Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Characterization of the Interaction between P143 and LEF-3 from
Two Different Baculovirus Species:
Choristoneura fumiferana
Nucleopolyhedrovirus LEF-3 Can Complement
Autographa
californica
Nucleopolyhedrovirus LEF-3 in Supporting
DNA 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 have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the
identifi-cation, nucleotide sequences, and transcription patterns of theChoristoneura fumiferananucleopolyhedrovirus
(CfMNPV) homologues ofp143andlef-3and demonstrate that CfMNPV LEF-3 is also responsible for P143
localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution
of 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-3
complex is an important baculovirus replication factor.
The familyBaculoviridaerepresents a unique group of large
rod-shaped enveloped viruses carrying a double-stranded cir-cular DNA genome and replicating only in invertebrates. Many of the advances in understanding the molecular biology of baculoviruses have resulted from studies of variants of the type
speciesAutographa californica nucleopolyhedrovirus (AcMNPV).
Nucleopolyhedroviruses (NPVs) replicate in cell nuclei and are characterized by the production of two virion phenotypes, the budded virions and the occlusion-derived virions. Both forms are produced following infection of cells in culture and are characteristic of late stages of the viral replication cycle following initiation of viral DNA replication at about 8 h postinfection (37). The early events prior to this time are characterized by the expression of several viral gene products, some of which have been shown to be essential for viral DNA
replication. Nine viral genes (ie-1,ie-2,p143,dnapol,lef-1,lef-2,
lef-3, pe38, and p35) are involved in directing replication of plasmids 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 thep143gene: its
nucle-otide sequence and the identification of the lesion in the 1,221-amino-acid open reading frame (ORF) (143 kDa) responsible for the temperature-sensitive DNA negative phenotype (29).
The p143gene is essential for viral DNA replication in vivo
since no replication occurs in cells infected at the nonpermis-sive temperature with ts8 (29).
Biochemical characterization of extracts from AcM
NPV-in-fected cells showed that P143 copurified through hydroxylapa-tite and coeluted from single-stranded DNA cellulose with another viral protein called LEF-3, suggesting a possible direct interaction between P143 and LEF-3 (22, 39). LEF-3, also demonstrated to be essential for DNA replication in transient assays, is a single-stranded DNA binding protein (14) that forms a homotrimer in solution (11). We have also clearly
demonstrated that with AcMNPV, LEF-3 is necessary for the
transport 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 a non-sequence-specific manner (22), a characteristic of some replication proteins including DNA helicases, DNA poly-merases, primases and their accessory factors, DNA ligases and DNA topoisomerases (4).
P143 may also play a role in the species specificity of
bacu-lovirus replication. Although P143 from AcMNPV and that
fromBombyx moriNPV (BmNPV) share about 95% identity in their amino acid sequences, substituting a small number of amino acids that are different between the two P143 proteins
(AcMNPV P143 amino acids 564 and 577 with the BmNPV
P143 amino acids 565 and 578) dramatically altered the host
range of AcMNPV. These changes permitted AcMNPV to
replicate more efficiently inB. moricell lines and to killB. mori
larvae (3, 18). In addition, several attempts have been made to
complement AcMNPV P143 with homologous genes from
other baculovirus species but all of these have failed (5, 12, 15), suggesting that there are important differences in P143 from different viral species that regulate P143 function during rep-lication.
* Corresponding author. Mailing address: Department of Microbi-ology and ImmunMicrobi-ology, Queen’s University, Kingston, Ontario, Can-ada K7L 3N6. Phone: (613) 533-2463. Fax: (613) 533-6796. E-mail: [email protected].
329
on November 8, 2019 by guest
http://jvi.asm.org/
We have been investigating the genetic organization of a
baculovirus specific for the spruce budworm (Choristoneura
fumiferana), calledC. fumiferanaNPV (CfMNPV), because it has potential for use in forestry as a biological pest control agent against the spruce budworm. We have previously shown
that the replication of CfMNPV and AcMNPV is host cell
specific (25) and therefore decided to investigate the possible role of P143 and LEF-3 in this specificity. We now report the
identification and sequences of the CfMNPV p143 and lef-3
homologues and investigations into their interactions together
and in combination with their AcMNPV homologues in
deter-mining their intracellular localization as well as their ability to complement each other in transient DNA replication assays.
MATERIALS AND METHODS
Cell lines and virus.C. fumiferana124T cells (Cf124T) and CfMNPV (strain EC1) were propagated and maintained as previously described (25).Spodoptera frugiperda(Sf21) cells and AcMNPV strain HR3 were propagated and main-tained as previously described (29).
Sequence analysis and plasmid constructs.The location of the CfMNPVp143
gene was previously mapped by Southern hybridization to the right end of the
BamHI E fragment region (34). The complete CfMNPVp143sequence was constructed by using a series of synthetic oligonucleotides as primers on plasmid clones of the CfMNPVBamHI E (pCfBamE) andHindIII MN2 (pCfHindMN2) fragments. Both strands were completely sequenced (4,783 nt).
The location of the CfMNPVlef-3gene was predicted to lie downstream of the CfMNPVdnapolgene, previously shown to overlap the left end of the CfMNPV
EcoRI G fragment (26). Sequence analysis of the right end of CfMNPVEcoRI G revealed homology with the AcMNPVlef-3gene so the right end ofEcoRI G and the left end of the adjoiningEcoRI H fragments were sequenced with universal and synthesized oligonucleotide primers. Some reactions used pCfHindB as tem-plate in order to sequence through theEcoRI G-H junction site. The sequencing reactions were performed by the core facility for protein and DNA chemistry (CORTEC, Queen’s University). The sequences were compiled and analyzed with computer programs AssemblyLIGN and MacVector (Accelrys Inc.).
The CfMNPVp143ORF was subcloned by digesting pCfBamE withBamHI andPacI to release a 3,911-bp fragment containing the complete P143 ORF. This fragment was cloned into BamHI- and PacI-digested pNEB193 to generate pNEB193-Cfp143. The 3,924-bpBamHI-SalI fragment of pNEB193-Cfp143 was cloned either into BglII- and SalI-digested pIE1 h/PA (8) to generate pIE1hrCfp143 or intoSalI- andBamHI-digested pBluescript SK(⫺) to generate pCfP143-SB(3.9).
The completelef-3ORF was amplified by PCR using purified CfMNPV DNA as template with primers C-6493 (5⬘ -CGGGATCCTAAATCAGTTGGCAAG-3⬘) and C-6795 (5⬘-CGGGATCCACATGATGGCCACCAAAC-3⬘). The ampli-fication product was digested withBamHI and ligated intoBamHI-digested pBluescript SK(⫺) to generate pBSCfLEF-3. pBSCfLEF-3 was digested with
BamHI and the 1.3-kb fragment carrying the CfMNPVlef-3ORF was cloned into pIE1/hr/PA cut withBglII to generate pIE1hrCflef-3. The 1.3-kbBamHI frag-ment was also cloned into pGEX-3X (35) to generate pGEX3-CfLEF-3, in preparation for overexpression of CfMNPV LEF-3 inEscherichia coli.
The CfMNPVp143coding 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⬘) and C-14112 (5⬘-TCG AGA TCT CTT GTA CAG CTC GTC C-3⬘, where the underlined sequence generated a newBglII site at the C terminus of the GFP ORF). The product was digested with BamHI and BglII and ligated into pIE1hrCfp143 digested withBamHI to generate pIE1hrCfp143GFP.
Preparation of polyclonal antibodies to LEF-3.The CfMNPV LEF-3 protein was expressed as a glutathioneS-transferase (GST) fusion product by inducing JM109 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 mM EDTA, 0.4 M NaCl) and 2 mM mercaptoethanol. Following sonication, the suspension was centrifuged (8,000⫻gfor 10 min) and the supernatant was loaded onto an equilibrated glutathione agarose column (Sigma). After washing with 50 mM Tris (pH 8), the GST–CfLEF-3 fusion protein was eluted with 10 mM reduced glutathione in 50 mM Tris (pH 8). The AcMNPV LEF-3 protein was 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. Inclusion bodies containing LEF-3 protein were purified. New Zealand White rabbits received intramuscular injections of 100g of protein in Titremax (CedarLane Laboratories) and received boosters three times, every 3 weeks. The rabbit antiserum 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 (30g) or poly(A)⫹RNA (700 ng) was denatured with formaldehyde, electrophoresed
through agarose gels, and transferred by downward blotting (19) in 50 mM sodium 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 NaCl plus 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 h and then hybridized with32P-labeled riboprobes at 60°C for 24 h in solutions
containing 50% formamide, 5⫻SSC, 0.1% polyvinyl pyrrolidone, 0.1% Ficoll, 0.5% sodium dodecyl sulfate, 50 mM sodium phosphate (pH 6.5), and denatured herring testis DNA (100g/ml) (7). Following three washes of 30 min each in 0.1⫻SSC at 65°C, the membranes were exposed to X-ray film. The sizes of the transcripts were determined from RNA standards (Invitrogen).
A strand-specific riboprobe specific for the CfMNPVlef-3ORF was generated by linearizing pBSCfLEF-3 withXhoI and radiolabeling cRNA with [32P]UTP in
the presence of T3 RNA polymerase. A strand-specific riboprobe specific for the CfMNPVp143ORF was generated byBamHI digestion of pCfP143-SB(3.9) and radiolabeling cRNA with [32P]UTP in the presence of T7 RNA polymerase.
The 5⬘ and 3⬘ termini of thep143 andlef-3mRNAs, derived from total intracellular RNA harvested at 18 h postinfection, were identified using a 5⬘- and 3⬘-rapid amplification of cDNA ends (RACE) system following the manufactur-er’s protocols (Invitrogen). A cDNA of the 5⬘end of thep143mRNA, generated with 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 was prepared using the abridged universal amplification primer (Invitrogen) and the nested primer C-8114. The product of this reaction was purified and sequenced using a second nested primer, C-22303 (5⬘ -CACCATCCATTCTTGAACAGG-3⬘). A cDNA of the 5⬘end of thelef-3mRNA, generated with primer C-5774 (5⬘-CAGTTGGCAAGCGCGAGC-3⬘), was PCR amplified using the abridged anchor primer (Invitrogen) and primer C-21845 (5⬘-GTGTAGTAGTCGTCGT CGGTGTTGG-3⬘). Then, a second PCR was prepared using the abridged uni-versal amplification primer and the nested primer C-6721 (5⬘-GTAACACTCT TGCTCAACC-3⬘). The product of this reaction was purified and sequenced using a nested primer C-10435 (5⬘-GCAATCGTTTACGTGCTC-3⬘). A cDNA of the 3⬘end of thep143mRNA was generated with an oligo(dT)-containing adaptor primer (Invitrogen) and primer C-0089 (5⬘-CTCTGGCGTATCTAAC GCAG-3⬘). The product was PCR amplified with primer C-22080 (5⬘-CAAGA CGCTGCTGGACAACGAC-3⬘) and the abridged universal amplification primer (Invitrogen). The product of this reaction was purified and sequenced with the nested primer C-22081 (5⬘-CACAACTACGACGAGCGTGG-3⬘). A cDNA of the 3⬘ end of thelef-3mRNA was generated with an oligo(dT)-containing adaptor primer (Invitrogen) and primer C-21846 (5⬘-CCAACACCG ACGACGACTACTACAC-3⬘). The product was PCR amplified with primer C-21912 (5⬘-GGAATTCAATGGAGGAAGACGACAGC-3⬘) and the abridged universal amplification primer (Invitrogen). The product of this reaction was purified and sequenced with the nested primer C-22437 (5⬘-GTTGGGTTTGC TGAAATACG-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) for immunoblotting. The immunoblot membranes were blocked with 5% skim milk powder overnight and then probed with a 1:10,000 dilution of rabbit polyclonal antibodies, washed, incubated with a 1:30,000 dilution of donkey anti-rabbit immunoglobulin conjugated to horseradish peroxidase, and visualized with a chemiluminescent detection system (NEN).
Sf21 cells on coverslips, either infected with whole virus or transfected with plasmid DNA, were prepared for immunofluorescence by washing with PBS, fixing with 10% paraformaldehyde for 10 min at room temperature, washing, and then permeabilizing in 100% methanol for 20 min at⫺20°C. Following three washes with PBS-T (PBS plus 0.1% Tween 20), the cells were blocked for 1 h in 1% goat serum in PBS-T, then incubated with rabbit-polyclonal anti-AcMNPV P143 (1:1,000) and/or mouse-monoclonal anti-AcMNPV LEF-3 (1:1,000)
on November 8, 2019 by guest
http://jvi.asm.org/
bodies for 1 h at room temperature. Following a wash with PBS-T, the coverslips were incubated for 1 h in goat anti-rabbit secondary antibody conjugated with Alexa Fluor 568 (Molecular Probes) and/or goat-anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes). The coverslips were again washed with PBS-T, then mounted on glass microscope slides in 50% glycerol. The slides were examined with a Meridian InSight Plus confocal microscope and a KX85 camera (Apogee Instruments). Color images were generated and ana-lyzed with Max Im DL version 2.00 (Cyanogen Productions) (Cancer Research Labs at Queen’s University).
Transient DNA replication assays.Sf21 cells (106cells) in 35-mm-diameter
dishes were washed three times with 1 ml of TC-100 medium and then replaced with TC-100 (1.5 ml per dish). A 20⫻stock of DOPE/DDAB (6, 36) liposome chemicals was mixed by vortexing with 1 ml of sterile water. An equal molar amount of plasmids expressing all of the AcMNPV genes essential for viral DNA replication (AcMNPV replication library: pAcie1, pAclef-1, pAclef-2, pAclef-3, pAcdnapol, pAcp143, pAcp35, and pAcie2pe38) (40) was mixed with a 1:6 ratio of DOPE/DDAB liposome reagent and diluted to a final volume of 200l with TC-100. In some experiments, the plasmids pAcLEF-3 and pAcp143 were re-placed with CfMNPV-expressing plasmid pIE1hrCflef-3, pIE1hrCfp143, or pIE1hrCfp143GFP. After incubation of the transfection mixture for 30 min at room temperature, 500l of TC-100 medium was added to the DNA-DOPE mixture, and the entire mixture was added to washed Sf21 monolayers and incubated at 28°C for 6 h. After incubation, the cells were washed three times with TC-100 medium, covered with fresh TC-100 supplemented with 10% fetal calf serum, and incubated at 28°C for 48 h. The replication of plasmid DNA was monitored byDpnI digestion of the total intracellular DNA as previously de-scribed (38).
Nucleotide sequence accession numbers.The sequence of the CfMNPVp143
gene region has been deposited with GenBank under the accession number AF127530. The sequence of the CfMNPVlef-3gene region has been deposited with GenBank under the accession number AF127908.
RESULTS
Identification and sequence of the CfMNPVp143andlef-3
genes. The promoter region of the CfMNPV p143 gene was
previously located within theHindIII MN2 region, a region at
the right end of the CfMNPVBamHI E fragment (34). Plasmid
clones of these fragments were used as templates with a variety of synthetic primers to complete 4,788 bp of sequence that revealed 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% identical
with the amino acid sequence of Orgyia pseudotsugata NPV
(OpMNPV) P143 and 57% identical with AcMNPV P143
(Table 1). Although recognizable, the CfMNPV P143 gene was
only 21 to 36% identical to the homologous genes of other NPV and granulovirus (GV) P143s (Table 1). All of the NPV P143s were conserved in size, ranging from 1,218 to 1,223 amino acids; however, the GV P143s were smaller (1,124 to 1,159 amino acids). Comparisons of the P143 amino acid
se-quences revealed that CfMNPV P143 retains the conserved
helicase motifs (motifs I, Ia, II, III, IV, V, and VI) that we
previously 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 baculovirus P143 proteins. Additional regions of P143 contain conserved amino acids, including a region previously shown to extend the
host range of AcMNPV toB. moricells (AcMNPV amino acids
551 to 578, CfMNPV amino acids 558 to 584). This
27-amino-acid region is 74% identical between AcMNPV and CfMNPV
and is highly conserved between all P143 proteins identified to date.
The CfMNPVlef-3gene was predicted to map downstream
of the DNA polymerase gene previously identified near the
right end of the CfMNPVEcoRI G fragment (26). Sequence
analysis of this region identified a 1,119-bp ORF (373 amino
acids, 43.0 kDa) predicted to code for the CfMNPVlef-3
ho-mologue. ORFs corresponding toiap2(252 amino acids, 28.1
kDa) andvlf-1(374 amino acids, 43.2 kDa) homologues were
also identified in this region (Fig. 1). CfMNPV LEF-3 is about
75% identical in amino acid sequence with the OpMNPV
LEF-3 protein but exhibits much lower levels of similarity with other baculovirus LEF-3 proteins (Table 2). In addition, the LEF-3 proteins varied considerably in size from 297 amino
acids (Plutella xylostellaGV [PxGV]) to 422 (Spodoptera exigua
NPV). The sequences of the upstream genesslp,dnapol, and
part oforf71have been previously described (26, 27).
Transcription analysis ofp143andlef-3.The expression of
the CfMNPVp143andlef-3genes was investigated by
North-ern blot analysis using gene-specific riboprobes hybridized to
poly(A)⫹RNA prepared from Cf124T cells at various times
after CfMNPV infection. A 4.4-kb transcript was detected by
6 h postinfection with ap143-specific probe spanning the
com-plete p143 ORF (Fig. 2B). This transcript continued to
in-crease in abundance from 12 through 48 h postinfection and appeared to be the major mRNA from this region. A
compar-ison of thep143promoter 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⬘RACE
using primers shown in Fig. 2A. Two different mRNA rations were used to generate cDNAs and both cDNA prepa-rations were subjected to PCR and sequence analysis. The results were identical. Two PCR products, produced with
FIG. 1. Location of identifiable ORFs in the sequenced region of
p143andlef-3. The regions of CfMNPV that were sequenced to identify thelef-3(above) andp143(below) genes are indicated and oriented on the CfMNPV genomeEcoRI restriction fragment map. The presence of ORFs with predicted functions is indicated as filled arrows above the scale in base pairs. ORFs with homologues in OpMNPV and AcMNPV and their numbers but no specific function are indicated as open arrows below the scale line.
on November 8, 2019 by guest
http://jvi.asm.org/
primer C-8114 (Fig. 2C), were sequenced identifying a strong start site at 188 (more abundant PCR product) and a weaker site 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 the
AcMNPVp143transcription start site was previously mapped
by primer extension analysis about 30 nucleotides closer to the translation start codon (30). The major start site mapped 10 nucleotides upstream of a CAGT sequence that is conserved
between CfMNPV and OpMNPV. Although the sequence
downstream ofp143is relatively A/T rich, there is no potential
polyadenylation signal sequence (AATAAA) in the sequenced
region downstream of the p143 ORF. However, 3⬘ RACE
sequencing of the PCR produced with primer C-22080 mapped
thep143polyadenylation addition site 142 nucleotides
down-stream of the stop codon, following a highly AT-rich region of almost 30 nucleotides. Together these data indicate a
mini-mum size of 4,015 nucleotides for thep143transcript, in good
agreement with the size of thep143mRNA seen on Northern
blots.
A 1.6-kb transcript was detected at 6 h postinfection with a CfMNPVlef-3-specific probe spanning the completelef-3ORF plus 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 at
24 and 48 h postinfection. Thelef-3transcription start site was
mapped by 5⬘ RACE and sequencing of the PCR product
produced with primer C-6721 (Fig. 2C). A single site at the beginning of the sequence AACATTGA 279 nucleotides
up-stream of thelef-3ORF 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 of
OpMNPV (1) and AcMNPV (23) revealed a conserved TATA
box sequence about 25 nucleotides upstream of the
transcrip-tion start site. The CfMNPV and OpMNPVlef-3transcription
start sites mapped within one nucleotide of each other, just upstream of a conserved CATTGA sequence. However, the
transcription start site for the AcMNPVlef-3 gene has been
mapped 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 of the translation stop codon and 14 nucleotides downstream of a polyadenylation addition signal. The determined size for the
lef-3 mRNA (1,528 nucleotides) was in good agreement with the estimated size of the transcript (1.6 kb) observed on North-ern blots.
Protein expression of CfMNPVlef-3.The expression of the
CfMNPVlef-3 gene was investigated by immunoblotting to
de-termine the time and level of protein expression in CfM
NPV-infected cells. Cf124T cells, NPV-infected with CfMNPV, were
har-vested at various time points postinfection and the infected cell samples were analyzed by immunoblotting using a rabbit
poly-clonal antibody directed against CfMNPV LEF-3. A 44-kDa
band, first detected by 8 h postinfection, increased in expression
levels through to 24 h postinfection (Fig. 4). The CfMNPV LEF-3
protein increased in expression until at least 48 h postinfection (data not shown). The observed molecular mass coincided closely
with the predicted molecular mass of 43.0 kDa for the CfMNPV
[image:4.603.42.549.81.198.2]LEF-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) Op(1,223)MNPV Ld(1,218)MNPV Se(1,222)MNPV (1,124)PxGV (1,158)TnGV XcGV b
(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)
aThe number of amino acid residues in the individual P143 proteins is given in parentheses. bXcGV,Xestia c-nigrumGV.
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)
aThe number of amino acid residues in the individual LEF-3 proteins is given in parentheses. bHaSNPV,Helicoverpa armigeraSNPV.
on November 8, 2019 by guest
http://jvi.asm.org/
[image:4.603.43.547.606.716.2]replication, the CfMNPVlef-3gene was expressed prior to the reported time of initiation of viral DNA replication (25). For
comparison, an immunoblot of AcMNPV-infected Sf21 cells was
prepared. AcMNPV LEF-3 was easily detectable at 4 h
postin-fection confirming that the virus replication cycle proceeds faster
for AcMNPV than CfMNPV as previously noted (25). Similar
blots were also probed with polyclonal antibodies against the
AcMNPV P143 protein but no signal was detected, indicating that
these antibodies did not cross-react with CfMNPV P143 (data not
shown).
Interaction between AcMNPV and CfMNPV P143 and
LEF-3. We have previously shown that in AcMNPV-infected
Sf21 cells, LEF-3 is essential for the translocation of P143 into the nucleus (39). It was important to demonstrate that this
function is required in other baculovirus systems since thelef-3
gene is not conserved in all baculoviruses. In addition, because we were interested in investigating the interaction between the
CfMNPV LEF-3 and P143 proteins in the absence of other
viral proteins, plasmids expressing the CfMNPV p143
(pIE1hrCfp143) orlef-3(pIE1hrCflef-3) genes, both driven by
the AcMNPV immediate-early promoter-1 (ie-1), were
con-structed and individually transfected into Sf21 cells. To confirm
the expression of CfMNPV LEF-3 from this plasmid, Sf21 cell
extracts obtained at 24 h posttransfection were analyzed by
immunoblotting. CfMNPV-infected Cf124T cells were used as
a positive control. A 44-kDa CfMNPV protein was observed in
both pIE1hrCflef-3-transfected Sf21 cells and in CfM
NPV-infected Cf124T cells (Fig. 5A). Larger amounts of LEF-3 were observed in Sf21 cells transfected with the expression vector
than were observed in CfMNPV-infected Cf124T cells, clearly
demonstrating the expression of large amounts of LEF-3 in the transfected cells. No band was detected in the control mock-infected cells.
Attempts were made to monitor the expression of CfMNPV
P143 in pIE1hrCfp143-transfected Sf21 cell extracts by
immu-noblotting using a polyclonal antibody against AcMNPV P143
but 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 reading frames, and the location of the strand-specific riboprobes used in the Northern analysis for the CfMNPVp143andlef-3genes. Also shown are the names and locations of the primers used in the 5⬘and 3⬘RACE analysis to map the 5⬘and 3⬘ends of thep143andlef-3mRNAs. (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 gels were probed with strand-specific riboprobes corresponding to thep143andlef-3genes. Similarly prepared poly(A)⫹RNA from mock-infected cells
was 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 analysis of thep143andlef-3mRNA 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).
on November 8, 2019 by guest
http://jvi.asm.org/
FIG. 3. Promoter sequence forp143andlef-3. An alignment of the promoter regions of thep143(A) andlef-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 CfMNPV
p143andlef-3genes, as determined by sequence analysis of PCR products, are shown with arrows. (C) The sequences of the 3⬘ends of thep143
andlef-3mRNAs as determined by 3⬘RACE and sequence analysis of PCR products are shown below the appropriate genomic sequence.
on November 8, 2019 by guest
http://jvi.asm.org/
CfMNPV P143, a plasmid was constructed where the CfMNPV
p143gene was fused in frame with the GFP reporter gene and
the fusion product was driven by the AcMNPVie-1promoter
(pIE1hrCfp143GFP). A protein of the predicted size was ob-served in extracts of pIE1hrCfp143GFP-transfected cells when probed with an antibody directed against GFP (Fig. 5B). In-tracellular location of the fusion protein was monitored by direct fluorescence microscopy. When pIE1hrCfp143GFP was transfected on its own, GFP fluorescence was only observed in the 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 AcMNPVie-1promoter was functional in both
Sf21 and Cf124T cells since both Lef-3 and P143 were ex-pressed under the control of this promoter in Cf124T cells, a result that has not been previously demonstrated. When pIE1hrCfp143GFP was transfected into Cf124T cells that were
subsequently infected with CfMNPV, CfMNPV P143-GFP
lo-calized to the nucleus (Fig. 6C). This result demonstrated that the fusion of GFP to P143 did not interfere with its recognition
and successful translocation to the nucleus by the CfMNPV
LEF-3 protein. We confirmed that this translocation was
me-diated by the CfMNPV LEF-3 protein by cotransfecting
pIE1hrCfp143GFP and pIE1hrCflef-3 into Cf124T or Sf21 cells (Fig. 6D). GFP fluorescence was observed in the nuclei of cotransfected cells, clearly demonstrating that GFP-tagged
CfMNPV P143 could interact with CfMNPV LEF-3. These
results also demonstrate that no C. fumiferana cell-specific
factors were required for the interaction between CfMNPV
P143 and LEF-3 since correct nuclear localization occurred in both Cf124T and Sf21 cells.
We then investigated the interaction of P143 and LEF-3
de-rived from the two different species of baculoviruses, AcMNPV
and CfMNPV. Cf124T cells or Sf21 cells were transfected with
pIE1hrCfp143GFP, then infected with AcMNPV. GFP
fluores-cence was detectable in the cytoplasm in both cell lines (Fig. 6E
and G), indicating that AcMNPV LEF-3 did not transport
CfMNPV P143 to the nucleus in either cell line. These results
suggest that a specific interaction between homologous P143 and LEF-3 is required for the correct nuclear transport of P143. This hypothesis was confirmed by cotransfecting Sf21 cells
with pIE1hrCfp143GFP and plasmids expressing either Ac
M-NPV LEF-3 or CfMNPV LEF-3. Nuclear fluorescence of P143,
indicating transport to the nucleus, was only observed when
CfMNPV LEF-3 was present (Fig. 6F and H). Because the
[image:7.603.92.492.80.277.2]correct localization of P143 and LEF-3 to the nucleus is re-quired for baculovirus DNA replication, we then investigated the interaction of the heterologous gene products to determine whether this interaction was necessary for DNA replication.
[image:7.603.49.277.465.640.2]FIG. 4. Temporal expression of CfMNPV LEF-3 in infected cells. Cf124T cells, infected with CfMNPV, were harvested at the indicated times after infection (A). Whole-cell extracts were resolved by SDS– 10% PAGE, blotted onto nitrocellulose filters, and then probed with polyclonal antibodies against CfMNPV LEF-3. CfMNPV LEF-3 was first clearly detectable at 8 h postinfection. For comparison, a similar blot of extracts prepared from AcMNPV-infected Sf21 cells and probed with LEF-3-specific polyclonal antibody is shown. (B) AcMNPV LEF-3 was first detectable at 4 h postinfection.
FIG. 3—Continued.
on November 8, 2019 by guest
http://jvi.asm.org/
Heterologous proteins in DNA replication.DNA replication assays were performed by transfecting Sf21 cells with a series
of plasmids that together express nine AcMNPV genes (ie-1,
ie-2,p143,dnapol,lef-1,lef-2,lef-3,pe38, andp35) necessary for viral replication. Total intracellular DNA was harvested at 48 h
posttransfection and digested withDpnI to distinguish between
unreplicated input plasmid DNA and newly replicated DNA.
As we have previously shown (40), when all nine AcMNPV
genes are expressed together, they support the replication of any plasmid DNA, including those expressing the viral proteins (Fig. 7). If any of the plasmids, including those expressing P143 or LEF-3, was eliminated from the mixture, no plasmid
replica-tion occurred (Fig. 7A, lanes 8 and 9). Replacement of AcMNPV
p143 with its CfMNPV homologue did not restore replication
function (Fig. 7A, lane 10) but replacement of AcMNPVlef-3by
its CfMNPV homologue did (Fig. 7A, lane 11). Replacement of
both AcMNPVp143andlef-3genes with their CfMNPV
homo-logues also restored plasmid DNA replication (Fig. 7A, lane 12).
These results demonstrated that CfMNPV LEF-3 did interact
with either AcMNPV P143 or CfMNPV P143 and complemented
viral DNA replication in the presence of the other AcMNPV
genes. However, CfMNPV P143 alone did not support plasmid
DNA 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 the
nuclei of cells expressing AcMNPV LEF-3, these results suggest
that only a small fraction of the expressed P143 is required to support 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 this fusion protein in DNA replication by replication assays. The plas-mid pIE1hrCfp143GFP worked as well as a plasplas-mid expressing
normal CfMNPV P143 in supporting DNA replication (Fig. 7B,
[image:8.603.60.263.67.334.2]lanes 9 and 10). Thus, these data show for the first time that cross species complementation of P143 in baculovirus transient repli-cation assays can occur, even with distantly related NPVs, if P143 is correctly transported to the nucleus.
[image:8.603.300.543.312.603.2]FIG. 5. Expression of LEF-3 and P143-GFP following transfection or infection and detected by immunoblotting. (A) Whole-cell extracts (5⫻104cells per lane) were prepared from mock-infected Sf21 cells (lane 1), mock-infected Cf124T cells (lane 2), CfMNPV-infected Cf124T cells (lane 3), or pIE1hrCflef-3-transfected Sf21 cells (lane 4) at 24 h posttransfection or postinfection. The extracts were analyzed by SDS–10% PAGE, transferred to a nitrocellulose membrane and probed with LEF-3-specific polyclonal antibody. The relative mobility of molecular weight markers is shown on the left and the immunore-active proteins are labeled on the right. (B) Whole-cell extracts (5⫻ 104cells per lane) were prepared from pIE1hrCfp143GFP-transfected Sf21 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, transferred to a nitrocellulose membrane and probed with anti-GFP monoclonal antibody. The relative mobility of the molecular weight markers is shown on the left and the immunoreactive proteins are labeled on the right.
FIG. 6. Intracellular localization of P143 and LEF-3 following transfection detected by immunofluorescence. Cf124T (A, C, D, and E) or Sf21 (B and F to H) cells, transfected with plasmids expressing CfMNPV P143 fused to GFP (pIE1hrCfp143GFP) (A to H), CfMNPV LEF-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 observed directly 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 stained with DAPI. Only infection with CfMNPV or cotransfection with CfMNPV LEF-3 resulted in nuclear GFP fluorescence from CfMNPV P143-GFP.
on November 8, 2019 by guest
http://jvi.asm.org/
DISCUSSION
The baculovirus protein P143 was first shown to be essential for viral replication by analysis of a temperature sensitive
AcMNPV mutant defective in viral DNA replication (13, 29).
We later showed that another viral protein, LEF-3, identified as a single-stranded DNA binding protein (14) with DNA-destabilizing properties (33), was an essential transporter for
localizing 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 play a role in species specificity of virus infection. Substitution of as few as two amino acids within a specific region of P143
be-tween the very closely related baculoviruses AcMNPV and
BmNPV altered the replication efficiency of AcMNPV inB.
moricells (3, 17). However, the basis of the block of AcMNPV
replication inB. moricells was not investigated. To investigate
the possible role of P143 in regulating viral DNA replication in
other host species, we identified, cloned and sequencedp143
andlef-3from CfMNPV. The CfMNPV P143 predicted amino
acid 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, but
CfMNPV LEF-3 was still most similar to the OpMNPV
homo-logue (75% identical) and only 39% identical with AcMNPV
LEF-3. The transcription patterns of both the CfMNPVp143
and lef-3 genes were consistent with their being early genes, essential for viral DNA replication. Both gene transcripts were detectable by 6 h postinfection, well before the time of increase
in CfMNPV DNA replication (25). In addition, the transcripts
for both genes increased in abundance up to 48 h postinfection, suggesting that they were transcribed for extended periods in Cf124T cells. These data support our previous studies, which
revealed a slower replication cycle of CfMNPV in these cells
than AcMNPV in Sf21 cells (25). The similarity between the
CfMNPV genes and their OpMNPV homologues was also
ev-ident in the sequence and location of their transcription start
sites. We identified the CfMNPV LEF-3 transcription start site
by 5⬘ RACE to be located 25 nucleotides downstream of a
potential TATA box sequence starting at the first A in the sequence AACATTGA. This corresponds exactly with the
identified OpMNPV LEF-3 start site (1). The AcMNPV LEF-3
transcription start site was mapped about 14 nucleotides
down-stream of this region (23). Two CfMNPV P143 transcription
start sites were identified by 5⬘RACE, at 188 and 371
nucle-otides upstream of the translation start codon. Both of these
sites represent conserved regions in OpMNPV and AcMNPV,
although the AcMNPV p143 transcription start site was
[image:9.603.49.279.83.600.2]mapped about 30 nucleotides closer to the translation start codon (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 a collection of plasmids, which together expressed the AcMNPV genes necessary for plasmid DNA replication (ie-1,dnapol,lef-1,lef-2,p35,
pe38, andie-2) exceptp143andlef-3. In separate transfections, this library was supplemented with plasmids expressing the AcMNPVp143
(Acp143), AcMNPV lef-3 (Aclef3), CfMNPV p143 (Cfp143) or CfMNPV lef-3(Cflef3) genes. Following incubation for 48 h, total intracellular DNA was prepared and digested withEcoRI (⫺DpnI) to linearize the plasmids or with EcoRI and DpnI (⫹DpnI) to detect replicated plasmid DNA. Southern blots of these restriction digestion DNA 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, and AcMNPV P143 and CfMNPV LEF-3 (A). Similar assays were also done 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.
on November 8, 2019 by guest
http://jvi.asm.org/
DNA replication are different from those of other early genes, many of which have transcription starts sites beginning with CAGT, likely reflecting different regulatory pathways for these genes. A CAGT sequence located 10 nucleotides downstream
of the mappedp143 transcription start site is also present in
OpMNPV. However, our 5⬘RACE analysis indicated that this
site was not used as a transcription start site.
Antibodies raised against CfMNPV LEF-3 reacted with a
44-kDa polypeptide expressed in virus-infected Cf124T cells from about 8 h postinfection, correlating well with the
expres-sion of the CfMNPV lef-3 transcript. Immunofluorescence
studies showed that CfMNPV LEF-3 was always observed in
the nucleus indicating that it carries the necessary signals re-quired for nuclear localization. Unfortunately, our polyclonal
antibodies directed against AcMNPV P143 did not cross-react
with the CfMNPV gene product and we have had no success at
overexpressing this protein to prepare CfMNPV P143-specific
antibodies so we could not study CfMNPV P143 expression
directly in virus-infected cells. We developed an alternative
method by preparing a plasmid which expressed a CfMNPV
P143-GFP fusion protein. The expression of the fusion protein was monitored by fluorescence of the GFP reporter compo-nent. These studies revealed that P143 remained cytoplasmic when expressed on its own, but was nuclear when coexpressed
with 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 the presence of LEF-3 although the specific role that LEF-3 plays in this process is unknown. Because LEF-3 may exist as a homotrimer (11), it is too large to diffuse through nuclear pores on its own, so it likely carries a nuclear localization target signal, which provides a signal sequence for LEF-3 interaction with cellular importin complexes for delivery to the nuclear pores and nuclear import (16). Because P143 does not appear to carry a nuclear signal sequence, the interaction between P143 and LEF-3 must establish a complex that is then recog-nized by this host transporting machinery. We have initiated studies to identify possible cellular components of this com-plex.
CfMNPV P143-GFP was also localized to the nuclei of Sf21
cells in the presence of CfMNPV LEF-3, suggesting that noC.
fumiferana-specific cell factors are essential for the correct translocation 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 in
Sf21 cells, suggesting that virus species specificity is important to the interaction of P143 and LEF-3. Other researchers have
attempted to rescue AcMNPV P143 with a heterologous P143
from OpMNPV, SeMNPV or Trichoplusia niGV, but these
experiments were unsuccessful (2, 5, 15). In these cases, rescue of P143 function was monitored by transient DNA replication assays. Based on those published results, we hypothesized that one reason these experiments failed was the lack of the ho-mologous LEF-3, which would recognize and transport P143 to its site of action in the nucleus. Our replication assay results
confirmed this hypothesis. As expected, CfMNPV P143 did
not rescue DNA replication in the presence of all the other
AcMNPV replication genes. However, replacing both
AcMNPV P143 and LEF-3 with their CfMNPV counterparts
restored the replication function in the presence of the
remain-der of the AcMNPV replication proteins. These results suggest
that a major factor in baculovirus replication is represented by the P143–LEF-3 complex. Our transient replication assays
demonstrated that replacement of AcMNPV LEF-3 with
CfMNPV 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 function of P143, possibly in conjunction with LEF-3, during DNA replication. However, no specific role of LEF-3 in baculovirus DNA replication has been demonstrated, so it is still not clear what its actual function is. We also confirmed that adding the GFP tag to P143 did not disrupt its ability to function during viral DNA replication since this construct was still able to rescue DNA replication in the transient assays. These results demonstrate that this fusion protein will be a useful tool for investigating the in vivo localization of P143 during viral rep-lication. We are continuing to investigate the interaction of these and other viral proteins in the assembly of a functional replication 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 Institute of Health Research.
REFERENCES
1. Ahrens, C. H., C. Carlson, and G. F. Rohrmann.1995. Identification, se-quence, and transcriptional analysis oflef-3, a gene essential forOrgyia pseudotsugatabaculovirus DNA replication. Virology210:372–382. 2. Ahrens, C. H., and G. F. Rohrmann.1996. The DNA polymerase and
helicase genes of a baculovirus ofOrgyia pseudotsugata. J. Gen. Virol.77: 825–837.
3. Argaud, O., L. Croizier, M. Lo´pez-Ferber, and G. Croizier.1998. Two key mutations in the host-range specificity domain of the p143 gene of Autogra-pha californicanucleopolyhedrovirus are required to killBombyx morilarvae. J. Gen. Virol.79:931–935.
4. Bell, S. P., and A. Dutta.2002. DNA replication in eukaryotic cells. Annu. Rev. Biochem.71:333–374.
5. Bideshi, D. K., and B. A. Federici.2000. TheTrichoplusia nigranulovirus helicase is unable to support replication ofAutographa californica multicap-sid nucleopolyhedrovirus in cells and larvae ofT. ni. J. Gen. Virol.81:1593– 1599.
6. Campbell, M. J.1995. Lipofection reagents prepared by a simple ethanol injection technique. BioTechniques18:1027–1032.
7. Carstens, E. B.1982. Mapping the mutation site of anAutographa californica
nuclear polyhedrosis virus polyhedron morphology mutant. J. Virol.43:809– 818.
8. Cartier, J. L., P. A. Hershberger, and P. D. Friesen.1994. Suppression of apoptosis in insect cells stably transfected with baculovirusp35: dominant interference by N-terminal sequencesp351–76. J. Virol.68:7728–7737.
9. Chomczynski, P.1993. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15:532–537.
10. Chomczynski, P., and N. Sacchi.1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Bio-chem.162:156–159.
11. Evans, J. T., and G. F. Rohrmann.1997. The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution. J. Virol. 71:3574–3579.
12. Evans, J. T., G. S. Rosenblatt, D. J. Leisy, and G. F. Rohrmann.1999. Characterization of the interaction between the baculovirus ssDNA-binding protein (LEF-3) and putative helicase (P143). J. Gen. Virol.80:493–500. 13. Gordon, J. D., and E. B. Carstens.1984. Phenotypic characterization and
physical mapping of a temperature-sensitive mutant ofAutographa califor-nicanuclear polyhedrosis virus defective in DNA synthesis. Virology138: 69–81.
14. Hang, X., W. Dong, and L. A. Guarino.1995. Thelef-3gene ofAutographa californicanuclear polyhedrosis virus encodes a single-stranded DNA-bind-ing protein. J. Virol.69:3924–3928.
15. Heldens, J. G. M., Y. Liu, D. Zuidema, R. W. Goldbach, and J. M. Vlak.1997.
on November 8, 2019 by guest
http://jvi.asm.org/
Characterization of a putativeSpodoptera exiguamulticapsid nucleopolyhe-drovirus helicase gene. J. Gen. Virol.78:3101–3114.
16. Jans, D. A., C-Y. Xiao, and M. H. C. Lam.2000. Nuclear targeting signal recognition: a key control point in nuclear transport? BioEssays22:532–544. 17. Kamita, S. G., and S. Maeda.1996. Abortive infection of the baculovirus
Autographa californicanuclear polyhedrosis virus in Sf-9 cells after mutation of the putative DNA helicase gene. J. Virol.70:6244–6250.
18. Kamita, S. G., and S. Maeda.1997. Sequencing of the putative DNA heli-case-encoding gene of theBombyx morinuclear polyhedrosis virus and fine-mapping of a region involved in host range expansion. Gene190:173–179. 19. Koetsier, P. A., J. Schorr, and W. Doerfler.1993. A rapid optimized protocol
for downward alkaline Southern blotting of DNA. BioTechniques15:260– 262.
20. Kool, M., C. H. Ahrens, R. W. Goldbach, G. F. Rohrmann, and J. M. Vlak. 1994. Identification of genes involved in DNA replication of theAutographa californicabaculovirus. Proc. Natl. Acad. Sci. USA91:11212–11216. 21. Koonin, E. V.1993. A common set of conserved motifs in a vast variety of
putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res.21:2541– 2547.
22. Laufs, S., A. Lu, L. K. Arrell, and E. B. Carstens.1997.Autographa califor-nicanuclear polyhedrosis virusp143gene product is a DNA-binding protein. Virology228:98–106.
23. Li, Y., A. L. Passarelli, and L. K. Miller.1993. Identification, sequence, and transcriptional mapping oflef-3, a baculovirus gene involved in late and very late gene expression. J. Virol.67:5260–5268.
24. Liu, G., and E. B. Carstens.1999. Site-directed mutagenesis of the AcMNPV
p143gene: effects on baculovirus DNA replication. Virology253:125–136. 25. Liu, J. J., and E. B. Carstens.1993. Infection ofSpodoptera frugiperdaand
Choristoneura fumiferanacell lines with the baculovirusChoristoneura fumif-erananuclear polyhedrosis virus. Can. J. Microbiol.39:932–940.
26. Liu, J. J., and E. B. Carstens.1995. Identification, localization, transcription and sequence analysis of theChoristoneura fumiferananuclear polyhedrosis virus DNA polymerase gene. Virology209:538–549.
27. Liu, J. J., and E. B. Carstens.1996. Identification, molecular cloning, and transcription analysis of theChoristoneura fumiferananuclear polyhedrosis virusspindle-likeprotein gene. Virology223:396–400.
28. Lu, A.1993. Nucleotide sequence, transcriptional mapping, regulation, and overexpression of an essential early baculovirus gene. Ph.D. thesis. Queen’s University, Kingston, Ontario, Canada.
29. Lu, A., and E. B. Carstens.1991. Nucleotide sequence of a gene essential for viral DNA replication in the baculovirusAutographa californicanuclear polyhedrosis virus. Virology181:336–347.
30. Lu, A., and E. B. Carstens.1992. Transcription analysis of theEcoRI D region of the baculovirusAutographa californicanuclear polyhedrosis virus identifies an early 4-kilobase RNA encoding the essential p143 gene. J. Virol. 66:655–663.
31. Lu, A., and L. K. Miller.1995. The roles of eighteen baculovirus late ex-pression factor genes in transcription and DNA replication. J. Virol.69:975– 982.
32. McDougal, V. V., and L. A. Guarino.2001. DNA and ATP binding activities of the baculovirus DNA helicase P143. J. Virol.75:7206–7209.
33. Mikhailov, V. S.2000. Helix-destabilizing properties of the baculovirus sin-gle-stranded DNA-binding protein (LEF-3). Virology270:180–189. 34. Qiu, W., J. J. Liu, and E. B. Carstens.1996. Studies of Choristoneura
fumiferananuclear polyhedrosis virus gene expression in insect cells. Virol-ogy217:564–572.
35. Smith, D. B., and K. S. Johnson.1988. Single-step purification of polypep-tides expressed inEscherichia colias fusions with glutathioneS-transferase. Gene67:31–40.
36. Staggs, D. R., D. W. Burton, and L. J. Deftos.1996. Importance of liposome complexing volume in transfection optimization. BioTechniques21:792–794. 37. Tjia, S. T., E. B. Carstens, and W. Doerfler.1979. Infection ofSpodoptera frugiperdacells withAutographa californicanuclear polyhedrosis virus. II. The viral DNA and the kinetics of its replication. Virology99:391–409. 38. Wu, Y., and E. B. Carstens.1996. Initiation of baculovirus DNA replication:
early promoter regions can function as infection-dependent replicating se-quences in a plasmid-based replication assay. J. Virol.70:6967–6972. 39. Wu, Y., and E. B. Carstens.1998. A baculovirus single-stranded DNA
bind-ing protein, LEF-3, mediates the nuclear localization of the putative helicase P143. Virology247:32–40.
40. Wu, Y., G. Liu, and E. B. Carstens.1999. Replication, integration, and packaging of plasmid DNA following cotransfection with baculovirus viral DNA. J. Virol.73:5473–5480.
