Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression.

0022-538X/93/063481-08$02.00/0

Copyright © 1993, American

Society

forMicrobiology

Identification and Characterization

of

lef-1,

a

Baculovirus

Gene

Involved in Late and Very Late Gene Expression

A. LORENA PASSARELLI'ANDLOIS K.

MILLER'12*

Department of Genetics' andDepartmentofEntomology,2 The University of Georgia,

Athens,

Georgia

30602

Received30 October 1992/Accepted5March 1993

An Autographa californica nuclear polyhedrosis virus (AcMNPV) gene required in transient expression

assays for late andverylate viral geneexpressionwasidentified, sequenced, and transcriptionally mapped.

This gene, designated late expression factor 1 (lef-1), was located between 7.4 and 8.7 map units of the

AcMNPVphysicalmap.Itwasidentified by cotransfecting Spodopterafrugiperda cultured cells withacollection ofoverlappingcloned DNA fragmentscovering theentire AcMNPVgenomeandareportergenecontrolledby

anearly, late,orverylate AcMNPVpromoter.Omissionof the DNAfragmentcontaininglef-lcurtailedmost

late andverylategeneexpressionbutnotearlygeneexpression.lef-1 wasfoundtobeanearlygenetranscribed

as a 1.8-kb RNA in the presence of the protein synthesis inhibitor cycloheximide. The C terminus of the

predicted polypeptide product,LEF-1, contained asequence motifcharacteristicof nucleoside

triphosphate-binding sites.

Upon infectionofpermissive culturedcells with the bac-ulovirusAutographa californica nuclear polyhedrosis virus (AcMNPV), three major classes of viral transcripts are detected: early, late, andverylate (forarecentreview, see reference23). Earlygenes aretranscribedbyan a-amanitin-sensitive RNA polymerase (8, 14), apparently host RNA polymerase II (13), andtranslated toprovidefactors

neces-saryfor DNAreplicationandtransactivation of lategenes.

Basal transcription of early genes does not require active

viralproteinsynthesis (6,13, 28, 34), althoughtheproductof the ie-1 gene isrequiredforexpressionofsomeearlygenes intransient expressionassays(9, 10).Following theonsetof

DNAreplicationat approximately6 hpostinfection (p.i.),a

drasticchangeingeneexpression is observed. Lateandvery

late gene transcription involving an ax-amanitin-resistant

RNApolymerase activity (8, 14)commences,whereaslevels

ofhosttranscripts arereduced (18). The latetranscriptional phase provides structural proteins for nucleocapsid assem-bly and budded virus production. Thevery late

transcrip-tionalphaseisdelayeduntilapproximately18to24 hp.i. (33)

and provides gene products involved in occlusion suchas polyhedrin.

We are interested inidentifying andcharacterizing bacu-lovirus genes involved in the transitions betweenearly and late and between late andvery late viralgene expression. Thus,wedevelopedatransientexpressionassaytoidentify

viralgenesinvolved inexpressionfrom thelatecapsid (vp39)

and the very late polyhedrin (polh) promoters (25). This assay has already been successfully applied in assigning a

role to the 630-nucleotide (nt) open reading frame (ORF) (26),renamedlef-2,in late andverylategeneexpression (25)

and inidentifyingadditional roles fortwobaculovirus

regu-latory proteins,IE-1 and IE-N(3, 9-11, 25).Inthisstudy,we

have used the transientexpressionassayto identifyanovel

earlygene, lef-1, involved in expressionofa reporter gene driven by a late or very late baculovirus promoter. The

predictedLEF-1polypeptidecontains266amino acids with

* Correspondingauthor.

an estimated molecular mass of 31 kDa and a C-terminal nucleoside triphosphate-binding motif.

MATERIALS ANDMETHODS

Virus and cells. The Spodoptera frugiperda (fall

army-worm) IPLB-SF-21 (SF-21) cell line (35)wasgrownat27°C

in TC-100 medium (GIBCO/BRL, Gaithersburg, Md.)

sup-plemented with 10% fetal bovine serumand0.26% tryptose brothasdescribedpreviously(23). Wild-type AcMNPVL-1 (16), usedto obtainviral DNA fortransfections,was

prop-agated in SF-21 cells or Trichoplusia ni (cabbage looper)

larvae infected peros (23).

Recombinant plasmids. The threereporterplasmids used, phcwt, pCAPCAT, and pETCAThr5, have been previously

described(20,25, 33). Briefly,thereporterplasmidscontain the AcMNPV promoter from the polyhedrin gene (for phcwt), the major capsid protein gene (forpCAPCAT), or

the proliferating cell nuclear antigen homologous gene, etl (for pETCAThr5), controlling the reporter gene encoding

chloramphenicol acetyltransferase (CAT). The enhancer-likeelementorhomologous region (hr5)ispresentupstream ofeachpromotersequence.

Subclones ofpBamC, a pBR322-based (2) plasmid con-taining the AcMNPV BamHI Cfragment (4.8 to 11.1 map units [m.u.]),were cloned into pBluescript (Stratagene, La

Jolla, Calif.). pBCPsB contains the PstI-BamHI fragment (7.35 to 11.1 m.u.), pBCBPscontains the BamHI-PstI

frag-ment (4.8 to 7.35 m.u.), pBCPsE contains the PstI-EcoRI

fragment (7.35 to8.65m.u.), pBCPsScontains the PstI-SalI

fragment (7.35 to 8.3 m.u.), and pBCSEcontains the

SalI-EcoRI fragment (8.3 to 8.65 m.u.). pBCNE contains a

NruI-EcoRIfragment (7.5 to8.65m.u.) comprisingthelef-1 ORFandtwosmall antisenseORFs. pBCHE, containingthe

HpaI-EcoRIfragment (7.9to8.65m.u.),wasconstructedto

synthesize a lef-1-specific probe used in Northern (RNA)

blot analysis.

DNA cotransfections and transient expression assays. Ap-proximately 2 x 106 SF-21 cells per 60-mm-diameter dish

were used per cotransfection by the calcium phosphate coprecipitation method (23). Each cotransfection mixture

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fresh complete TC-100 medium was added to each mono-layer, and the cells were incubated at 27°C for 24 h for

cotransfections containing pETCAThr5 or48 h for cotrans-fections containing pCAPCATor phcwtbefore harvesting.

CATreactions were performed as previously described (7, 27), using 1/10 of theharvested cell lysate perreaction.

Nucleotide sequencing. pBluescript plasmids containing

theregionbetweenPstI(7.35 m.u.) andSall (8.3 m.u.)were

used togenerate nested setsofdeletions, usingexonuclease IIIand mungbeannuclease asdescribedby Stratagene (La Jolla, Calif.)based ontheoriginalmethodby Henikoff(12).

Selected deletion clones, providing sequence information

from bothDNAstrands across the entireregion, wereused as double-stranded DNA templates and sequenced by the dideoxyribonucleotide chaintermination method (30), using

T7 and T3 primers purchased from Stratagene. Since the

sequence from the Sall (8.3 m.u.) site to the EcoRI (8.65 m.u.) site had been previously determined (22), only one

DNA strand of thisregionwas sequenced, and thejunction

at theSalIsite (8.3 m.u.) wasverified.

RNAisolation. For RNA studies, SF-21 monolayers (5 x 106 cells per 100-mm-diameter dish) were infected with

AcMNPVatamultiplicityof20 PFUpercell. The viruswas

allowedtoadsorb for1 hatroomtemperature,and timezero was defined as the time when the virus was removed and

incubation at 27°C started. The protein synthesis inhibitor cycloheximidewasaddedat100p,g/mltothecells for 30min

before infection and maintained thereafter. The DNA

syn-thesis inhibitor aphidicolin wasadded at5 ,ug/ml following adsorption.TotalRNAwasisolatedatselectedhoursp.i. by

theguanidinium isothiocyanate method(4).

Northern blotanalysis. Samples oftotal RNAl,g(15 per

lane) were denatured by glyoxalation (17), separated by agarose gel electrophoresis, transferred to a nylon mem-brane, and hybridized to a strand-specific DNA probe at

42°C. The probe, which was complementary to the lef-1 transcript, was made from single-stranded pBCHE DNA. The T7 oligonucleotide primer was annealed to pBCHE single-stranded DNA to synthesize a radiolabeled DNA

strandcomplementarytothelef-1 transcript, usingthelarge fragment ofDNApolymeraseI to extend the primer in the

presenceof[Ot-32P]dCTP (29).

Si nuclease protection oflef-l transcripts. Total RNA(30 ,ugperlane)wasusedforS1nucleaseprotectionassays(36).

To construct the plasmidused as aprobe for mapping the

3' end(s) of the lef-1 transcript(s), pBamC was digested

with NdeI, radiolabeled exclusively at one end with T4

DNA polymerase and [a-32P]dCTP, and redigested with

MscI togenerateafragment3.9kbpinlength with onlyone

radiolabeled end. The labeled fragment of interest was

purified by agarose gel electrophoresis and annealed to

total RNA in

formamide-piperazine-N,N'-bis(2-ethanesulfon-ic acid) (PIPES) buffer (80% deionized formamide, 40 mM PIPES [pH 6.4], 0.4 M EDTA) at 52°C. After S1 nuclease

digestion, theprotected fragmentsweredenatured, separated on a polyacrylamide-urea gel, and visualized by autoradi-ography.

Primer extension analysis. The oligodeoxyribonucleotide primer 5'-TCGACGCGCTTCTGCG-3', complementary to sequencesstartingattheSall site (8.3 m.u.) downstreamof thefirstmethioninecodon ofthelef-1 ORF,waslabeledwith [_y-32P]ATP (New England Nuclear, Boston, Mass.) andT7

BRL). A sequencing ladder of this regionwas generated by using the labeled primer in dideoxyribonucleotide chain termination DNA sequencing reactions.The extended prod-ucts were denatured, separated on a polyacrylamide-urea gel, and visualized by autoradiography.

Nucleotide sequence accession number. The GenBank

ac-cession number for the AcMNPV lef-I gene sequence is

L09723.

RESULTS

Experimental

strategy.

To identify genesinAcMNPVthat regulate different temporal classes of promoters, a plasmid containing the CAT reporter gene controlled by an early,

late, orvery late AcMNPV promoter was cotransfectedinto SF-21 cells with a series of cloned, overlapping fragments

comprising allof the AcMNPV DNA genome (Fig. 1D)(25)

toprovide all of theviral factors required for transcription.

Upon cotransfection of SF-21cells with the entirecollection

ofcloned viral DNAs and areporter plasmid, reporter gene

expression was expected from each temporal class of

pro-moter. However, omission of any one of the AcAMNPV

fragments which provides a function necessary for

expres-sion from the promoter tested should eliminate CAT gene

expression.

Identification of agenein the region between 4.9 and 14.7

m.u. required for vp39 andpolh promoter-mediated expres-sion.The reporterplasmids used, pETCAThr5, pCAPCAT, andphcwt, contain the CAT reporter gene fused to the early

etl(6), the late vp39 (32), and the very latepolh promoters, respectively. The etl promoter is dependent solely on the IE-1 gene product for expression in transient expression assays,but IE-1 is not sufficient for expressionfrom the vp39 or polh promoter (25).

Transfection of each reporter plasmid alone into SF-21

cells gave no CAT activity, but highlevels were observed

whentheplasmids werecotransfectedwith intactAcMNPV

DNA (Fig. 1A to C; compare lanes 1 and 2).Cotransfection

of each reporter plasmid with the overlapping library of

AcMNPV clones gave equal or slightly lower levels of

expression fromtheearly andlatepromoterstested (Fig. 1A

and B, lanes 3). CATexpressiondrivenby thepolh promoter

was much lower in cotransfections with the library than those with AcMNPV (Fig. 1C; compare lanes 2 and 3). Higher expression from thepolh promoter was observed if cells were harvested at 72 h postcotransfection, but the levels of expression with the library of clones were still lower than thatobserved with intact AcMNPV DNA under these assayconditions.

Removal of theIE15 clone, which containsie-1 from the AcMNPV clone library, abolished expression from all three temporal classes of promoter, as expected (Fig. 1, lane 16) (25). However, omission of several clones (i.e., BC5, PstH5, and HC10) from the library greatly diminished expression from the late and very late promoters but not the early promoter (Fig. 1, lanes 4, 11, and 13). Although omission of the IE15 clone gave no detectable levels of CAT activity, omission of BC5, PstH5, orHC10 gave very slight levels of CAT activity when autoradiograms were overexposed. Omission of several other library clones (i.e., PstHl,

pXmaB, and HK5) also diminished expression from the late andvery late promoters (Fig.1B and C, lanes 10, 14, and 15).

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---FIG. 1. CAT expression from early (A), late (B), and very late (C)AcMNPVpromoters in thepresence or absence ofdifferent regions of the AcMNPV genome(D). SF-21 cells were transfected withpETCAThr5 (A), pCAPCAT (B), or phcwt (C), and lysates were collected after 24hforpETCAThr5or 48 h forpCAPCAT and phcwt. Transfections werecarriedout with the reporter plasmid only (lane 1), intact wild-type AcMNPV DNA(lane 2), the entire overlapping AcMNPV clone library (lane 3), or theAcMNPV library lacking the specified clones (lanes 4 to 16). The acetylated products (Ac Cm) and unacetylated substrate (Cm) of each enzymatic reactionwere separated by thin-layer chromatography and visualized byautoradiography. (D) Locations of the cloneswithrespect to the AcMNPV genome are schematically presented aslines below theEcoRIrestriction map of theAcMNPV genome as reported by O'Reilly et al. (23). The solid lines indicate the regionknown to be covered by the clone; the broken lines at the ends of the solid lines denote the ambiguity of the endpoints of each clone.

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Ps

Ps

r-

'~~~~~~~~~~~-Ps E

IP

Ps S

pBCSE

B

0

r-l

S E

0

1kb

AcCm

** -Cm

BC5 clone BC5 subclone

1 2 3 4 5 6 7 8

BamC BCPsB BCBPs BCPsE BCPsS BCSE

FIG. 2. CAT expression from thepCAPCATreporterplasmid, usingsubclones of the AcMNPVgenomein theregionfrom 4.8to11.1m.u.

toreplace the BC5 cloneof theoverlappingAcMNPVclonelibraryincotransfections.(A)The subclones used intransientexpressionassays.

The numbers indicateapproximatemapunits in theAcMNPVgenome;restrictionenzymesitesareabbreviatedasfollows: B, BamHI; Ps, PstI;S,Sall;E, EcoRI. Onlyoneof theSalland EcoRI sites inthisregionis shown.(B)AssayinwhichSF-21 cellswerecotransfected with

pCAPCATand theentire AcMNPVlibrary (lane 1)orcellsweretransfected withpCAPCATandthe AcMNPVlibraryexcept for theBC5

clone andaBC5 subclonewasaddedasindicated(lanes2to8).Theacetylated products (AcCm)andunacetylatedsubstrate(Cm)ofeach enzymaticreactionwereseparated by thin-layerchromatographyandvisualizedby autoradiography.

This study focused on determining the gene(s) within the

BC5 clonerequiredforexpressionfrom the late andverylate promoters(Fig. 1,lane4). TheBC5 clonemapsbetween 4.9 and 14.7m.u. ontheAcMNPVgenome.

A clonecontaining the BamHI Cfragment ofAcMNPV, pBamC,which extends from4.8to11.1m.u. onthegenome,

was foundto substitute forBC5 in the transient expression

assay (Fig. 2B, lane 3). pBamC was then subcloned into

smaller fragments, which were tested by similar transient

expression assays for the ability to restore CAT activity

from thevp39promoterinthe absence of BC5. The subclone

pBCPsE (7.35 to 8.65 m.u.) was the smallest subclone capable ofsubstituting for BC5 (Fig. 2). Subclones ofthis plasmidcut attheSalI site, pBCPsS (7.35 to 8.3 m.u.) and pBCSE (8.3to8.65 m.u.),wereunabletoreplace BC5 (Fig. 2B, lanes 7 and 8). This finding suggested that a gene

necessary forexpression from the vp39 promoter spanned

the SalI site at 8.3 m.u. The same results were obtained

when thevery latepromoter reporter plasmid, phcwt, was used in transient expression assays with these subclones (datanotshown).

Nucleotide sequence of thelef-) gene. The nucleotide

se-quencecorresponding to theregion between 7.35 and 8.65

m.u. was determined in both directions. This region con-tainedseveral putative ORFs larger than 50 amino acids,as indicated in Fig. 3A. The largest ORF, designatedlef-1 for

late expression factor 1,was 266 codons and spanned the Sall site at 8.3 m.u.; since disruption of this site reduced

CATactivity (Fig. 2B,lane 7),thepolypeptideencodedby

this ORFwaslikelytoberequiredforvp39 expression. The three smallORFs contained withinpBCPsSwerenotable to reconstitute CAT activity in the absence of BC5 (Fig. 2B,

lane 7). To rule out the possibility that the partial ORF downstream oflef-1 wasnecessaryforexpressionfromthe vp39promoter, asubclone(NruIat7.5m.u.toEcoRIat8.65

m.u.)disruptingthispartial ORFwasusedto substitute for

BC5 in transient expression assays. This clone, pBCNE,

was able to complement CAT activity (data not shown), indicating that the lef-1 ORF and not the flanking and

overlappingORFswasinvolved invp39- and polh-mediated

expression.

The nucleotide sequence of lef-1 is shown in Fig. 3B.

Comparison ofthe predicted polypeptide sequenceoflef-1 with those in published data bases revealed no obvious homologyto anyother knownpolypeptide. Analysis of the

derived amino acid sequence of lef-I for known protein

sequence motifs revealed the presence of a nucleoside triphosphate-binding site similarto the ATP-binding site of

severalproteinkinases (15). Itconsists ofatriad of appro-priately spaced glycines followed by a lysine 21 residues

downstreamneartheC terminus(Fig. 3B, amino acids 237to

263).

Northernblotanalysisoflef-1 transcription.The temporal regulation oflef-I transcriptionwasexamined by Northern

blot analysis. SF-21 cells were infected with AcMNPV,

harvested at various times p.i., and lysed, and total RNA wasextracted. Aprotein synthesis inhibitor, cycloheximide,

anda DNAsynthesis inhibitor, aphidicolin, wereadded to pBamC rB

B pBCPsB

pBCBPs rF B pBCPsE

pBCPsS

B

B

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A

Ps Be (7.35)

Nr H

l

100bp

< LEF-1

Bs

I

N S Be

(8.3)

E (8.65)

ILD

IED

i EGT

vMLB

EcoRI

TGACATTTTTCGGCAAGGGCTTCAATATACACTTTGTACACTATATGGTGGCTGTAAGCTGGCGTAGGAAACACGGCCAATATATTGGCCGCATTTACAGCAGTAAGCGTAGACAGCAGT

GCAAGCCAGCAGAGAATAGTCATTTTGCTTCAAACCGAATAACTGTACTGGTAATTTGATTATGTAACAAATTTTAAAGGGTGACCTTTTTATAGCCAAAAATTAACACAATCGAGGTCA

SallI

AGTTCAAAGGGCAACATGTTAGTGTGCAATTATACGCAGAAGCGCGTCGACATGATGTGGGACGCCATTGCGTACAACGACAGCCGCAAGTACGCGTTCATGACGGTCAACGCGCGCTGG

M L V C N Y T Q K R V D M M W D A I A Y N D S R K Y A F M T V N A R W

ATTCACGCCGACAGATATTTTGATACCTCCGCACAATTGTATAGTTATATTGTGCAAAACAAAGTGTCCGACGTGCACGTCAAACCGTTGGACGACGGCGGCGGCAGGGAATGGGTCGTA

I H A D R Y F D T S A Q L Y S Y I V Q N K V S D V H V K P L D D G G G R E W V V

GACGCCGATTACAAAAATTATGTTGACGAACACGATTTAATGCTGAAAATTTACATTGGCGCCACGGCGTTTCT'GTTGTTTTACACGGAAGAGAACGTGTCAAGAGTCATGTATACCGGC

D A D Y K N Y V D E H D L M L K I Y I G A T A F L L F Y T E E N V S R V M Y T G

AACCGTGGATTTCATTGTGGTTAAAATTCACCGACAAGTTTAAAATCACGTCCGCTCAAAATGTTCGCGTGCATCGGTACAAGGCTTTCGAGAAACCTGCAAAGTTGGACAGTGATTAC

N R G F H L W L K F T D K F K I T S A Q N V R V H R Y K A F E K P A K L D S D Y

ATTCAGCCGGGCAGTTTTGCGCATTGTGTGAGAGAGGCCGTACGTTTGTACGTTCCGCATATGCAAGATTCAAACTTGGACGCGCTCACGTTGCAGTATTGGCCGGACGTGGACAGGGAT

I Q P G s F A H C V R E A v R L Y V P H M Q D S N L D A L T L Q Y W P D V D R D

ATTTTTTGTAACGTTAACAAACAAATACGCGCACCGTACAGCTATAATTATAAGGGAACGAAATTTTCTCGTTGTATAACAAAGGAATTGCTAGACAAGCTAAAGCAATGCTATCCTGGT 10180

I F C N V N K Q I R A P Y S Y N Y K G T K F S R C I T K E L L D K L K Q C Y P G 235

TATGGAACTGGTGGATGTGGTCCGGTGACAACGACAACGACGCCATCGCCGCCGAAGATCGGTTCAATGCAGACGACTACAAAAAGTACCACATAAACGCCCAACAATGGTCGCACATCG

Y G T G G c G P v T T T T T P S P P K I G S M Q T T T K S T T .

TTAAATGGGACTCATTCAAATGCAACACGCACAGTTTCAAGTACAGATACGTGCACAGCGACACGAACGCAAAATGCTACAATGTGATAGATTTTGCAAAGGTCTTGAAATTGCGCACG

1200 266

1320

ACGACATACTTGATTGCAATTGGGACGGCGATCAAGTTTACCATTTAAACGAAATTGTTTTTCACAAACAGAGATCCAAACGCGATCTCAACTCGTTGGGCGCATTGTTCGCGACCAAGC 14140

ACGGGTTGTTGGAAATTTTGATGCGGTTAAATTTTGCCAACAAAAGCAACGCGTTGCTGCACATTCAAACTGAAGGCGAGCGTGATGATTTGCGCGACAAGATTGAATCTGTTTTAAAAC 11560

ATGTAAAGAAACTGAATGCAAACAGCGAAAAATTTATGGTCACCCACGAGACGTTCAAGAACGAGGTGGGCAACAGGTTTGAGCAGTTTGAATTGCGTTTGCACGAACTCGACGCCAAAC 168 0

TTAACATGCTGCA 1694

PstI

FIG. 3. Distributionof ORFsand nucleotide sequenceof theAcMNPV region from 7.35 to8.65m.u. (A) Distribution of ORFs in the

PstI-EcoRI (7.35to8.65 m.u.) subfragment of AcMNPV. EGT is theecdysteroid UDP-glucosyltransferasegene(21); the barlabeled vMSLB

indicatesone endpoint of the deletionpresentinthe mutantvirus vMSLB (24).Abbreviations for restriction enzyme sites:Ps, PstI; Be,

BstEII; Nr, NruI; H, HpaI; N, NarI;S, SalI; Bs, BstXI; E, EcoRI. (B) Nucleotidesequenceoflef-I from 8.65to7.35m.u. and the derived polypeptidesequence.The thickand thinarrowsindicate positions of the 5' ends mapped for thelef-I andegttranscripts,respectively. The

brokenoverline shows thepositionof thetranslationalstartcodon for EGT. Thenucleotides complementarytotheoligonucleotide used in primerextensionanalysisareoverlined. Theamino acidsconstituting the putative nucleosidetriphosphate-binding site haveanasterisk. Key

restriction endonuclease sitesareunderlined and indicated.

somecellstoassesstherequirementsforproteinand DNA

synthesis forlef-1 transcription. The strand-specific probe

used is represented byan arrow inFig. 4A. Atranscript of

approximately 1.8 kbwas observedat 3, 6, 9,and 12 h p.i. (Fig. 4B). Thetranscriptwasalso observed in thepresence ofcycloheximide. In thepresenceofaphidicolinat12hp.i.,

thelevels of thetranscriptwerelow. Thistranscriptwasnot observed at very late times (e.g., 24 and 48 h p.i.) or in mock-infected cells.Thus,lef-1 appearstobeanearlygene, and lef-1 transcription occurs in the absence of protein synthesisorDNAreplication. Upon longerexposuresof the

autoradiogram,aminortranscriptofapproximately2.8 kb in lengthwas observed at early times and at 12 h p.i. in the

presenceofcycloheximide;wehavenotfurtherexploredthe natureof this early transcript.

Mapping the 5' termini oflef-l transcripts. The 5' termini of the major lef-1 RNAwere mapped by primer extension analysis. A16-ntoligomer complementaryto thelef-1

tran-scriptandcoincidingwith theSallsiteat8.3m.u. (Fig. 3B) was5' end labeled and annealedtototal RNA isolated from uninfected SF-21 cells or cells infected with AcMNPV harvestedatvarious hoursp.i.Theannealedoligonucleotide was then extended with reverse transcriptase, and the

ex-tended products were analyzed and compared with a

se-quenceladder of thesameregioninadenaturing

polyacryl-amidegel.Twomajorextensionproductsof117 and 119 nt

B

120

240 360

480 35 600 75 720 115 840 155 960 195

I I

I I

I I I I

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[image:5.612.69.572.79.506.2]

J

H N S Be

(8.3)

Bs E

(8.65)

mi 3 6 9 12 12C12A 24 48

11.20-

9.16-

7.13-

5.09-4.07-_

3.05-2.04'- it

t

1.640

1.0

FIG. 4. Northern blot analysis of lef-1 transcripts. (A) The

strand-specific DNAfragment used for Northern blot analysis is

illustrated byan arrowunder theopen arrowrepresentingthelef-l

ORF. Numbers indicateapproximate mapunits, and letters show

key restriction sites as designatedinFig. 3A. (B)Total RNAwas

extracted from SF-21 cells either mock infected(mi)orinfected with AcMNPVatthe timesindicated inhoursabove the lanes. RNA from

samples treated with cycloheximide(12C)oraphidicolin(12A)was

extractedat12hp.i. Molecular weightmarkersareindicatedtothe left inkilobasepairs.

inlength, correspondingto nt292and294ofFig. 3B,were

observed (Fig. 5). These virus-specific extension products wereobserved forRNA isolatedat3, 6, 9,and 12 hp.i.inthe presenceor absenceofcycloheximide. The sameextension

products werealso observed at 12hp.i. in thepresence of

aphidicolin, albeit atmuch lowerlevels. Atvery latetimes (24 and 48 h p.i.), the extension products of these RNAs

weregreatly reduced.Usinganoligonucleotide

complemen-tary to sequences downstream of the fourth methionine of thelef-I ORF (i.e.,aprimerspanningnt473to488;Fig. 3B),

the only extension products observed also mapped to the samesitesasdescribed above,thusrulingoutthepossibility

that thelef-1 transcript(s) initiated within sequences corre-sponding to the predicted LEF-1 N terminus (data not

shown).

The 5' ends of the transcripts were also mapped by S1 nuclease protection analysis using a probe uniquely end labeledatthe 5' endofthe Sall site shown in Fig.3(datanot

shown). Twofragments 117 and 119 ntlong, corresponding againto nt292 and294 in Fig.3B,wereprotected byRNA

frominfected butnotfrom mock-infected cells.

Mappingthe 3' termini oflef-I transcripts. The 3' end of

themajorlef-1 transcriptwaslocalized by using afragment

3'end labeledattheNdeI siteandextendingtosequencesin

theplasmidvector(Fig.6A). Fragments 1.4to1.25kbp long wereprotected at6through 12 h p.i. andat 12 hp.i. in the presence of cycloheximide (Fig. 6B). The amount of

pro-3'

A

am

_A

Ut___

T --__a

_Tm _-

-A / :Ct

-A M

A /"a

5' _

JL..

.._

3=__'..

FIG. 5. Primer extension analysis of lef-1 transcripts.An oligo-nucleotideprimer (overlined in Fig. 3B) coinciding with theSallsite at8.3m.u. was5' endlabeled, annealedtototal RNAisolatedfrom mock-orAcMNPV-infected SF-21cells, andextended withreverse

transcriptase. The same primerwas used to derive a sequencing ladder(G, A,T, C)fromaplasmidcontainingtheprimer-extended region. Lanedesignationsarethesameasdescribed forFig.4B.The

sequencespanningthetranscriptionalstartsite isrepresentedtothe leftof the sequencing ladder, and the mapped 5' ends of the RNAs

areindicatedbyarrows.

tectedfragmentwasreduced at 12 hp.i. in thepresenceor absence ofaphidicolin.Similarexperimentsusingafragment 3' end labeledattheSalI site (8.3 m.u.)andextendingtothe

PstI site(7.35 m.u.)and then intoplasmidvectorsequences confirmed thatlef-1 transcriptsextendbeyond the PstI site at 7.35 m.u. (data not shown). Extension beyond thePstI site is also consistent with the lack of a polyadenylation

signalinthesequenced regiondownstream of thelef-1ORF. The 5' and 3' mapping dataare consistent with the 1.8-kb lef-1 RNAobservedin Northernblots.

DISCUSSION

Werecently developedanassaydesignedtoidentifygenes

necessaryfor late andverylateAcAMNPVgene expression (25). Ourassay uses alibrary ofclonescovering the entire

AcMNPV genome that can activate expression from a

re-porterCATgeneunderthe controlof thelatevp39promoter

or the very late polh promoter. The assay is based on deletinganindividualclone from thelibrary anddetermining

the smallest region(s) which can functionally substitute to activatereportergeneexpression. Thus,theassaymarkedly differs fromaprevious approachwhichusedaCATreporter gene under the control of a putative late promoter of a relativelyuncharacterizedgeneandmonitoredCAT expres-sion after adding only limited regions of the AcAMNPV

genome (11). One of the two ORFs mapped by this latter

Ps Be

(735)

Nr

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khp

A

11I

BSM ,4-8

B

@LEF Zii1EGT >

l

I I

E E Ps Nd S E M S

f7e.351 8.311.651

mi 3 6 9 12 12C 12A 24 48

3.05 2.04

-I.64

I.02

-0.52

-FIG. 6. Mapping the 3' end of the lef-1 transcript. (A) The probe

usedtomapthe3' endof thelef-I transcript. Thearrowindicates

the 5'-to-3'directionoftheprobe, and the asteriskrepresentsthe position of the label. Key restriction sitesareabbreviatedasin Fig. 3A.Additional sites: M, MscI; Nd, NdeI. Keymapunitsareshown

inparentheses belowtherestrictionsite.The size anddirection of

thelef-1 andegtORFsareindicated above therestrictionmap. (B) S1 protection analysis ofthe3'end of thelef-l RNA. Total RNA

was isolated from uninfected and AcMNPV-infected SF-21cells,

annealedtotheend-labeledprobe,and treated withS1 nuclease,and

protected fragmentswereseparated in a polyacrylamide-urea gel.

Lanedesignationsarethesame asdescribed for Fig. 4B. Thearrow

totheright indicatesthemigration of the untreated labeled probe.

Molecularweightmarkersareindicatedtothe leftinkilobasepairs.

technique was subsequently found to be nonessential for

virus replication in vivo and in vitro (24). The use of well-characterized promotersofgenes ofknown functional importance provides assurancethatwe are locating

neces-sarylate expressionfactors.

We have found that expression from the vp39 andpolh

promoters inourassayrequiresanumber of AcMNPVgenes distributedthroughoutthegenome. Inaprevious study, we defined threegenesinthe 83.7-to7.5-m.u.regioninvolved in lategene expression (25), and in thisstudy, we extendour analysistoa clone from the library thatflanksthis region.

One gene, which we have named lef-1, was found to be

required for substantial levels ofexpression from the late

vp39and theverylatepolhpromoters.

The derived polypeptide sequence of lef-1 contains 266 amino acids andpredictsa31-kDapolypeptide. TheLEF-1 sequence has no apparent homology to other published

sequences. The C terminus contains a nucleoside triphos-phate-bindingsite motif similartothetype found inprotein

kinases (G-X-G-X2-G-X15_20-K). The regioncontaining this sequence motif is rich in serine, threonine, andproline.

lef-1istranscribedas anearly1.8-kbRNA whichappears

to be a bicistronic mRNA, with a second ORF located

downstream andoverlapping thelef-1 ORF. Levels of the 1.8-kb transcript begin to decline during the late phase of

infection (e.g., 12 h p.i.) and are very low by 24 h p.i. This

declinemaydepend on early protein synthesis; at 12 h p.i.,

lef-l RNA is more abundant in the presence than in the absence of cycloheximide. The very low abundance of lef-1 RNAin the presence of aphidicolin at 12 h p.i. may be due to amorerapid decline oflef-IRNAin the absence of viral DNA replication and late gene products or the failure to expresslef-1 efficiently in the absence of DNA replication.

Using primer extension andSi nuclease protection anal-yses, we mapped the putative 5' ends of the lef-1 transcript

to sites 82 and 84 nt upstream of the translational start codon. The adjacent egt gene is transcribed in the opposite

direction from lef-1 (22), and the 5' ends of the egt RNA overlap with the 5' end(s) of the lef-1 RNA (Fig. 3B).

Overlappingantisense RNAs may result in inaccurate

map-ping of 5' termini by S1 nuclease protection and primer extension analyses (19). If this is also the case here, then the

lef-1 5' end(s) may extend further upstream than mapped here. Closer examination of the region within the egt ORF reveals the presence of two CAGT sequences (nt 221 and 237;Fig. 3B) with a TATA box at nt 201. A number of other earlybaculovirus genes have been reported to initiate at a

CAGT sequence usually located approximately 20 to 30nt

downstream ofaTATAbox(1, 5, 9, 10, 31). There areno

ATGcodons between the two CAGT sequences and the lef-I 5' endsthatwehave mapped. However, thereisanATG in the RNA leader atnt302 which is immediately followed by

aterminationcodon.

Atthe presenttime, we assume thatlef-1is essential to the virusin vivo. However, anAcMNPVmutant isolatedafter

serial passage through Manduca sexta larvae, vMSLB,

containsadeletion thatstarts at nt360 upstream of thelef-1

ORF(Fig. 3) and extendsthroughegt (24). Thus, thelef-1

ORF remains intact in vMSLB, but the promoter region including the putative transcriptional start sites has been

deleted;itispossiblethatlef-1is stilladequatelytranscribed invMSLB. Theprecise role of lef-Iin late geneexpression

remainstobe determined; genesidentifiedinthisassay may

include genesrequired forexpressionfromearly promoters other than the etl promoter, genesinvolvedin DNA replica-tion, and genesdirectlyinvolved in late gene expression.

This transient expression assay is clearly apowerful tool fordefiningtheAcMNPVgenesinvolved inexpressionfrom thevp39andpolh promotersand may be extendedtostudy

other viral gene promoters as well. We are presently at-tempting to

identify

all ofthe AcMNPV genesrequired for

expressionfrom thevp39 andpolh promoters.

ACKNOWLEDGMENTS

We thank D. R. O'Reilly for providing plasmids pBCPsE and pBCPsB, andwethankS.Kumar forpBamC.

This workwassupportedin partbyPublicHealth Servicegrant

Al 23719 from the National Institute of Allergy and Infectious Diseases.

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