characterization of the interaction between p143 and...

JOURNAL OF VIROLOGY, Jan. 2004, p. 329–339 Vol. 78, No. 10022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.1.329–339.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of the Interaction between P143 and LEF-3 fromTwo Different Baculovirus Species: Choristoneura fumiferanaNucleopolyhedrovirus LEF-3 Can Complement Autographa

californica Nucleopolyhedrovirus LEF-3 in SupportingDNA Replication

Tricia Chen, Daniela Sahri, and Eric B. Carstens*Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada K7L 3N6

Received 9 July 2003/Accepted 16 September 2003

The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We havedemonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, isrequired for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several otherearly viral proteins, are also essential for DNA replication in transient assays. We now describe the identifi-cation, nucleotide sequences, and transcription patterns of the Choristoneura fumiferana nucleopolyhedrovirus(CfMNPV) homologues of p143 and lef-3 and demonstrate that CfMNPV LEF-3 is also responsible for P143localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical forcross-species complementation of DNA replication. Support for this hypothesis was generated by substitutionof heterologous P143 and LEF-3 between two different baculovirus species, Autographa californica nucleopoly-hedrovirus and CfMNPV, in transient DNA replication assays. The results suggest that the P143–LEF-3complex is an important baculovirus replication factor.

The family Baculoviridae represents a unique group of largerod-shaped enveloped viruses carrying a double-stranded cir-cular DNA genome and replicating only in invertebrates. Manyof the advances in understanding the molecular biology ofbaculoviruses have resulted from studies of variants of the typespecies Autographa californica nucleopolyhedrovirus (AcMNPV).Nucleopolyhedroviruses (NPVs) replicate in cell nuclei andare characterized by the production of two virion phenotypes,the budded virions and the occlusion-derived virions. Bothforms are produced following infection of cells in culture andare characteristic of late stages of the viral replication cyclefollowing initiation of viral DNA replication at about 8 hpostinfection (37). The early events prior to this time arecharacterized by the expression of several viral gene products,some of which have been shown to be essential for viral DNAreplication. Nine viral genes (ie-1, ie-2, p143, dnapol, lef-1, lef-2,lef-3, pe38, and p35) are involved in directing replication ofplasmids carrying viral DNA inserts in transfected cells (20, 31,38). These data supported earlier genetic analysis of a condi-tional lethal AcMNPV mutant defective in DNA replication(13), which led to the description of the p143 gene: its nucle-otide sequence and the identification of the lesion in the 1,221-amino-acid open reading frame (ORF) (143 kDa) responsiblefor the temperature-sensitive DNA negative phenotype (29).The p143 gene is essential for viral DNA replication in vivosince no replication occurs in cells infected at the nonpermis-sive temperature with ts8 (29).

Biochemical characterization of extracts from AcMNPV-in-fected cells showed that P143 copurified through hydroxylapa-tite and coeluted from single-stranded DNA cellulose withanother viral protein called LEF-3, suggesting a possible directinteraction between P143 and LEF-3 (22, 39). LEF-3, alsodemonstrated to be essential for DNA replication in transientassays, is a single-stranded DNA binding protein (14) thatforms a homotrimer in solution (11). We have also clearlydemonstrated that with AcMNPV, LEF-3 is necessary for thetransport of P143 from the cytoplasm to the nucleus (39).These results have been confirmed by a yeast two-hybrid anal-ysis of P143 and LEF-3 that also revealed an interaction be-tween these two proteins (12). P143 also binds to DNA in anon-sequence-specific manner (22), a characteristic of somereplication proteins including DNA helicases, DNA poly-merases, primases and their accessory factors, DNA ligasesand DNA topoisomerases (4).

P143 may also play a role in the species specificity of bacu-lovirus replication. Although P143 from AcMNPV and thatfrom Bombyx mori NPV (BmNPV) share about 95% identity intheir amino acid sequences, substituting a small number ofamino acids that are different between the two P143 proteins(AcMNPV P143 amino acids 564 and 577 with the BmNPVP143 amino acids 565 and 578) dramatically altered the hostrange of AcMNPV. These changes permitted AcMNPV toreplicate more efficiently in B. mori cell lines and to kill B. morilarvae (3, 18). In addition, several attempts have been made tocomplement AcMNPV P143 with homologous genes fromother baculovirus species but all of these have failed (5, 12, 15),suggesting that there are important differences in P143 fromdifferent viral species that regulate P143 function during rep-lication.

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Queen’s University, Kingston, Ontario, Can-ada K7L 3N6. Phone: (613) 533-2463. Fax: (613) 533-6796. E-mail:[email protected].

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We have been investigating the genetic organization of abaculovirus specific for the spruce budworm (Choristoneurafumiferana), called C. fumiferana NPV (CfMNPV), because ithas potential for use in forestry as a biological pest controlagent against the spruce budworm. We have previously shownthat the replication of CfMNPV and AcMNPV is host cellspecific (25) and therefore decided to investigate the possiblerole of P143 and LEF-3 in this specificity. We now report theidentification and sequences of the CfMNPV p143 and lef-3homologues and investigations into their interactions togetherand in combination with their AcMNPV homologues in deter-mining their intracellular localization as well as their ability tocomplement each other in transient DNA replication assays.

MATERIALS AND METHODS

Cell lines and virus. C. fumiferana 124T cells (Cf124T) and CfMNPV (strainEC1) were propagated and maintained as previously described (25). Spodopterafrugiperda (Sf21) cells and AcMNPV strain HR3 were propagated and main-tained as previously described (29).

Sequence analysis and plasmid constructs. The location of the CfMNPV p143gene was previously mapped by Southern hybridization to the right end of theBamHI E fragment region (34). The complete CfMNPV p143 sequence wasconstructed by using a series of synthetic oligonucleotides as primers on plasmidclones of the CfMNPV BamHI E (pCfBamE) and HindIII MN2 (pCfHindMN2)fragments. Both strands were completely sequenced (4,783 nt).

The location of the CfMNPV lef-3 gene was predicted to lie downstream of theCfMNPV dnapol gene, previously shown to overlap the left end of the CfMNPVEcoRI G fragment (26). Sequence analysis of the right end of CfMNPV EcoRIG revealed homology with the AcMNPV lef-3 gene so the right end of EcoRI Gand the left end of the adjoining EcoRI H fragments were sequenced with universaland synthesized oligonucleotide primers. Some reactions used pCfHindB as tem-plate in order to sequence through the EcoRI G-H junction site. The sequencingreactions were performed by the core facility for protein and DNA chemistry(CORTEC, Queen’s University). The sequences were compiled and analyzed withcomputer programs AssemblyLIGN and MacVector (Accelrys Inc.).

The CfMNPV p143 ORF was subcloned by digesting pCfBamE with BamHIand PacI to release a 3,911-bp fragment containing the complete P143 ORF. Thisfragment was cloned into BamHI- and PacI-digested pNEB193 to generatepNEB193-Cfp143. The 3,924-bp BamHI-SalI fragment of pNEB193-Cfp143 wascloned either into BglII- and SalI-digested pIE1 h/PA (8) to generatepIE1hrCfp143 or into SalI- and BamHI-digested pBluescript SK(�) to generatepCfP143-SB(3.9).

The complete lef-3 ORF was amplified by PCR using purified CfMNPV DNAas template with primers C-6493 (5�-CGGGATCCTAAATCAGTTGGCAAG-3�) and C-6795 (5�-CGGGATCCACATGATGGCCACCAAAC-3�). The ampli-fication product was digested with BamHI and ligated into BamHI-digestedpBluescript SK(�) to generate pBSCfLEF-3. pBSCfLEF-3 was digested withBamHI and the 1.3-kb fragment carrying the CfMNPV lef-3 ORF was cloned intopIE1/hr/PA cut with BglII to generate pIE1hrCflef-3. The 1.3-kb BamHI frag-ment was also cloned into pGEX-3X (35) to generate pGEX3-CfLEF-3, inpreparation for overexpression of CfMNPV LEF-3 in Escherichia coli.

The CfMNPV p143 coding region was cloned in frame with the green fluo-rescence protein (GHP) by amplifying the GFP region of pEGFP-1 (Clontech)with primers C-3417 (5�-GAG AAA GGC GGA CAG GTA TCC-3�) andC-14112 (5�-TCG AGA TCT CTT GTA CAG CTC GTC C-3�, where theunderlined sequence generated a new BglII site at the C terminus of the GFPORF). The product was digested with BamHI and BglII and ligated intopIE1hrCfp143 digested with BamHI to generate pIE1hrCfp143GFP.

Preparation of polyclonal antibodies to LEF-3. The CfMNPV LEF-3 proteinwas expressed as a glutathione S-transferase (GST) fusion product by inducingJM109 cells transformed with pGEX3-CfLEF-3 with 0.4 mM IPTG (isopropyl-�-D-thiogalactopyranoside) for 16 h at 37°C. The cells were collected by centrif-ugation and suspended in equilibration buffer (50 mM Tris [pH 7.5], 2 mMEDTA, 0.4 M NaCl) and 2 mM mercaptoethanol. Following sonication, thesuspension was centrifuged (8,000 � g for 10 min) and the supernatant wasloaded onto an equilibrated glutathione agarose column (Sigma). After washingwith 50 mM Tris (pH 8), the GST–CfLEF-3 fusion protein was eluted with 10mM reduced glutathione in 50 mM Tris (pH 8). The AcMNPV LEF-3 proteinwas expressed as a His-tagged fusion product by cloning the open reading frame

into pRSET-B (Invitrogen) to produce pRSETB-Aclef3 and inducing trans-formed BL21(DE3)pLysS cells with 0.4 mM IPTG for 2 h at 37°C. Inclusionbodies containing LEF-3 protein were purified. New Zealand White rabbitsreceived intramuscular injections of 100 �g of protein in Titremax (CedarLaneLaboratories) and received boosters three times, every 3 weeks. The rabbitantiserum was collected 3 days after the last boost.

RNA transcription. Total intracellular RNA was extracted from either mock-or CfMNPV-infected Cf124T cells at various times postinfection using guani-dine-phenol (9, 10). Poly(A)� RNA was selected from total RNA on oligo(dT)-cellulose using the Micro-Fast Track kit (Invitrogen). Total RNA (30 �g) orpoly(A)� RNA (700 ng) was denatured with formaldehyde, electrophoresedthrough agarose gels, and transferred by downward blotting (19) in 50 mMsodium hydroxide to positively charged nylon membranes (Nytran Plus; Schlei-cher and Schuell). The blots were neutralized in 5� SSC (1� SSC is 0.15 M NaClplus 0.015 M sodium citrate) for 15 min, and the RNA was fixed to the mem-brane by baking for 2 h at 80°C. The blots were prehybridized at 60°C for 24 hand then hybridized with 32P-labeled riboprobes at 60°C for 24 h in solutionscontaining 50% formamide, 5� SSC, 0.1% polyvinyl pyrrolidone, 0.1% Ficoll,0.5% sodium dodecyl sulfate, 50 mM sodium phosphate (pH 6.5), and denaturedherring testis DNA (100 �g/ml) (7). Following three washes of 30 min each in0.1� SSC at 65°C, the membranes were exposed to X-ray film. The sizes of thetranscripts were determined from RNA standards (Invitrogen).

A strand-specific riboprobe specific for the CfMNPV lef-3 ORF was generatedby linearizing pBSCfLEF-3 with XhoI and radiolabeling cRNA with [32P]UTP inthe presence of T3 RNA polymerase. A strand-specific riboprobe specific for theCfMNPV p143 ORF was generated by BamHI digestion of pCfP143-SB(3.9) andradiolabeling cRNA with [32P]UTP in the presence of T7 RNA polymerase.

The 5� and 3� termini of the p143 and lef-3 mRNAs, derived from totalintracellular RNA harvested at 18 h postinfection, were identified using a 5�- and3�-rapid amplification of cDNA ends (RACE) system following the manufactur-er’s protocols (Invitrogen). A cDNA of the 5� end of the p143 mRNA, generatedwith primer C-1360 (5�-CGCAAAGGCTGTTAAAGGTAG-3�), was PCR am-plified using the abridged anchor primer (Invitrogen) and primer C-22031 (5�-GGAATTCCAAACAGTTTAACGGGCGGC-3�). Then, a second PCR wasprepared using the abridged universal amplification primer (Invitrogen) and thenested primer C-8114. The product of this reaction was purified and sequencedusing a second nested primer, C-22303 (5�-CACCATCCATTCTTGAACAGG-3�). A cDNA of the 5� end of the lef-3 mRNA, generated with primer C-5774(5�-CAGTTGGCAAGCGCGAGC-3�), was PCR amplified using the abridgedanchor primer (Invitrogen) and primer C-21845 (5�-GTGTAGTAGTCGTCGTCGGTGTTGG-3�). Then, a second PCR was prepared using the abridged uni-versal amplification primer and the nested primer C-6721 (5�-GTAACACTCTTGCTCAACC-3�). The product of this reaction was purified and sequencedusing a nested primer C-10435 (5�-GCAATCGTTTACGTGCTC-3�). A cDNAof the 3� end of the p143 mRNA was generated with an oligo(dT)-containingadaptor primer (Invitrogen) and primer C-0089 (5�-CTCTGGCGTATCTAACGCAG-3�). The product was PCR amplified with primer C-22080 (5�-CAAGACGCTGCTGGACAACGAC-3�) and the abridged universal amplificationprimer (Invitrogen). The product of this reaction was purified and sequencedwith the nested primer C-22081 (5�-CACAACTACGACGAGCGTGG-3�). AcDNA of the 3� end of the lef-3 mRNA was generated with an oligo(dT)-containing adaptor primer (Invitrogen) and primer C-21846 (5�-CCAACACCGACGACGACTACTACAC-3�). The product was PCR amplified with primerC-21912 (5�-GGAATTCAATGGAGGAAGACGACAGC-3�) and the abridgeduniversal amplification primer (Invitrogen). The product of this reaction waspurified and sequenced with the nested primer C-22437 (5�-GTTGGGTTTGCTGAAATACG-3�).

Immunoblotting and immunofluorescence. Infected cell extracts were ana-lyzed by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis (SDS–10% PAGE). Gels were either stained with Coomassie brilliant blue or electro-phoretically transferred to nitrocellulose membranes (Hybond-C) forimmunoblotting. The immunoblot membranes were blocked with 5% skim milkpowder overnight and then probed with a 1:10,000 dilution of rabbit polyclonalantibodies, washed, incubated with a 1:30,000 dilution of donkey anti-rabbitimmunoglobulin conjugated to horseradish peroxidase, and visualized with achemiluminescent detection system (NEN).

Sf21 cells on coverslips, either infected with whole virus or transfected withplasmid DNA, were prepared for immunofluorescence by washing with PBS,fixing with 10% paraformaldehyde for 10 min at room temperature, washing, andthen permeabilizing in 100% methanol for 20 min at �20°C. Following threewashes with PBS-T (PBS plus 0.1% Tween 20), the cells were blocked for 1 h in1% goat serum in PBS-T, then incubated with rabbit-polyclonal anti-AcMNPVP143 (1:1,000) and/or mouse-monoclonal anti-AcMNPV LEF-3 (1:1,000) anti-

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bodies for 1 h at room temperature. Following a wash with PBS-T, the coverslipswere incubated for 1 h in goat anti-rabbit secondary antibody conjugated withAlexa Fluor 568 (Molecular Probes) and/or goat-anti-mouse secondary antibodyconjugated with Alexa Fluor 488 (Molecular Probes). The coverslips were againwashed with PBS-T, then mounted on glass microscope slides in 50% glycerol.The slides were examined with a Meridian InSight Plus confocal microscope anda KX85 camera (Apogee Instruments). Color images were generated and ana-lyzed with Max Im DL version 2.00 (Cyanogen Productions) (Cancer ResearchLabs at Queen’s University).

Transient DNA replication assays. Sf21 cells (106 cells) in 35-mm-diameterdishes were washed three times with 1 ml of TC-100 medium and then replacedwith TC-100 (1.5 ml per dish). A 20� stock of DOPE/DDAB (6, 36) liposomechemicals was mixed by vortexing with 1 ml of sterile water. An equal molaramount of plasmids expressing all of the AcMNPV genes essential for viral DNAreplication (AcMNPV replication library: pAcie1, pAclef-1, pAclef-2, pAclef-3,pAcdnapol, pAcp143, pAcp35, and pAcie2pe38) (40) was mixed with a 1:6 ratioof DOPE/DDAB liposome reagent and diluted to a final volume of 200 �l withTC-100. In some experiments, the plasmids pAcLEF-3 and pAcp143 were re-placed with CfMNPV-expressing plasmid pIE1hrCflef-3, pIE1hrCfp143, orpIE1hrCfp143GFP. After incubation of the transfection mixture for 30 min atroom temperature, 500 �l of TC-100 medium was added to the DNA-DOPEmixture, and the entire mixture was added to washed Sf21 monolayers andincubated at 28°C for 6 h. After incubation, the cells were washed three timeswith TC-100 medium, covered with fresh TC-100 supplemented with 10% fetalcalf serum, and incubated at 28°C for 48 h. The replication of plasmid DNA wasmonitored by DpnI digestion of the total intracellular DNA as previously de-scribed (38).

Nucleotide sequence accession numbers. The sequence of the CfMNPV p143gene region has been deposited with GenBank under the accession numberAF127530. The sequence of the CfMNPV lef-3 gene region has been depositedwith GenBank under the accession number AF127908.

RESULTS

Identification and sequence of the CfMNPV p143 and lef-3genes. The promoter region of the CfMNPV p143 gene waspreviously located within the HindIII MN2 region, a region atthe right end of the CfMNPV BamHI E fragment (34). Plasmidclones of these fragments were used as templates with a varietyof synthetic primers to complete 4,788 bp of sequence thatrevealed a large open reading frame (ORF) of 3,684 bp, pre-dicted to code for a protein of 1,228 amino acids (141.7 kDa)(Fig. 1). The product of this ORF was about 85% identicalwith the amino acid sequence of Orgyia pseudotsugata NPV(OpMNPV) P143 and 57% identical with AcMNPV P143(Table 1). Although recognizable, the CfMNPV P143 gene wasonly 21 to 36% identical to the homologous genes of otherNPV and granulovirus (GV) P143s (Table 1). All of the NPVP143s were conserved in size, ranging from 1,218 to 1,223amino acids; however, the GV P143s were smaller (1,124 to1,159 amino acids). Comparisons of the P143 amino acid se-quences revealed that CfMNPV P143 retains the conservedhelicase motifs (motifs I, Ia, II, III, IV, V, and VI) that wepreviously predicted in the AcMNPV P143 protein (24, 29).The motifs A, B, and C, characterized by superfamily 3 heli-case (21), were also highly conserved among all the baculovirusP143 proteins. Additional regions of P143 contain conservedamino acids, including a region previously shown to extend thehost range of AcMNPV to B. mori cells (AcMNPV amino acids551 to 578, CfMNPV amino acids 558 to 584). This 27-amino-acid region is 74% identical between AcMNPV and CfMNPVand is highly conserved between all P143 proteins identified todate.

The CfMNPV lef-3 gene was predicted to map downstreamof the DNA polymerase gene previously identified near the

right end of the CfMNPV EcoRI G fragment (26). Sequenceanalysis of this region identified a 1,119-bp ORF (373 aminoacids, 43.0 kDa) predicted to code for the CfMNPV lef-3 ho-mologue. ORFs corresponding to iap2 (252 amino acids, 28.1kDa) and vlf-1 (374 amino acids, 43.2 kDa) homologues werealso identified in this region (Fig. 1). CfMNPV LEF-3 is about75% identical in amino acid sequence with the OpMNPVLEF-3 protein but exhibits much lower levels of similarity withother baculovirus LEF-3 proteins (Table 2). In addition, theLEF-3 proteins varied considerably in size from 297 aminoacids (Plutella xylostella GV [PxGV]) to 422 (Spodoptera exiguaNPV). The sequences of the upstream genes slp, dnapol, andpart of orf71 have been previously described (26, 27).

Transcription analysis of p143 and lef-3. The expression ofthe CfMNPV p143 and lef-3 genes was investigated by North-ern blot analysis using gene-specific riboprobes hybridized topoly(A)� RNA prepared from Cf124T cells at various timesafter CfMNPV infection. A 4.4-kb transcript was detected by6 h postinfection with a p143-specific probe spanning the com-plete p143 ORF (Fig. 2B). This transcript continued to in-crease in abundance from 12 through 48 h postinfection andappeared to be the major mRNA from this region. A compar-ison of the p143 promoter regions of CfMNPV with OpMNPV(2) and AcMNPV (30) showed that all three encode minicis-trons upstream of the start codon for the P143 ORF (Fig. 3A).CfMNPV and OpMNPV both encode a 12-amino-acid mini-cistron while AcMNPV encodes a five amino acid minicistron.The P143 transcription start sites were identified by 5� RACEusing primers shown in Fig. 2A. Two different mRNA prepa-rations were used to generate cDNAs and both cDNA prepa-rations were subjected to PCR and sequence analysis. Theresults were identical. Two PCR products, produced with

FIG. 1. Location of identifiable ORFs in the sequenced region ofp143 and lef-3. The regions of CfMNPV that were sequenced to identifythe lef-3 (above) and p143 (below) genes are indicated and oriented on theCfMNPV genome EcoRI restriction fragment map. The presence ofORFs with predicted functions is indicated as filled arrows above the scalein base pairs. ORFs with homologues in OpMNPV and AcMNPV andtheir numbers but no specific function are indicated as open arrows belowthe scale line.

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primer C-8114 (Fig. 2C), were sequenced identifying a strongstart site at 188 (more abundant PCR product) and a weakersite at 371 nucleotides upstream of the translation start codon.Both of these sites initiated within the sequence CAAT (Fig.3A) and are conserved in OpMNPV and AcMNPV but theAcMNPV p143 transcription start site was previously mappedby primer extension analysis about 30 nucleotides closer to thetranslation start codon (30). The major start site mapped 10nucleotides upstream of a CAGT sequence that is conservedbetween CfMNPV and OpMNPV. Although the sequencedownstream of p143 is relatively A/T rich, there is no potentialpolyadenylation signal sequence (AATAAA) in the sequencedregion downstream of the p143 ORF. However, 3� RACEsequencing of the PCR produced with primer C-22080 mappedthe p143 polyadenylation addition site 142 nucleotides down-stream of the stop codon, following a highly AT-rich region ofalmost 30 nucleotides. Together these data indicate a mini-mum size of 4,015 nucleotides for the p143 transcript, in goodagreement with the size of the p143 mRNA seen on Northernblots.

A 1.6-kb transcript was detected at 6 h postinfection with aCfMNPV lef-3-specific probe spanning the complete lef-3 ORFplus 187 downstream nucleotides. This mRNA increased dra-matically in abundance from 12 through 48 h postinfection(Fig. 2B). In addition, many larger transcripts were detected at24 and 48 h postinfection. The lef-3 transcription start site wasmapped by 5� RACE and sequencing of the PCR productproduced with primer C-6721 (Fig. 2C). A single site at thebeginning of the sequence AACATTGA 279 nucleotides up-stream of the lef-3 ORF and 26 nucleotides downstream of a

putative TATA box (Fig. 3B) was identified (Fig. 3B). A com-parison of the CfMNPV lef-3 promoter region with those ofOpMNPV (1) and AcMNPV (23) revealed a conserved TATAbox sequence about 25 nucleotides upstream of the transcrip-tion start site. The CfMNPV and OpMNPV lef-3 transcriptionstart sites mapped within one nucleotide of each other, justupstream of a conserved CATTGA sequence. However, thetranscription start site for the AcMNPV lef-3 gene has beenmapped about 14 nucleotides further downstream (23) (Fig.3B). The 3� RACE and sequencing of the PCR product pro-duced with primer C-22437 (Fig. 2C) mapped the lef-3 tran-script polyadenylation site to 125 nucleotides downstream ofthe translation stop codon and 14 nucleotides downstream of apolyadenylation addition signal. The determined size for thelef-3 mRNA (1,528 nucleotides) was in good agreement withthe estimated size of the transcript (1.6 kb) observed on North-ern blots.

Protein expression of CfMNPV lef-3. The expression of theCfMNPV lef-3 gene was investigated by immunoblotting to de-termine the time and level of protein expression in CfMNPV-infected cells. Cf124T cells, infected with CfMNPV, were har-vested at various time points postinfection and the infected cellsamples were analyzed by immunoblotting using a rabbit poly-clonal antibody directed against CfMNPV LEF-3. A 44-kDaband, first detected by 8 h postinfection, increased in expressionlevels through to 24 h postinfection (Fig. 4). The CfMNPV LEF-3protein increased in expression until at least 48 h postinfection(data not shown). The observed molecular mass coincided closelywith the predicted molecular mass of 43.0 kDa for the CfMNPVLEF-3 gene. As expected for a protein required for viral DNA

TABLE 1. Similarities between P143 amino acid sequences

Protein

% Identity (similarity) to:

AcMNPV(1,221)a

BmNPV(1,222)

OpMNPV(1,223)

LdMNPV(1,218)

SeMNPV(1,222)

PxGV(1,124)

TnGV(1,158)

XcGVb

(1,159)

CfMNPV 57 (15) 56 (15) 85 (6) 36 (21) 36 (20) 22 (16) 21 (18) 21 (18)AcMNPV 95 (1) 58 (14) 39 (21) 40 (18) 24 (16) 23 (17) 24 (17)BmNPV 57 (14) 39 (21) 41 (18) 24 (17) 23 (17) 24 (17)OpMNPV 35 (21) 36 (19) 22 (16) 21 (18) 21 (18)LdMNPV 48 (19) 24 (15) 23 (17) 24 (17)SeMNPV 24 (17) 24 (15) 24 (15)PxGV 47 (19) 47 (18)TnGV 88 (6)

a The number of amino acid residues in the individual P143 proteins is given in parentheses.b XcGV, Xestia c-nigrum GV.

TABLE 2. Similarities between LEF-3 amino acid sequences

Protein% Identity (similarity) to:

AcMNPV (385)a BmNPV (385) OpMNPV (373) LdMNPV (374) SeMNPV (422) PxGV (297) HaSNPV (379)b XcGV (351)

CfMNPV 39 (21) 39 (22) 75 (10) 22 (17) 24 (17) 12 (14) 23 (20) 9 (19)AcMNPV 91 (2) 39 (22) 26 (18) 25 (16) 15 (15) 23 (20) 11 (18)BmNPV 39 (23) 25 (18) 25 (17) 15 (14) 23 (19) 12 (18)OpMNPV 24 (17) 24 (19) 12 (15) 22 (21) 9 (20)LdMNPV 26 (21) 11 (13) 24 (21) 13 (18)SeMNPV 10 (13) 26 (15) 12 (15)PxGV 12 (15) 18 (15)HaSNPV 12 (17)

a The number of amino acid residues in the individual LEF-3 proteins is given in parentheses.b HaSNPV, Helicoverpa armigera SNPV.



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replication, the CfMNPV lef-3 gene was expressed prior to thereported time of initiation of viral DNA replication (25). Forcomparison, an immunoblot of AcMNPV-infected Sf21 cells wasprepared. AcMNPV LEF-3 was easily detectable at 4 h postin-fection confirming that the virus replication cycle proceeds fasterfor AcMNPV than CfMNPV as previously noted (25). Similarblots were also probed with polyclonal antibodies against theAcMNPV P143 protein but no signal was detected, indicating thatthese antibodies did not cross-react with CfMNPV P143 (data notshown).

Interaction between AcMNPV and CfMNPV P143 andLEF-3. We have previously shown that in AcMNPV-infectedSf21 cells, LEF-3 is essential for the translocation of P143 intothe nucleus (39). It was important to demonstrate that thisfunction is required in other baculovirus systems since the lef-3gene is not conserved in all baculoviruses. In addition, becausewe were interested in investigating the interaction between theCfMNPV LEF-3 and P143 proteins in the absence of otherviral proteins, plasmids expressing the CfMNPV p143

(pIE1hrCfp143) or lef-3 (pIE1hrCflef-3) genes, both driven bythe AcMNPV immediate-early promoter-1 (ie-1), were con-structed and individually transfected into Sf21 cells. To confirmthe expression of CfMNPV LEF-3 from this plasmid, Sf21 cellextracts obtained at 24 h posttransfection were analyzed byimmunoblotting. CfMNPV-infected Cf124T cells were used asa positive control. A 44-kDa CfMNPV protein was observed inboth pIE1hrCflef-3-transfected Sf21 cells and in CfMNPV-infected Cf124T cells (Fig. 5A). Larger amounts of LEF-3 wereobserved in Sf21 cells transfected with the expression vectorthan were observed in CfMNPV-infected Cf124T cells, clearlydemonstrating the expression of large amounts of LEF-3 in thetransfected cells. No band was detected in the control mock-infected cells.

Attempts were made to monitor the expression of CfMNPVP143 in pIE1hrCfp143-transfected Sf21 cell extracts by immu-noblotting using a polyclonal antibody against AcMNPV P143but no cross-reactivity was seen (data not shown). There-fore, in order to monitor the expression and localization of

FIG. 2. Expression and mapping of P143 and LEF-3 transcripts. The upper diagrams (A) show the orientation of the mRNAs, the open readingframes, and the location of the strand-specific riboprobes used in the Northern analysis for the CfMNPV p143 and lef-3 genes. Also shown are thenames and locations of the primers used in the 5� and 3� RACE analysis to map the 5� and 3� ends of the p143 and lef-3 mRNAs. (B) Poly(A)�

RNA, prepared from CfMNPV-infected Cf124T cells at the times indicated was resolved by 0.6% agarose gel electrophoresis. Blots of these gelswere probed with strand-specific riboprobes corresponding to the p143 and lef-3 genes. Similarly prepared poly(A)� RNA from mock-infected cellswas included as controls (M). The exposures were long, to enable the detection of virus-specific mRNA at the early time point (6 h postinfection).The sizes of the detectable transcripts are indicated on the right side of each blot. (C) PCR products generated from the 5�and 3� RACE analysisof the p143 and lef-3 mRNA were separated on agarose gels. Sequence analysis of these products revealed the 5� transcription start site and 3�polyadenylation site for each gene (shown in Fig. 3).



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FIG. 3. Promoter sequence for p143 and lef-3. An alignment of the promoter regions of the p143 (A) and lef-3 (B) genes from CfMNPV,OpMNPV, and AcMNPV is shown. TATA-box-like sequences are shaded, the location of published transcription start sites are underlined,minicistron coding regions are boxed and the translation start codons are in bold. The locations of the transcription start sites for the CfMNPVp143 and lef-3 genes, as determined by sequence analysis of PCR products, are shown with arrows. (C) The sequences of the 3� ends of the p143and lef-3 mRNAs as determined by 3� RACE and sequence analysis of PCR products are shown below the appropriate genomic sequence.



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CfMNPV P143, a plasmid was constructed where the CfMNPVp143 gene was fused in frame with the GFP reporter gene andthe fusion product was driven by the AcMNPV ie-1 promoter(pIE1hrCfp143GFP). A protein of the predicted size was ob-served in extracts of pIE1hrCfp143GFP-transfected cells whenprobed with an antibody directed against GFP (Fig. 5B). In-tracellular location of the fusion protein was monitored bydirect fluorescence microscopy. When pIE1hrCfp143GFP wastransfected on its own, GFP fluorescence was only observed inthe cytoplasm in both Sf21 and Cf124T cells (Fig. 6A and B),supporting our previous data with AcMNPV that, on its own,P143 remains cytoplasmic (39). These results also demon-

strated that the AcMNPV ie-1 promoter was functional in bothSf21 and Cf124T cells since both Lef-3 and P143 were ex-pressed under the control of this promoter in Cf124T cells, aresult that has not been previously demonstrated. WhenpIE1hrCfp143GFP was transfected into Cf124T cells that weresubsequently infected with CfMNPV, CfMNPV P143-GFP lo-calized to the nucleus (Fig. 6C). This result demonstrated thatthe fusion of GFP to P143 did not interfere with its recognitionand successful translocation to the nucleus by the CfMNPVLEF-3 protein. We confirmed that this translocation was me-diated by the CfMNPV LEF-3 protein by cotransfectingpIE1hrCfp143GFP and pIE1hrCflef-3 into Cf124T or Sf21cells (Fig. 6D). GFP fluorescence was observed in the nuclei ofcotransfected cells, clearly demonstrating that GFP-taggedCfMNPV P143 could interact with CfMNPV LEF-3. Theseresults also demonstrate that no C. fumiferana cell-specificfactors were required for the interaction between CfMNPVP143 and LEF-3 since correct nuclear localization occurred inboth Cf124T and Sf21 cells.

We then investigated the interaction of P143 and LEF-3 de-rived from the two different species of baculoviruses, AcMNPVand CfMNPV. Cf124T cells or Sf21 cells were transfected withpIE1hrCfp143GFP, then infected with AcMNPV. GFP fluores-cence was detectable in the cytoplasm in both cell lines (Fig. 6Eand G), indicating that AcMNPV LEF-3 did not transportCfMNPV P143 to the nucleus in either cell line. These resultssuggest that a specific interaction between homologous P143 andLEF-3 is required for the correct nuclear transport of P143.This hypothesis was confirmed by cotransfecting Sf21 cellswith pIE1hrCfp143GFP and plasmids expressing either AcM-NPV LEF-3 or CfMNPV LEF-3. Nuclear fluorescence of P143,indicating transport to the nucleus, was only observed whenCfMNPV LEF-3 was present (Fig. 6F and H). Because thecorrect localization of P143 and LEF-3 to the nucleus is re-quired for baculovirus DNA replication, we then investigatedthe interaction of the heterologous gene products to determinewhether this interaction was necessary for DNA replication.

FIG. 4. Temporal expression of CfMNPV LEF-3 in infected cells.Cf124T cells, infected with CfMNPV, were harvested at the indicatedtimes after infection (A). Whole-cell extracts were resolved by SDS–10% PAGE, blotted onto nitrocellulose filters, and then probedwith polyclonal antibodies against CfMNPV LEF-3. CfMNPV LEF-3was first clearly detectable at 8 h postinfection. For comparison, asimilar blot of extracts prepared from AcMNPV-infected Sf21 cellsand probed with LEF-3-specific polyclonal antibody is shown. (B)AcMNPV LEF-3 was first detectable at 4 h postinfection.

FIG. 3—Continued.



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Heterologous proteins in DNA replication. DNA replicationassays were performed by transfecting Sf21 cells with a seriesof plasmids that together express nine AcMNPV genes (ie-1,ie-2, p143, dnapol, lef-1, lef-2, lef-3, pe38, and p35) necessary forviral replication. Total intracellular DNA was harvested at 48 hposttransfection and digested with DpnI to distinguish betweenunreplicated input plasmid DNA and newly replicated DNA.As we have previously shown (40), when all nine AcMNPVgenes are expressed together, they support the replication ofany plasmid DNA, including those expressing the viral proteins(Fig. 7). If any of the plasmids, including those expressing P143or LEF-3, was eliminated from the mixture, no plasmid replica-tion occurred (Fig. 7A, lanes 8 and 9). Replacement of AcMNPVp143 with its CfMNPV homologue did not restore replicationfunction (Fig. 7A, lane 10) but replacement of AcMNPV lef-3 byits CfMNPV homologue did (Fig. 7A, lane 11). Replacement ofboth AcMNPV p143 and lef-3 genes with their CfMNPV homo-logues also restored plasmid DNA replication (Fig. 7A, lane 12).

These results demonstrated that CfMNPV LEF-3 did interactwith either AcMNPV P143 or CfMNPV P143 and complementedviral DNA replication in the presence of the other AcMNPVgenes. However, CfMNPV P143 alone did not support plasmidDNA replication in the presence of the AcMNPV gene products.Because the immunofluorescence data discussed above demon-strated that CfMNPV P143 fluorescence was not detectable in thenuclei of cells expressing AcMNPV LEF-3, these results suggestthat only a small fraction of the expressed P143 is required tosupport DNA replication in the nucleus. The immunofluores-cence analysis was done with a plasmid that expressed a P143-GFP fusion protein, so we confirmed the functionality of thisfusion protein in DNA replication by replication assays. The plas-mid pIE1hrCfp143GFP worked as well as a plasmid expressingnormal CfMNPV P143 in supporting DNA replication (Fig. 7B,lanes 9 and 10). Thus, these data show for the first time that crossspecies complementation of P143 in baculovirus transient repli-cation assays can occur, even with distantly related NPVs, if P143is correctly transported to the nucleus.

FIG. 5. Expression of LEF-3 and P143-GFP following transfectionor infection and detected by immunoblotting. (A) Whole-cell extracts(5 � 104 cells per lane) were prepared from mock-infected Sf21 cells(lane 1), mock-infected Cf124T cells (lane 2), CfMNPV-infectedCf124T cells (lane 3), or pIE1hrCflef-3-transfected Sf21 cells (lane 4)at 24 h posttransfection or postinfection. The extracts were analyzed bySDS–10% PAGE, transferred to a nitrocellulose membrane andprobed with LEF-3-specific polyclonal antibody. The relative mobilityof molecular weight markers is shown on the left and the immunore-active proteins are labeled on the right. (B) Whole-cell extracts (5 �104 cells per lane) were prepared from pIE1hrCfp143GFP-transfectedSf21 cells (lane 1), pAcGFP-transfected Sf21 cells (lane 2), and mock-transfected Sf21 cells (lane 3) harvested at 24 h posttransfection.Whole-cell extracts were analyzed by SDS–11.25% PAGE, transferredto a nitrocellulose membrane and probed with anti-GFP monoclonalantibody. The relative mobility of the molecular weight markers isshown on the left and the immunoreactive proteins are labeled on theright.

FIG. 6. Intracellular localization of P143 and LEF-3 followingtransfection detected by immunofluorescence. Cf124T (A, C, D, andE) or Sf21 (B and F to H) cells, transfected with plasmids expressingCfMNPV P143 fused to GFP (pIE1hrCfp143GFP) (A to H), CfMNPVLEF-3 (pIE1hrCflef-3) (D and H), or AcMNPV LEF-3 (pAcLEF-3)(G), were mock infected or infected with CfMNPV (C) or AcMNPV(E and F). At 24 h posttransfection, the cells were either observeddirectly for GFP fluorescence or were also processed for immunoflu-orescence using antibodies directed against CfMNPV LEF-3(CfLEF3) or AcMNPV LEF-3 (AcLEF3). Nuclear DNA was stainedwith DAPI. Only infection with CfMNPV or cotransfection withCfMNPV LEF-3 resulted in nuclear GFP fluorescence from CfMNPVP143-GFP.



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DISCUSSION

The baculovirus protein P143 was first shown to be essentialfor viral replication by analysis of a temperature sensitiveAcMNPV mutant defective in viral DNA replication (13, 29).We later showed that another viral protein, LEF-3, identifiedas a single-stranded DNA binding protein (14) with DNA-destabilizing properties (33), was an essential transporter forlocalizing AcMNPV P143 to the nuclei of infected cells (39).We predicted that a major function of P143 would be to pro-vide DNA unwinding activity during viral DNA replication(29). The helicase activity of P143 has recently been confirmed(32). There is also some evidence to suggest that P143 may playa role in species specificity of virus infection. Substitution of asfew as two amino acids within a specific region of P143 be-tween the very closely related baculoviruses AcMNPV andBmNPV altered the replication efficiency of AcMNPV in B.mori cells (3, 17). However, the basis of the block of AcMNPVreplication in B. mori cells was not investigated. To investigatethe possible role of P143 in regulating viral DNA replication inother host species, we identified, cloned and sequenced p143and lef-3 from CfMNPV. The CfMNPV P143 predicted aminoacid sequence was highly similar to that of OpMNPV (85%identical) but only 58% identical with that of AcMNPV P143.LEF-3 is less highly conserved among baculoviruses, butCfMNPV LEF-3 was still most similar to the OpMNPV homo-logue (75% identical) and only 39% identical with AcMNPVLEF-3. The transcription patterns of both the CfMNPV p143and lef-3 genes were consistent with their being early genes,essential for viral DNA replication. Both gene transcripts weredetectable by 6 h postinfection, well before the time of increasein CfMNPV DNA replication (25). In addition, the transcriptsfor both genes increased in abundance up to 48 h postinfection,suggesting that they were transcribed for extended periods inCf124T cells. These data support our previous studies, whichrevealed a slower replication cycle of CfMNPV in these cellsthan AcMNPV in Sf21 cells (25). The similarity between theCfMNPV genes and their OpMNPV homologues was also ev-ident in the sequence and location of their transcription startsites. We identified the CfMNPV LEF-3 transcription start siteby 5� RACE to be located 25 nucleotides downstream of apotential TATA box sequence starting at the first A in thesequence AACATTGA. This corresponds exactly with theidentified OpMNPV LEF-3 start site (1). The AcMNPV LEF-3transcription start site was mapped about 14 nucleotides down-stream of this region (23). Two CfMNPV P143 transcriptionstart sites were identified by 5� RACE, at 188 and 371 nucle-otides upstream of the translation start codon. Both of thesesites represent conserved regions in OpMNPV and AcMNPV,although the AcMNPV p143 transcription start site wasmapped about 30 nucleotides closer to the translation startcodon (30). Together, these results support our previous hy-pothesis that the promoter structures of genes involved in viral

FIG. 7. Transient plasmid DNA replication in the presence of het-erologous P143 and LEF-3 proteins. Sf21 cells were transfected with acollection of plasmids, which together expressed the AcMNPV genesnecessary for plasmid DNA replication (ie-1, dnapol, lef-1, lef-2, p35,pe38, and ie-2) except p143 and lef-3. In separate transfections, thislibrary was supplemented with plasmids expressing the AcMNPV p143(Acp143), AcMNPV lef-3 (Aclef3), CfMNPV p143 (Cfp143) orCfMNPV lef-3 (Cflef3) genes. Following incubation for 48 h, totalintracellular DNA was prepared and digested with EcoRI (�DpnI) tolinearize the plasmids or with EcoRI and DpnI (�DpnI) to detectreplicated plasmid DNA. Southern blots of these restriction digestionDNA preparations were probed with labeled pUC19 DNA. Replica-tion of input plasmid DNA was detected in the presence of plasmids

expressing AcMNPV P143 and LEF-3, CfMNPV P143 and LEF-3, andAcMNPV P143 and CfMNPV LEF-3 (A). Similar assays were alsodone with a plasmid expressing the CfMNPV P143-GFP fusion protein(B). This protein also supported plasmid DNA replication in the pres-ence of CfMNPV LEF-3.



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DNA replication are different from those of other early genes,many of which have transcription starts sites beginning withCAGT, likely reflecting different regulatory pathways for thesegenes. A CAGT sequence located 10 nucleotides downstreamof the mapped p143 transcription start site is also present inOpMNPV. However, our 5� RACE analysis indicated that thissite was not used as a transcription start site.

Antibodies raised against CfMNPV LEF-3 reacted with a44-kDa polypeptide expressed in virus-infected Cf124T cellsfrom about 8 h postinfection, correlating well with the expres-sion of the CfMNPV lef-3 transcript. Immunofluorescencestudies showed that CfMNPV LEF-3 was always observed inthe nucleus indicating that it carries the necessary signals re-quired for nuclear localization. Unfortunately, our polyclonalantibodies directed against AcMNPV P143 did not cross-reactwith the CfMNPV gene product and we have had no success atoverexpressing this protein to prepare CfMNPV P143-specificantibodies so we could not study CfMNPV P143 expressiondirectly in virus-infected cells. We developed an alternativemethod by preparing a plasmid which expressed a CfMNPVP143-GFP fusion protein. The expression of the fusion proteinwas monitored by fluorescence of the GFP reporter compo-nent. These studies revealed that P143 remained cytoplasmicwhen expressed on its own, but was nuclear when coexpressedwith CfMNPV LEF-3 or in CfMNPV-infected Cf124T cells.These biochemical data confirm our previous immunofluores-cence data that the nuclear localization of P143 requires thepresence of LEF-3 although the specific role that LEF-3 playsin this process is unknown. Because LEF-3 may exist as ahomotrimer (11), it is too large to diffuse through nuclearpores on its own, so it likely carries a nuclear localization targetsignal, which provides a signal sequence for LEF-3 interactionwith cellular importin complexes for delivery to the nuclearpores and nuclear import (16). Because P143 does not appearto carry a nuclear signal sequence, the interaction betweenP143 and LEF-3 must establish a complex that is then recog-nized by this host transporting machinery. We have initiatedstudies to identify possible cellular components of this com-plex.

CfMNPV P143-GFP was also localized to the nuclei of Sf21cells in the presence of CfMNPV LEF-3, suggesting that no C.fumiferana-specific cell factors are essential for the correcttranslocation of the P143–LEF-3 complex to the nucleus. How-ever, AcMNPV LEF-3 or whole AcMNPV virus infection re-sulted in cytoplasmic fluorescence of CfMNPV P143-GFP inSf21 cells, suggesting that virus species specificity is importantto the interaction of P143 and LEF-3. Other researchers haveattempted to rescue AcMNPV P143 with a heterologous P143from OpMNPV, SeMNPV or Trichoplusia ni GV, but theseexperiments were unsuccessful (2, 5, 15). In these cases, rescueof P143 function was monitored by transient DNA replicationassays. Based on those published results, we hypothesized thatone reason these experiments failed was the lack of the ho-mologous LEF-3, which would recognize and transport P143 toits site of action in the nucleus. Our replication assay resultsconfirmed this hypothesis. As expected, CfMNPV P143 didnot rescue DNA replication in the presence of all the otherAcMNPV replication genes. However, replacing bothAcMNPV P143 and LEF-3 with their CfMNPV counterpartsrestored the replication function in the presence of the remain-

der of the AcMNPV replication proteins. These results suggestthat a major factor in baculovirus replication is represented bythe P143–LEF-3 complex. Our transient replication assaysdemonstrated that replacement of AcMNPV LEF-3 withCfMNPV LEF-3 also restored replication function. This sug-gests that there are less stringent requirements for P143–LEF-3 interaction in nuclear localization than for the functionof P143, possibly in conjunction with LEF-3, during DNAreplication. However, no specific role of LEF-3 in baculovirusDNA replication has been demonstrated, so it is still not clearwhat its actual function is. We also confirmed that adding theGFP tag to P143 did not disrupt its ability to function duringviral DNA replication since this construct was still able torescue DNA replication in the transient assays. These resultsdemonstrate that this fusion protein will be a useful tool forinvestigating the in vivo localization of P143 during viral rep-lication. We are continuing to investigate the interaction ofthese and other viral proteins in the assembly of a functionalreplication complex in vivo.

ACKNOWLEDGMENTS

We gratefully acknowledge Don Back for the Northern blotting,Linda Guarino for the monoclonal antibody against AcMNPV LEF-3,and Marilyn Garrett and Colin Inalsingh for technical assistance.

This research was supported by a grant from the Canadian Instituteof Health Research.

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