Identification, sequence, and transcriptional mapping of lef-3, a baculovirus gene involved in late and very late gene expression.

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JOURNALOFVIROLOGY,Sept. 1993,p.5260-5268 0022-538X/93/095260-09$02.00/0

Identification, Sequence, and Transcriptional Mapping of lef-3,

a

Baculovirus Gene

Involved

in

Late and

Very

Late Gene

Expression

YONGHONG LI,' A. LORENAPASSARELLI,1ANDLOIS K. MILLER'2*

Departmentsof Genetics' andEntomology,2*Universityof Georgia, Athens, Georgia 30602 Received 2 April 1993/Accepted 4 June 1993

Atrans-actinggenerequired for lateviralgeneexpression in transient expressionassays wasidentifiedin the genome ofAutographa

californica

nuclear

polyhedrosis

virus. A genomic library of A. californica nuclear

polybhedrosisvirusDNAlackingaclonespanning the regionfrom43to48mapunits(mu)wasunabletoactivate geneexpressionfromareporterplasmidwhenthereportergene wasdriven byabaculovirus lateor verylate promoter in transient expression assays. The genomic region responsible for activating reporter gene

expressionwasfurthermappedto43.4to45.2muof the viralgenome. Thenucleotidesequenceof thisregion

wasdetermined and showntocontainseveral smallopenreading frames (ORFs) andonemajor ORF, named

lef-3, for late expression factor 3. The kf-3 ORFwaspredictedtoencodeapolypeptideof 385 aminoacids and

withamolecularmassof44,529daltons. No homologtothelef-3 ORFwasfoundinexistingdata bases. Further

analysis showed that only the lef-3 ORF in the region from 43.4 to 45.2 mu was necessary for late gene activation in theassaysused.Thetemporal regulation oflef-3 transcriptionwasstudied by Northern (RNA) blot hybridization; lef-3 wasfound tobe anearly genethatwastranscribedprimarily as a 2.0-kb mRNA. Primerextensionanalysisand Si nucleaseprotectionassaysrevealed that lef-3 transcription initiated about 280

bpupstreamof the firstATG codon andterminatednear apolyadenylation signal, 130bpdownstream of the

lastcodonof thekf-3 ORF.

Although baculoviruses are now widelyused asvectors for theexpression ofa diversityofheterologousgenes, the

fundamental nature of the RNA polymerase(s) and other factorsinvolved intranscriptionfromlate andverylateviral promoters remains largely unknown. On the basis of the differentialsensitivityofearlyand late baculovirus

transcrip-tion to a-amanitin in nuclear run-on assays (10, 16), it is

generally thoughtthatanRNApolymerase switching mech-anism isinvolved in the transition between thesetwophases

oftranscription. This view is furthersupported by biochem-ical evidence of avirus-induced RNApolymerase activity appearing bytheonsetof the latephaseof infection(8, 43)

andbymolecular evidence that theprimarydeterminantsof late andverylatepromotersarelocatedatthetranscriptional initiation site, invariablyaTAAGsequence, andwithin the sequencesspecifyingtheuntranslated leaderregions (24, 25, 32, 41, 42).Thenatureof thenewpolymeraseand thenature of the factors thatrecognizeordistinguishtheseunusual late andverylatepromotersremaintobe determined.

During infection, the transition from the early to the late

transcriptional phasesisdependentonproteinsynthesisand DNAreplication.In thepresenceofaphidicolin, aninhibitor of both hostand viral DNApolymeraseactivities(23), early

butnotlateorverylategene transcription isobserved (34, 36).Amutantdefective in the viralproliferatingcellnuclear

antigen (pcna) homolog is delayed in late gene expression (6),andamutantdefective in the DNA helicase motif ofp143

is blocked in late gene expression (21). The basis for this

dependence of lategene expression on DNAreplication is

notknown,noris it known whetherthisdependence would also be observed in transientexpression assayswith naked viral andreporterDNAs. Transientexpressionofareporter

*Corresponding author.

gene under the control of late and very late promoters requirestheexpressionofmanyviralgenes(27, 28).For the

polyhedrin promoter, expression fromreporterplasmids is

independent of homologous repeat (hr) sequences (33), whichhave been assigned the functions of enhancers of early

geneexpression (11, 13)andoriginsof DNAreplication (17, 29). Expression from polyhedrin promoter-based reporter plasmidsis also knowntorespond authenticallytopromoter mutations when theplasmids are cotransfected with intact

Autographa californica nuclear polyhedrosis virus (AcM NPV) DNA in transientexpression assays(25, 33).

Werecently developedamethod foridentifyingAcMNPV genesthatarerequiredintransientassaysfortheexpression

ofareportergene underthe control of the latevp39 (major capsid protein gene)orverylatepolyhedringenepromoters (27). The method uses an overlapping set (library) of 12 clonesthatcoverstheentire AcMNPVgenomeand thatcan substitute for intactAcMNPV DNA intrans-activatingthe

expression ofreporter genes under late andvery late pro-moter control. Removal of some of the clones from the

libraryresults in thereductionortotalelimination of expres-sion fromthereporterplasmids. By removingonecloneata time and substituting smaller plasmid clones for thelarger clone, we found it possibleto identify preciselythe genes requiredfortrans-activating reportergene expression.

Thus far,we have identified four genes,ie-1, ie-n, lef-1, andlef-2,thatarenecessaryand sufficienttosubstitute for 2 of the 12library clones, coveringtheregionfrom 83.7to8.7 map units (mu) of the AcMNPV genome, in supporting

transient expression from late andvery late reporter

plas-mids (27, 28). ie-1 and ie-n were previously identified as

genes involved in the trans-activation of some early viral promoters intransient expression assays (3, 12, 14),sothe role ofthesetwogenesin the transientexpressionassayfor late gene expression may simply be toactivate earlygene

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LATE GENE EXPRESSION FACTOR 3 OF AcMNPV 5261

expression. However,lef-1 and lef-2 have no apparent effect on expression from the early pcna (also known as eti) promoter,which serves as a control in our assays, and are therefore more likely to be involved in late gene expression as components of viral transcriptional or replication com-plexes. In this study, we used the transient expression assay forlate expression factor identification to define a third gene located between 43.4 and 45.2 mu of theACMNPVgenome. The sequence and transcriptional regulation of this gene

(lef-3)weredetermined.

MATERIALS AND METHODS

Cells and virus. Spodoptera

frugiperda

IPLB-SF21

(SF-21) cells (39) were used as host cells for propagating AcM-NPV; the L-1 variant of AcMNPV (20) was used as the wild-type strain. The cells were maintained as described before (26) in TC100 medium (GIBCO BRL, Grand Island, N.Y.) supplemented with10% fetal bovine serum and 0.26% tryptosebroth.

Transient expression assays. SF-21 cells(2 x 106cells per 60-mmplate)werecotransfectedwitha reporterplasmidand either AcMNPV DNA or clonedsegmentsrepresentingall or

most of the viral genome. The reporter plasmids were

pETCAThr5 (27), pCAPCAT (37), andphcwt(33),in which the chloramphenicol acetyltransferase (CAT) gene was un-der thecontrolofearly

(eti),

late(capsidprotein genevp39),

and very late (polyhedrin gene) viral promoters, respec-tively. For each transfection, 2 ,g of the reporter plasmid wascombined with0.5 ,g of viral DNA or 0.5 ,g of each of the clones from the AcMNPV library. When one of the cloned DNAswasomitted, 0.5 pgofplasmidoran equiva-lentamountofagel-purifiedDNAfragmentwassubstituted inthe transfection mixture. The total amount of DNA used in eachcotransfection was 8.0pg; salmon sperm DNA was used tobalance DNA concentrations whenever necessary. The transfection was performedby the calcium phosphate method (26). The cells were collected 24 h after transfec-tion for the early promoter and 48 h after transfection for both the late and the very late promoters andweredisrupted

bythreerepeatedfreeze(liquid nitrogen)-thaw (37°C)cycles. Cell debris was pelleted, and 1/10 of the supernatant was

used to assayfor CATactivity as described previously (9,

33).

DNA cloning and sequencing. The DNA fragment

corre-sponding to theAcMNPV genome from the EcoRI site at 43.4 mu to theSstI site at 45.4 mu (see Fig. 2A) was cloned into thepBluescriptSKvector(Stratagene,LaJolla,Calif.).

TheEcoRI-MluIfragmentof thisclone was then subcloned into the EcoRI and SmaI sites of thepBluescript KSvector

(Stratagene) byfillingin theM1uI site of the fragmentwith the large fragment of DNA polymerase I (Klenow) and

ligating the fragment to the vector. The resulting clone, pSDEM2,wasusedtogeneratetwo setsof nested deletions

with exonuclease III and mung bean nuclease(15) byuseof

the

KpnI

orSstIsitesasdeletionblockpoints,inaccordance with protocols recommended by Stratagene. pSDEM2 and the setsof deletion cloneswere sequencedfrom the T3 and T7 primer sites by the dideoxynucleotidechaintermination

method (35)with asequencingkit (US Biochemical, Cleve-land, Ohio) and double-stranded DNA plasmid templates

(38). Sequenceswere compiled and analyzed byuse of the programsof Pustell and Kafatos(31)and the merger program ofIntelliGenetics. The deduced openreadingframes(ORFs)

within thesequencedregionwere comparedwith the

trans-lated sequences from GenBank (release 73) and U-EMBL (release 31) by use of the FASTA program (30) and the BLAST (1) network service at the National Center for Biotechnology Information.

RNA isolation. Total RNA was isolated from mock- or AcMNPV-infected SF-21 cells at various timespostinfection (p.i.) by the guanidinium isothiocyanate method(7).Avirus inoculum(10PFU percell) was allowed to adsorb to SF-21 monolayers (5 x 106 cells per 100-mm plate) at room temperature.The inoculum wasremovedand replaced with fresh medium after a 1-h adsorption period. The infected cells were then incubated at 27°C; the beginning of the incubation was considered zero time p.i. For inhibition of DNA synthesis, aphidicolin (5

,ug/ml)

was included in the medium added to 0 h. For blocking ofprotein synthesis,

cycloheximide(100 ,ug/ml)wasaddedtothe medium 30 min

before inoculation.

Northern (RNA) blot

analysis.

Total RNA (10 ,ug) from mock- or AcMNPV-infected cellswas denatured by glyox-alation (22), electrophoretically separated in 1.0% agarose, and transferred to Zeta-Probeblottingmembranes (Bio-Rad Laboratories, Richmond, Calif.). The 1-kb DNA ladder

(GIBCO BRL)usedasthe size markerwasalsoglyoxalated.

Blots were hybridized at 50°C to a strand-specific RNA

probe(hybridizationbuffer:50% formamide,1.5xSSPE[1x

SSPEis 180 mMNaCl, 10 mMNa2HPO4,1 mM

EDTA],

0.1 mg of tRNA perml, and 0.5% salmon sperm (DNA). For construction of a lef-3-specific probe, deletion plasmid

pKHD31, generated for sequencing of the lef-3 gene, was

linearized with ClaI and transcribed in the presence of

[32P]UTP

with T3 RNApolymerase.

Primerextension

analysis.

Asynthetic23-mer

oligonucle-otide (5'-GCTCATCAATTIACCACTAATCC-3') near the Nterminus oflef-3 was radiolabeled atthe 5' end with T4

polynucleotide kinase, annealedto10p,g of totalRNA,and extended withMoloney murine leukemiavirusreverse

tran-scriptase under the same conditions as those described earlier(6). Theextensionproductswere resolvedon an8% polyacrylamide-urea gel along with a DNA

sequencing

ladder generated from plasmid pSDEM2 by use of the

radiolabeledoligonucleotide primerdescribed above. S1 nuclease protection analyses. The termini oflef-3

tran-scriptsweredeterminedby S1nucleaseprotection analyses (40) with 10,ug of total cell RNA per reaction. The DNA

fragment used as a probe to map the 5' end oflef-3 was

prepared bydigesting plasmid pKHD31 (one of the nested deletionplasmids generatedforsequencingof thelef-3 gene)

with ClaI, dephosphorylating the ends with calf intestinal

phosphatase, radiolabeling the 5' ends with T4

polynucle-otide kinase, and recleaving the plasmid with SstI, a site

within the vector. The resulting 984-bp fragment was

iso-lated andgelpurified.The DNAfragmentusedtomapthe 3' end oflef-3was madeby

digesting plasmid

pSDEM2with

ClaI, radiolabeling the 3' ends with the Klenow DNA

polymerase fragment, recleaving the plasmid with EcoRI,

and gel purifying the resulting 1,408-bp fragment. DNA-RNAhybridizationwascarriedoutwith80%formamide-40 mMpiperazine-N,N'-bis(2-ethanesulfonic acid)

(PIPES)-0.4

MNaCl-1mMEDTAat50°Covernightfor 5' mappingand 60%formamide-40mMPIPES-0.4 MNaCl-1 mM EDTAat 52°Covernightfor 3' mapping.

Nucleotide sequence accession number. The sequence

re-ported in this paper was

deposited

in GenBank under the accession number L18873.

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FIG. 1. LocalizationtoPstH5 ofthegene(lef-3) requiredfor late

geneexpression of AcMNPV. (A) Restrictionmapof theAcMNPV

genomefrom 33.0to57.0muandkey restriction sitesintheregion:

H, HindIll;P,PstI;S, SmaI; Bg, BglII.mu aregivenunder the lines

representingthe AcMNPVgenome, and below themu are arrows

representingthepositions of knowngenes.The three overlapping genomicclones of thelibraryin thisregionof thegenome, PstHI, PstH5, and HC9,areindicatedby bars, with theopen area

repre-sentingtheregionknowntobe included in the clone and the shaded

areas representing additional regions that may be included. (B) Autoradiogram of CAT transient expression assays. SF-21 cells

were cotransfected with a reporter plasmid (pETCAThr5, pCAP CAT,orphcwt)and additional viral DNAasindicated above each lane: wt, wild-type AcMNPV DNA; L, entire library of cloned

overlapping AcMNPV DNA fragments; L*, ACMNPV genomic library lacking PstH5;0,reporter DNAonly. Cm, chloramphenicol;

AcCm,various forms ofacetylated chloramphenicol.

RESULTS

Identification of late expression factor gene 3 Ief-3. The reporter plasmids and clone library used in the transient assays to define AcMNPV genes involved in late gene expression were described previously (27). In brief, three differentreporterplasmidswereused. All three containeda

portion of the hr5 sequence and the CAT gene under the control ofaviral promoter. Theearlypromoterof theviral pcna (also known as eti) homolog (6) was the promoter driving CAT expression in reporter plasmid pETCAThr5 (27)and servedessentiallyas acontrolinthese experiments; it isdependent onie-1 expression and is stimulated byie-n

expression (28). The vp39 promoter, including 462 bp up-streamof thetranslationalstartcodon(36, 37), servedasthe

late promoter driving CAT expression in reporter plasmid pCAPCAT,and thepolyhedrinpromoterservedasthevery late promoter driving CAT expression in reporter plasmid phcwt (33).

CATactivitywasobtained for all three reporterplasmids

FIG. 2. Finemapping of lef-3 withinPstH5.(A) Diagram ofthe SmaI-D fragment(40.3 to49.4mu) clonedasplasmid pXmal7 (a)

and the derivedsubfragments (btoi).Therelative CATactivity for each transfection with the plasmidor asubfragment isgiven as a

plusor aminus, meaning that CATactivitywascomparabletothat of thepositiveorthenegative control, respectively. Restriction sites were SmaI(S),PstI(P),EcoRI(E), BglII (Bg), SstI (SI), SstII(SII), BamHI(B),SphI(Sp), MluI (M),andHpaI(H). (B) CATassaysfor cotransfections with pCAPCAT and the various DNAfragments, indicatedasletters above the lanes. The lanes labeledL - PstH5 (lanes 2to11) indicate that PstH5wasomittedfrom these transfec-tions. The lowercase letters above each lane (a to i) referto the plasmidorDNA fragmentshownin panelA and usedto replace PstH5.Lane 2(0)containsnoadditionalDNAsupplement. Lane 1 (L) contains the entire library of overlapping AcMNPV DNA clones.AcCm and Cmare asdefinedin thelegendtoFig. 1.

cotransfected with either wild-typeviral DNAor the

com-pletesetofoverlappingAcMNPV clones(Fig. 1B,lanes1, 2,

5, 6, 9, and10). Thus,aspreviously reported(27),theability

ofwild-typeAcMNPV DNA to trans-activate the reporter

plasmidscould bebasicallyreconstitutedbysubstitutingthe entire set of viral DNA fragments for intact viral DNA.

When the PstH5 clone,which extends from

approximately

37to50mu(Fig. 1A),wasomitted from thecotransfections,

CATexpression from the earlypromoterwas not

substan-tially affected (Fig. 1B, compare lanes 2 and 4). However, the omission of the PstH5 clone from the cotransfections with the reporterplasmids carryingthe CAT gene under late and very late promoter control substantially reduced CAT

expression (Fig. 1B, compare lanes 6 and 8 and lanes 10 and

12). Thus,atleastonetrans-activatingfactor necessary for late and very late gene expression in these assays was

present exclusively in the PstH5 clone. The PstH5 clone

substantially overlaps two other clones of the genomic library,PstHlandHC9; theregion uniquetoPstH5extends fromapproximately43to48mu (Fig. 1A).

To delineate further the region containing the

PstH5-i

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FIG. 3. Distributionof translational termination codons and sequence of theregionfrom 43.4to45.2muofAcMNPV,containinglef-3. (A)Restriction map of theregion sequenced. Stopcodons within all sixpossible readingframesareindicated below the mapbyvertical lines. Thelef-3ORF extends in thecounterclockwise direction.(B)Nucleotide sequence andpredictedamino acid sequence for the AcMNPVlef-3 ORF. The nucleotide sequence fromMluI (45.2 mu)toEcoRI(43.4mu)ispresentedwithnumberingtothe left of the first nucleotide in each line. Thepredicted amino acid sequence forlef-3is shown below the nucleotide sequence with the standardsingle-lettersymbolfor each amino acid. CAGT motifsnearthe N terminus oflef-3arenotedwithacolon(:)abovethe sequence. The actualtranscriptionalinitiationsite determinedempiricallyisindicatedwith asterisks above the sequence. The underlined sequence represents theregioncomplementarytothe primer sequence used for 5'-endmapping. The potential polyadenylation signal (AATAAA) is doublyunderlined. The ClaI site used in Northern blotanalysisandS1nuclease assaysis underlinedwith abrokenline.

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FIG. 4. Assignmentoftrans-activati Schematic representation of ORFsand

midsusedtoreplace PstH5 intheCATa

amino acid (aa) residues are represer

relative CAT activity for each cotrans

phcwt and various DNAs is given asa

assay withreporter plasmid pCAPCA'

done with pCAPCAT as areporter pla indicated above each lane. Lane 1inclu

(L), while lanes 2to5included clonesL

DNA was added in lane 2, while lane,

plasmidsathroughc, diagrammatically aspanelB,exceptthatCAT activitywa,

fections withreporterplasmidphcwt.A inthe legendtoFig. 1.

specific trans-activating function,

the PstH5 clone could be replace

plasmid, pXmal7, which contains

AcMNPV (40.3 to 49.4 mu) and

region unique to PstH5 (Fig. 1A

pXmal7 was equivalent to PstH5

trans-activate the vp39 promoter

remainder of thelibraryclones(Fig. 3).Twosubfragments of pXmal7 e: BglIIsiteat44.6mutothe SmaIsit

2A, rowsbandc)wereunabletofi PstH5 (Fig. 2B, lanes 4 and 5).

extending from the SstI site at40.3

2A, row d) or SstII (Fig. 2A, ro

trans-activate lategeneexpression

lanes 6 and 7). A PstI-SstI

subfral

(Fig. 2A, rowf)aswellas an EcoR

to 45.4mu) (Fig.2A, rowg) alsote

expression assays (Fig. 2B, lanes E these fragments was mapped with

zymes (Fig. 2A). Further subfragm testedfor their abilitytosubstituteI

(Fig. 2B, lanes 10 and 11), and th(

c

waslocalized to the EcoRI-MluI(43.4to45.2mu) segment of

AcMNPV(Fig.2A,rowh).Asmallfragmentextending from

the SphI site to the HpaI site, however, was unable to restore expression when substituted for the PstH5 clone

(Fig. 2B, lane 11).

Nucleotide sequenceofkf-3 and the flanking regions. The EcoRI-MluI

fragment

that was found to restore late gene

expression

tothe AcMNPV

library

in the absence ofPstH5

was cloned and sequenced. The nucleotide sequence is -- presented in Fig. 3B. Computer analysis revealed a

major

--

- ORF in the counterclockwise direction and predicted a

polypeptideproductof 385 amino acids and withamolecular

massof44,529daltons. Thepredictedinitiation codon is ina favorable context (AATATGG) for efficient translation in accordance with Kozak's rules (PuNNATGPu) (18) and

other

abundantly expressed

AcMNPV genes

(26).

ThisORF wasdesignatedlef-3, for late expression factor 3. Thecoding

region of lef-3 has a high A+T content (63.8% A+T; 36.2%

G+C).

No apparentsignificant homology tolef-3wasfound 2 ' w; s whendata bases (GenBank release 73; U-EMBL release

31)

were searched by use of the Genetics Computer Group

* * w w v -

>-

FASTA program

(30)

and the BLAST

(1)

network serviceat the National Center for Biotechnology Information, nor

were anysignificant known sequence motifs found within the

lef-3 gene product, with the exception of five potential

N-glycosylation sites(IntelliGeneticsKeybank program,

re-: lease9), although no apparent signal sequence was found.

ing

function to the ORF. (A)

Identification

of the ORFwithin the

EcoRI-MluI

fragment

AcMNPV portions of plas- that is necessary for late and very late gene expression. In issay. ORFs of more than 80 addition tolef-3, sequence data revealed two smaller ORFs nted by open arrows. The in the minimal trans-activating fragment (EcoRI-MluI) that sfection with pCAPCAT or overlapped lef-3 and could potentially encode polypeptides

iplus or a minus. (B) CAT of 84 and 192 amino acids, as depicted in Fig. 4A. To T. All cotransfections were determine whether one of thesmaller ORFs was responsible smid. Additional DNAs are for trans-activating the late

promoter,

wetested the ability of ided the entire clone library several deletionplasmidstosubstitute for PstH5 in transient ackingPstH5. No additional expression plasmid containin

trantie

s 3 to 5 contained deletion expression assays. Only the plasmid containing the entire shown in panel A. (C) Same lef-3 ORFwas functionally similar to PstH5 in providing a

Ls

assayed following cotrans- trans-activating factor for the vp39 promoter(Fig. 4B). This LcCM and Cm are as defined plasmid (Fig. 4A, deletion clone a) was also able to

substi-tute for PstH5 in trans-activating the very late polyhedrin

promoter (Fig. 4C). Thetwo smallerdeletion plasmids (Fig. 4A, clones b andc) were unable tofunctionally substitute for we first determined that PstH5 when used together in thecotransfections, indicating -d in these assays by a that the two smaller ORFsand/or partial ORFs do not act in the SmaI-D fragment of concert (data not shown). Thus, lef-3 is necessary for encompasses the entire trans-activation, although wecannotpreclude thepossibility and Fig. 2A). Plasmid that the84-amino-acid-encoding ORF (Fig. 4A) is necessary in terms of its ability to for late gene expression in addition to lef-3. The data are in the presence of the consistent with prior experiments showing that the disrup-.2B, compare lanes1 and tion of either the BglII or the SphI site abolished trans-xtending from the unique activating function(Fig. 2, lanes 4, 5, and11); these sites are

;e

at 40.3 or49.4 mu(Fig. within the translated region and are upstream of thepoly(A) unctionally substitute for signal, respectively, of lef-3 (Fig. 4A) (see also the 3' However, subfragments mapping data below).

3mu to theBamHI (Fig. Northern (RNA) blot

analysis.

To examine the temporal

)w e) site were able to regulation of lef-3 transcription, we performed a Northern in these assays (Fig. 2B, blotanalysis with alef-3-specific RNA probe. The uniformly gment (42.0 to 45.4 mu) labeled, strand-specific RNA probe extended from

nucle-I-SstI subfragment (43.4 otide (nt) 940 to about nt 1840 in anorientation opposite that sted positive in transient of lef-3 mRNA (Fig. 3B and Fig. 5A). As shown in Fig. SB, and 9). The smallest of a primaryvirus-specific transcript of approximately 2.0 kb

i various restriction en- was observed in infected cells at 3 h p.i. The level of this

ients of this region were 2.0-kb RNA was highest at 6 h p.i., declined slightly at 12 h for PstH5 in these assays p.i., anddeclineddramatically but remained detectable at24

e trans-activating region and 48 h p.i. When cells were treated with the DNA J. VIROL.

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VOL.67,1993L~~~~~ATEGEN-E EXPRESSION FACTOR 3 OF AcMNPV 5265

A

B

M 3 6 12 A C 24 48

.RI ScI

*-.4

4AT :

Clal

_II,

:: gIiIr

I

4.11

30-o

2.0- P:

1.6-1.-0

-C

D

e0M 501-= em 489 404 00

331 OM

242 *a

190- eAm 147- Of 111- 4fb

67- *oa

P M 3 6 12 A C 24 48

4= -4 ao

1444 eMM4 _ _. -~

736 a.

476 "

FIG. 5. Transcriptional mappingoflef-3. (A)Location andnatureofprobesusedin the Northern blotanalysisand the 5' and 3' Si nuclease

protection assays, shown in relationship to lef-3 (represented by an open arrow), and key restriction enzyme sites within the region sequenced. Theorientation of the RNAprobeused in the Northern blotanalysis is indicatedbythe arrowheadatthe 3' end. Thepositions

of radiolabels in 5' and 3' probesare indicatedby asterisks. Thepotential poly(A) site (AATAAA) is indicatedbyaverticalarrow. The

transcriptionalmappingdataforlef-3aresummarizedbythefilledarrowatthetop,representingthesize, location,and orientation of thelef-3 transcript.skvectorrepresentspBluescript sequences. (B)Northern blotanalysisoflef-3transcripts.Total RNAwasisolated from either

mock-infected cells(laneM)orAcMNPV-infected cellsat3, 6, 12, 24,and 48 hp.i. (lanes3, 6, 12, 24,and48,respectively) and 12 hp.i.in

thepresence ofaphidicolin (lane A)orcycloheximide (lane C).The RNAwasdenatured,electrophoretically separated, andtransferredtoa

nylon blottingmembrane. The membranewashybridizedwith thelef-3strand-specificRNAprobeindicated inpanelA.Thesizes of DNA

markers, inkilobases, are indicatedonthe left. (C)5'Mappingoflef-3transcripts bySi nucleaseprotection analyses.A984-bp fragment,

5' 32plabeled attheCiaI-generatedend as shown inpanelA,was hybridizedto RNAsamplesasdescribed forpanelBand subjectedto

digestionwith Si nuclease. The Si nuclease-resistantproductswereresolvedona4.0% polyacrylamide gelandvisualizedby autoradiog-raphy. Molecularweightmarkers (laneM),innucleotides,werederived fromMspl-digestedpUC19. LaneP,undigested probe.Themajor protected fragmentisindicatedbyanarrowhead.(D)3'Mappingoflef-3transcripts bySi nucleaseprotection analyses.Theprobeused for

hybridization to total RNAwas a 1,408-bpEcoRI-CiaI fragment radiolabeled with 32Pat theCiIA-generated end as shown in panel A.

Molecular weight markers were derived from Taql-digested pUC19. Lane P, undigested probe. The nuclease-resistant fragment is

indicatedbyanarrowhead.

synthesis inhibitor aphidicolin,which inhibits late and very latetranscriptionbecause ofadependenceonDNA

replica-tion, the level of the 2.0-kb RNA decreased only a small

degree (Fig. SB, compare lanes 12 and A), indicating the

early nature of lef-3 transcription. Treatment of infected cells with the protein biosynthesis inhibitor cycloheximide significantlyelevated the levels oflef-3transcripts (Fig. SB, compare lanes 12 and C), a result also consistent with the classification oflef-3 as an earlygene. Aminortranscript,

slightly largerthan 2.0kb,wasalsoseenatalltimesp.i.;this

transcript had a temporal pattern similar to that for the 2.0-kbtranscript.

Mapping of the 5' and 3' ends of kf-3 transcripts. To

determine theinitiationsite(s)forlef-3transcriptioninSF-21 cells, we performed a primer extension analysis and nuclease protection assays. Examination of the sequence

nearthe 5' end of thetranscribedregionoflef-3revealedtwo

CAGTmotifs that are frequently found near the

transcrip-P M 3 6 12 A C 24 48

2 0

. . a

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5266 LI ET AL.

T C G A M 3 6 12 A C 2448

loo Fw

A

I

:5 f A|

T/

l

a I

ok- ..'

..- -w

vo:

ldk. 3

W--f4

*4-i@D "*ft

.i. IOW

FIG. 6. Primer extension analysis of the 5' end of the lef-3

mRNA. A 5'32P-labeled oligonucleotide(S'-GCTCATCAATITCA CACTAATCCr-3') was hybridized to total RNA from mock-in-fected cells(M) orfrom infected cells harvested atthe hours p.i.

indicated above thelanes, and the RNAwas reverse transcribed.

The lanes designated A and C between the 12- and 24-h lanes indicated thatthe RNAwasisolatedat12 hp.i. in thepresenceof

aphidicolin (A)orcycloheximide (C). The radiolabeled oligonucle-otidewasalsoused as aprimer with aDNAtemplatecontaining lef-3 andwas extended in thepresenceofdideoxyribonucleotides ddATP (A), ddTTJP (T), ddGTP (G), and ddCTP (C). The primer

extension productswereresolvedon an8.0%polyacrylamide gel.

Thetranscriptional startsite is shown ina region specified by the

nucleotidesequenceof the DNA strand complementarytothelef-3 mRNA.

tionalstart sites of early baculovirusgenes(2, 4, 5, 14, 19).

One CAGT is located 41 bp upstream of the translational start codon, while theother CAGT is located within theN terminus of the translated region and is followed, 57 bp

downstream,byanATG codon inafavorablecontext(AAA ATGA)fortranslationalinitiation (Fig. 3B).A23-ntprimer, corresponding to the sequence from nt 900to nt 922, was

thussynthesized and radiolabeled at the 5' end for the primer extensionanalysis. Three major distinct extension products, none of which mapped to a CAGT motif, were observed (Fig. 6). The longest and most abundant primer extension productcorresponded to a transcriptional start site about 280 bp upstreamof the first ATG codon oflef-3. The temporal pattern for this extension product was consistent with that

observedin Northernblots; the most obvious feature was an increase in the level of the product in the presence of

cycloheximide (Fig. 6, laneC). The shortestprimer exten-sion product would place the transcriptional start site at nt 750, 3 bp upstreamof the first CAGT motif. No extension products corresponding to the second CAGT motif were

observed in these primer extension reactions when gel electrophoresis was carried out to resolve shorter products (data notshown).

Si

nuclease protection analyses were also performed to mapthe 5'end of thelef-3mRNA. A 984-ntDNAfragment

radiolabeledattheClaI sitewasused as a probe (Fig.SA),

and afragment of about 400 to 450 nts was protected when this probe washybridized to total RNA and subjected toSi

nuclease digestion (Fig. SC). The transcriptional start site

deduced from the size of this protected fragment corre-sponds to that defined by the longest primer extension products. No protected fragment of a size corresponding to the shortestprimer extension product in the primer exten-sionanalysiswasobserved. The shortest extension product maytherefore represent a prematurely terminated product. Formapping of the 3' end of the lef-3 mRNA, a 1,408-bp

EcoRI-ClaI fragment 3' labeled at the CiaI-generated end was used as a probe in

Si

nuclease protection analyses of

totalRNA(Fig. SA). Asshown inFig. 5D, anSi

nuclease-resistantfragment of about 1,100 to 1,200 nts was found in

AcMNPV-infectedbut notmock-infected cells. The size of this fragment suggests that the AATAAA motifoccurring 130 bp downstream of the last codon oflef-3 is a

polyade-nylationsignal.

Taken together, the 5' and 3' mapping data showed that

lef-3was transcribed froma 1.6-kbregionof the AcMNPV genome.Thesizewasingoodagreementwith Northern blot data, which revealedamajor2.0-kbtranscript.

DISCUSSION

We havefound another gene ofAcMNPVthatisrequired

intransient assays for substantialexpressionfrom a reporter gene under late and very late promotercontrol; removal of this gene from a library of clones covering the AcMNPV genome results in a dramatic reduction in the level of

expression from late and very late gene reporter plasmids.

This gene, knownasthe lateexpressionfactor3 gene(lef-3),

is located inaregion of theAcMNPVgenome(43.4to45.2

mu) that has not been previously characterized in any baculovirus system. It is the fifth gene to be identified as

functional in late baculovirus gene expression in this sub-tractive assay system. Like lef-1 and lef-2, lef-3 affects

expression from the late vp39 and very late polyhedrin

promotersbut does notaffectexpressionfrom theearlypcna

(etl)

promoter, although we cannot currently eliminate the

possibilitythat it affectsexpressionfrom otherearly

promot-ers. It is most likely that lef-3 is involved in late gene

expression, but whether it acts at the level ofreplication, transcription, ortranslation hasnotbeendetermined. Since

these threelefgenesaffect late geneexpression, it islikely

that the effect thatweobservedonverylate geneexpression

is duetotheir activation of late genes.

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LATE GENE EXPRESSION FACTOR 3 OF AcMNPV 5267 Analysesoflef-3 transcription showed thatlef-3 isanearly

genewithamajor transcript of 2.0 kbandaminortranscript

of slightlylarger than 2.0kb. The steady-statelevels of lef-3 transcripts are highest between 3 and 12 h p.i. and are

reduced dramatically by 24 h p.i. This temporal pattern is

similarto that observed forlef-1. Aswith other

AcAMNPV

earlygenes, transcription occurs in the presence of

cyclo-heximideand aphidicolin. Theelevation oflef-3 RNA levels

at 12 h p.i. in the presence of cycloheximide but not

aphidicolin suggests that the shutoff oflef-3 transcription requires an early gene product, although there are other

possible explanations (e.g., the presence of cycloheximide mayincrease thestability oflef-3 RNA).

Both primer extension and Si nuclease analyses of the 5'

end of the lef-3 transcript indicated that transcriptional initiation occurs approximately 280 nts upstream of the

translational initiation codon for the predicted 385-residue gene product. This result was surprising in two respects.

Firstly, a 280-nt leader is unusually long for an mRNA,

particularlyfor AcMNPV mRNAs;AcMNPV ORFs thatare

transcribed with long leaders generally have translated

ORFs in the leaders. NoATG-initiated ORFs were found

withinthelef-3leadersequence, nor werestable secondary

structures observed by computer analysis. Secondly, one

CAGTmotifwasfoundapproximately 60 bpupstreamofthe

translational initiation codon of the 385-codon ORF, and

another,with an upstreamTATAbox, was found

approxi-mately100bp downstreamof thepredicted initiationcodon

(CAGTmotifs havebeen foundatthestartsites ofsomebut

notall early baculovirus RNAs [2, 4, 5, 14, 19]). However,

both S1 nuclease and primer extension data indicated that

neitherof these CAGT motifswasusedas atranscriptional

initiation site. The site determined for lef-3 transcriptional

initiation mapped approximately 25 bp upstream of aCT

TATsequence,suggesting thatalatetranscriptmayinitiate

ontheopposite strand (i.e., fromATAAG) and overlap the

5'endof the lef-3 RNA. Whetherorhowthis late transcript

mightaffect lef-3 expression isnot yet known. The overlap shouldnotaffectour5'transcriptional mapping data because

the5' end mappedtosimilarlocations when RNA from cells

infectedinthepresenceof cycloheximide oraphidicolinwas

used in primer extension andS1 nuclease analyses. The

nature of the less abundant and slightly longer transcript

observedin the Northern blots wasnot specifically defined in thesestudies.

It is likely that lef-3 playsacentral role inlate baculovirus

gene expression. Only a few of the 12 clones of the AcM

NPVlibraryhavesuchastrongeffectonexpression fromthe

late andverylate promoters when removed from the

tran-sient expression assays (28). The four clones showing the

strongest effects uponremoval areIE15, BC5, PstH5, and

HC10. Wehave found that ie-1, ie-n, and lef-2 are able to substitute for IE15 (27), lef-1 is able to substitute for BC5

(28), and lef-3 is abletosubstitute for

PstH5

in the subtrac-tiveassaysystem(this paper). Wearecurrently determining

thefunctionalgenes mappingto the

HC10

region. ACKNOWLEDGMENTS

We thankS.Hilliard for technical assistance.

Thisworkwassupported inpartbyPublic HealthServicegrant

AI23719 from the National Institute of Allergy and Infectious Diseases.

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