNPV on BmNPV

replica-tion and transcription. Infection of BmN cells with

eh2-AcNPVoreh-AcNPV resulted in typical viral growth curves

which reachedplateau maximums of about 4 x 108(Fig.1B)

and 6 x 107 PFU/ml (data not shown), respectively, at 72 h

p.i. Coinfectionof BmN cells with BmNPV andeh2-AcNPV

or eh-AcNPV also resulted in typical viral growth curves

which reachedplateaumaximums of about 7 x 108(Fig. 1B)

and3 x

108

PFU/ml (datanotshown), respectively.

Theproteinsynthesispatternsof BmN cellsinfected with

eh2-AcNPV(Fig. SA), eh2-AcNPV and BmNPV (Fig. 5B),

eh-AcNPV (data not shown), or eh-AcNPV and BmNPV

(data

not shown) were essentially identical to those of

BmNPV-infected (Fig. 3A) BmN cells, as described above.

Theputative polyhedrin protein band of eh2-AcNPV

(indi-catedbythearrowheads in Fig. 5) and eh-AcNPV (data not

shown)

appeared slightlylarger (31 kDa) than the polyhedrin

band of BmNPV (30 kDa). The 31-kDa band was also

observedbeginningat36 h p.i., whereas the 30-kDa putative

bandwasdetected beginning at 24 h p.i. (Fig. SB).

Asdescribedabove, transcription inAcNPV-infected and

in AcNPV- and BmNPV-coinfected BmN cells appeared to

occur normally and in a manner similar to that found in BmNPV-infected BmN cells. In order to analyze whether

transcriptionin eh-AcNPV-infected and in eh-AcNPV- and

BmNPV-coinfected BmN cellsoccurrednormally,Northern

blot analysiswas performed with the probes BE284BE and

OT2EH (Fig. 4). The transcription patterns of these

in-A 1 5 12 24 36 48 72

97-

66-

45-

.v-.-31-:.

21.5-14.4- -

-B 1 5 12 24 36 48 72

97-66-4

45- J

31- jg *

21.5-

14.4-FIG. 5. SDS-PAGE of pulse-labeled proteins from BmN cells that were infected with AcNPV (A) and BmNPV and

eh2-AcNPV (B). Proteins were pulse-labeled with [35S]methionine at 1,

5, 12, 24, 36, 48, or 72 h p.i. The arrowheads indicate the migration

of polyhedrin. The approximate sizes of marker proteins are indi-cated to the left in kilodaltons.

fections were very similar to those of BmNPV-infected,

AcNPV-infected, and BmNPV- and AcNPV-coinfected

BmN cells probed with OT2EH or BE284BE. The intensity of the OT2EH-probed patterns was, however,

approxi-matelyfive times higher than that of BmNPV-infected cells.

In eh-AcNPV-infected BmN cells, OT2EH hybridized to transcripts of 2,000 and 2,800 nt at 5 h p.i. and of 1,700,

2,000, 2,800, 3,600, 4,300, 7,000, and 8,000 nt at 24h p.i. In

eh-AcNPV- and BmNPV-coinfected BmN cells, OT2EH

hybridized to transcripts of 1,700, 2,000, and 2,800 nt at 5 h

p.i. and of 1,700, 2,000, 2,800, 3,600, and 4,300 nt at 24 h p.i. In eh-AcNPV-infected BmN cells, BE284BE hybridized to a 2,200-nt transcript (very weakly) at 5 h p.i. and to 1,300-, 2,200-, 3,300-, and 4,200-nt transcripts at 24 h p.i. In eh-AcNPV- and BmNPV-coinfected BmN cells, BE284BE hy-bridized to a 2,200-nt transcript (very weakly) at 5 h p.i. and to 1,300-, 2,200-, 3,300-, and 4,200-transcripts at 24 h p.i.

The 2,000- and 2,800-nt transcripts which hybridized to OT2EH and the 1,300- and 2,200-nt transcripts which hy-bridized to BE284BE were putatively transcribed from gp64,

94K, ORF1629, and polyhedrin, respectively, as described

above. The other transcripts hybridized by OT2EH (1,700, 3,600, 4,300, 7,000, and 8,000 nt) and BE284BE (3,300 and 4,200 nt) were in general similar to those examined previ-ously (27, 30).

DISCUSSION

In previous studies, BmN cells coinfected with BmNPV and AcNPV were found to exhibit a unique morphological response, i.e., atypical CPE (12). In the present study, we found that exchange of a 572-bp (or smaller) region of a single AcNPV gene with the corresponding region of Bm-NPV can eliminate these effects. Nucleotide sequence and physical map comparisons indicated that this gene putatively encodes a DNA helicase (20) which is expressed at an immediate-early stage of infection (14). Superinfection of BmN cells with AcNPV at 5 or 12 h post-BmNPV infection did not induce the unique CPE found following BmNPV and AcNPV coinfection, nor did it significantly affect BmNPV

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BmNcells, especially when probe OT2EH was used (Fig. 4A). This might be explained by (i) a higher efficiency of

AcNPVtranscriptional factors and/or(ii) a higher efficiency

of viralreplication by AcNPV-encoded polypeptides. These

effects might have been masked in AcNPV-infected or

AcNPV-BmNPV-coinfected BmNcells because of the CPE induced by AcNPV. These results indicated that the

prema-turedrop in proteinsynthesis was not due to the lack of viral

transcripts and that the inhibition of viral replication proba-bly occurred at the translational level.

A premature drop in protein synthesis and active viral

transcription has also been reported in

non-AcNPV-permis-sive gypsy moth (Lymantria dispar) cells infected with

AcNPV(7). Thiswasspeculated to be due to (i) the inability

of L. disparcells to translate theabundantlyexpressed viral

mRNAsand/or(ii)interferenceinthetranslational processes

by a viral structural polypeptide(s) in the infecting viral

particles. The normal transcription of late and very late

genes, e.g.,ORF1629 andpolyhedrin of AcNPV, indicated

thatbaculovirus early gene products required for the

trans-activation (e.g., IE-1 [6]) and transcription (e.g., a

virus-specificRNApolymerase[9])ofbaculoviruslate genes were

efficiently transcribed and translated. Furthermore, since translation of eh-AcNPV and eh2-AcNPV (Fig. 5A)

oc-currednormally in thenon-AcNPV-permissiveBmNcells, it

appears that an inability of the BmN cell translational

machinery to translate mRNAs from AcNPV isnotafactor

in the inhibition phenomena. This leads to the hypothesis

that the AcNPV DNA helicase gene itself may cause the

BmNtranslational machinerytobecome dysfunctional. Coinfectionof BmNPV and the AcNPVisolateLiin BmN

cells did not result in the complete inhibition of BmNPV

replication (BmNPV replicated to about 106 PFU/ml [data

not shown]), indicating that AcNPV isolates may vary in

their abilities to inhibit BmNPV replication. Morris and

Miller (26) have recently reported that a recombinant

AcNPV Li isolate carrying achloramphenicol

acetyltrans-ferase(CAT)genedrivenbyanAcNPV late gene promoter

canexpress CAT in BmN cells. Infection of BmN cells with

arecombinant AcNPV

Li

expressing the CAT gene under a

very late gene promoter, however, was shown to result in

very low CATactivity. Although directcomparison between

theexpression of CAT (by a viral promoter) and expression

of virus- and host-derived proteins by SDS-PAGE is

diffi-cult,thesephenomenaareconsistent with our observation of

virus-specific proteins inAcNPVOT2-infected and AcNPV OT2-BmNPV-coinfected BmN cells at 5 and 12 h p.i. (at

very lowlevels). On the basis of these data, it is difficult to

conclude at present whether the difference in the abilities of

that the host range of baculoviruses may be determined by

the ability or lack of ability of host cells to combat

virus-induced cytotoxicity. Virally encoded proteins with helicase activity (e.g., the putative RNA helicase of the turnip crinkle

virus [3]) and cell-specific host factors (e.g., those induced

by the parvovirus minute virus of mice [1]) have been

implicated in inducing

cytotoxicity

in several host systems

(20).

Aclear understanding of the mechanisms of host range

determination of baculoviruses is essential for their safe use

and acceptance as insecticides and expression vectors. At

present, the exact mechanism by which the putative AcNPV

DNA helicase inhibits viral replication in BmN cells is

unknown; however, our results indicate that the AcNPV

DNAhelicase itself may inhibit either directly or indirectly

thetranslational machinery of BmN cells by inducing

cyto-toxicity.

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