P34.8 (GP37) is not essential for baculovirus replication
Xiao-Wen Cheng1,
Peter J. Krell2 and
Basil M. Arif1
Laboratory for Molecular Virology, Great Lakes Forestry Center, 1219 Queen St E, Sault Ste Marie, Ontario, CanadaP6A 5M71
Department of Microbiology, University of Guelph, Guelph, Ontario, CanadaN1G 2W12
Author for correspondence: Basil Arif. Fax +1 705 759 5700. e-mail barif{at}nrcan.gc.ca
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Abstract
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Previous reports have indicated that p34.8 (gp37) may be essential for the replication of Autographa californica nucleopolyhedrovirus (AcMNPV) because no virus with inactivated p34.8 was isolated. We have ascertained the requirement for this gene by attempting to inactivate it with a large insertion [the gene encoding GFP (green fluorescent protein)] or by deleting all the conserved domains from the open reading frame (ORF). The gene encoding GFP was inserted into the NotI site of the p34.8 ORF and a viral plaque containing the insertion was propagated in SF-21 cells. Similarly, 531 bp (NotIXbaI) containing all conserved domains were deleted from the ORF. All mutants were authenticated by PCR amplification, restriction endonuclease analysis, DNA sequencing, and Southern and Northern blot analysis. It was found that inactivation of p34.8 of AcUW1-LacZ (AcMNPV containing a lacZ gene in the p10 locus) had no effect on the biological property of virus, such as virulence and kinetics. These two independent methods showed that p34.8 is not essential for replication and that this locus could provide another site for the engineering of baculoviruses.
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Introduction
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The baculovirus genome contains about 150 genes and open reading frames (ORF) some of which are referred to as auxiliary genes that are not essential for replication but give the virus a certain selective advantage in nature (OReilly, 1997
). The virus also contains essential genes that are required for replication and cannot be deleted from the genome. The p34.8 gene (homologous to gp37 and spindlin) of Autographa californica nucleopolyhedrovirus (AcMNPV) was thought to be essential because a p34.8-null mutant could not be isolated in cell lines (Wu & Miller, 1989
). Apart from this report, other attempts made to examine the requirement of p34.8 or its homologues for virus replication have not been published. Baculovirus p34.8 homologues that have been sequenced share 3040% amino acid identity with entomopoxvirus fusolins. Both proteins contain five highly conserved domains which are presumably involved in function (Gross et al., 1993
; Liu & Carstens, 1996
; Phanis et al., 1999
; Li et al., 2000
). Fusolin is not essential for EPV replication and appears to enhance the infectivity of baculoviruses (Gross et al., 1993
; Liu & Carstens, 1996
; Dall et al., 1993
; Gauthier et al., 1995
; Hayakawa et al., 1996
; Mitsuhashi et al., 1997
, 1998
; Afonso et al., 1999
). Recently, the homologue of p34.8 and its encoded product (termed spindlin) in Choristoneura fumiferana defective nucleopolyhedrovirus (CfDEFNPV) were characterized (Li et al., 2000
). Spindlin was shown to accumulate as bipyramidal crystals in the cytoplasm of infected cells. The function of P34.8 is still a matter of inference to fusolin and is yet to be elucidated. Since the two homologues are so closely related, the question arose whether the gene encoding fusolin could substitute p34.8 in AcMNPV. Therefore, the essentiallity of p34.8 first had to be confirmed. It has also been reported that P34.8 is associated with the occlusion bodies (OBs) of AcMNPV (Gross et al., 1993
). If this protein has a similar enhancing function as fusolin, then inactivation of p34.8 may lead to a reduced potency of the virus. In this paper, we report experiments designed to inactivate p34.8 and determine the effects on LD50 and virus replication kinetics.
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Methods
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Construction of transfer vectors to inactivate p34.8.
The virus used in these experiments was AcUW1-LacZ, which is an AcMNPV carrying a lacZ gene in the p10 locus (Pharmingen). The green fluorescent protein (GFP) ORF was excised from pEGFP-1 (Clontech) by digesting with BamHI and NotI (NotI site was blunted with Klenow). The fragment was purified on agarose gels and cloned into the BamHI and XhoI (XhoI site was blunted with Klenow) sites of pBlueBac4.5 (Invitrogen) to generate pBlueGFP (Fig. 1B
). The fragment PstI-H (5·4 kb), which contained p34.8 of AcUW1-LacZ, was cloned into pUC18 (pAcPstH, Fig. 1A
). This plasmid was cleaved with NotI (218 bp downstream of the p34.8 ORF translation initiation codon ATG) and blunted with Klenow. The plasmid pBlueGFP was digested with EcoRV and HindIII, and blunted with Klenow to retrieve the polyhedrin promoter for GFP expression. The GFP fragment was gel purified and cloned into the blunted NotI site of pAcPstH. The transfer vector (pAcGFP), carrying 2·3 kb and 3·1 kb flanking DNA, was sequenced (Fig. 1B
).
SF-21 cells were co-transfected with pAcGFP and AcUW1-LacZ DNAs (OReilly et al., 1992
). Plaque purification was carried out to isolate putative recombinants with GFP inserts. Expression of GFP as well as PCR amplification, restriction endonuclease (REN) analysis, DNA sequencing, and Southern and Northern blot analysis confirmed the authenticity of the recombinant viruses. The PCR primers used to amplify DNA from GFP plaques were located 20 bp downstream of the p34.8 ORF ATG translation initiation codon and 100 bp downstream of the TAA termination signal (forward primer, FP1, 5' CAT TGT TTG CTG CGA TTC AC 3'; reverse primer, RP1, 5' GCA TAG CAG ACT ACG AAT TTG 3'). AcGFP was propagated in SF-21 cells and analysed by REN and Southern blot hybridization.
To construct a transfer vector lacking all the conserved p34.8 domains, pPstH was digested with XbaI and blunted with Klenow. It was then cut with SalI to retrieve a 1·7 kb fragment encoding the C terminus of P34.8. The plasmid pAcGFP was digested with NotI, and blunted. It was then cut with SalI to retrieve a 5·8 kb fragment encoding the N terminus of P34.8 and part of a pUC18 sequence. The two fragments (1·7 and 5·8 kb) were ligated, propagated in DH5
cells and the clone generated termed pAcp34.8- (Fig. 1C
). SF-21 cells were co-transfected with pAcp34.8- and AcGFP DNAs. White plaques containing putative recombinants lacking all the five conserved domains were isolated and initially screened by PCR as indicated above.
REN, Southern and transcription analysis.
AcUW1-LacZ, AcGFP and pAcp34.8- DNAs were digested with PstI, separated on 0·7% agarose gels and transferred onto nylon membranes (Chomczynski, 1992
). The 0·5 kb NotIXbaI fragment internal to the p34.8 ORF was labelled with 32P by the random primer extension method (Sambrook et al., 1989
) and used as a probe in Southern blot analysis. For Northern blot analysis, SF-21 cells were infected, total RNA was extracted at various times thereafter (Chomczynski & Sacchi, 1987
) and analysed (5 µg) on 1% denaturing formaldehydeagarose gels. It was transferred to a nylon membrane and probed with 32P-labelled p34.8 ORF or with the NotIXbaI DNA fragment. In RTPCR analysis for transcription, a total RNA sample obtained at 36 h post-infection was treated with RNase-free DNase (Promega). The same pair of primers (FP1 and RP1) employed in the PCR screening of viral recombinants was used in the RTPCR to detect transcripts from the p34.8 promoter. The reverse primer (RP1) and a 23-mer forward primer (GFPF122) starting at 122 bp from ATG of the GFP ORF were used in the RTPCR to analyse transcripts initiated by the polyhedrin promoter. Negative controls not containing AMV reverse transcriptase were included. An Access RTPCR System from Promega was used in all the RTPCR analyses. The products were analysed on a 0·7% agarose gel.
Bioassay and viral kinetics.
OBs of AcUW1-LacZ and pAcp34.8- were purified by differential and sucrose gradient centrifugation and suspended in distilled water at a concentration of 3x106 OBs/ml (Cheng et al., 1990
). Samples containing 1·5x104, 3x104, 3x105, 1·6x106 and 3x106 OBs/ml were used to determine the LD50 in larvae (Wang & McCathy, 1993
). Briefly, 1 µl of virus suspension was applied onto a small diet plug and third-instar Trichoplusia ni larvae (50 animals per sample) were allowed to feed until all the diet was consumed, before transfer onto fresh diet. Larvae that did not consume all the diet within 24 h were discarded. Control larvae were treated with distilled water. Mortality was scored every day after infection until control larvae pupated. The dose-mortality data were analysed with the aid of the POLO-PC computer program (Le Ora Software Inc., Berkeley, CA, USA) which is based on the probit analysis method described by Finney (1971)
.
Kinetics of pAcp34.8- replication in vitro was compared with that of AcUW1-LacZ. SF-21 cells were infected with AcUW1-LacZ and pAcp34.8- at a multiplicity of 1 p.f.u. per cell. The virus was allowed to adsorb for 1 h and the cells were then washed with medium, re-furnished with fresh medium and incubated at 27 °C. Samples were removed from the infected cells at 12, 24, 36 and 48 h post-infection and assayed for budded viruses by the end-point dilution method (Summers & Smith, 1987
). Differences in virus replication in vitro were analysed by regression analysis by using the Minitab computer program.
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Results
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Inactivation of p34.8
A transfer vector (pAcGFP), carrying the green fluorescence protein (GFP, 0·92 kb) gene under the control of the polyhedrin promoter, was constructed and used to insert the gene in the NotI site of p34.8 (Methods, Fig. 1A
). SF-21 cells were co-transfected with the transfer vector and AcUW1-LacZ DNAs and the putative recombinant progeny virus was initially observed by GFP expression. Progeny virus was plaque purified and green fluorescent plaques were further screened by PCR amplification to determine the proper insertion and orientation of the GFP gene (Fig. 2
). One out of ten plaques yielded a single 1·9 kb PCR product suggestive of an authentic double-crossover recombinant free of the parental genotype. The rest of the plaques yielded both 1·0 kb and 1·9 kb PCR products indicative of a progeny contaminated with the parental genotype, or represented single-crossovers, or both (Fig. 2A
). Recovery of a recombinant virus with disrupted p34.8 suggested that this gene is not essential for virus replication. Sequencing of pAcGFP revealed that the insertion resulted in the introduction of three stop codons upstream of the GFP gene. However, another ORF that contained the five conserved domains was formed from an internal p34.8 ATG codon just downstream of the TAA of the GFP gene (Fig. 1B
). We could not rule out the possibility that the new ORF could somehow still be expressed. To exclude this possibility, it was decided to delete most of the p34.8 ORF, including the five conserved domains. A second transfer vector based on this locus, minus the internal 531 bp NotIXbaI fragment, was constructed (pAcp34.8-, Fig. 1C
, Methods). The deletion encompassed all the five conserved domains. SF-21 cells were co-transfected with this vector and AcGFP viral DNAs. The anticipated deletion mutant would have lost the gene encoding GFP as well as 531 bp from the p34.8 ORF. Progeny virus was plaque purified as GFP- phenotype (Fig. 1C
). A background of phenotypes expressing GFP was also observed. PCR amplification using a pair of p34.8 primers (FP1 and RP1) resulted in the expected 470 bp long product (Fig. 1C
, Fig. 2B
). Amplification of the same region in AcUW1-LacZ with intact p34.8 yielded exclusively a 1 kb product, compared to 1·9 kb expected for p34.8 interrupted with GFP (Fig. 2B
). This showed that during homologous recombination between the transfer vector and AcGFP virus, a double-crossover had occurred which deleted the GFP gene and all the five p34.8 conserved domains. The 470 bp PCR product was cloned into pGEM-T vector (Promega) and sequenced (Fig. 1C
). The sequence confirmed that all the conserved domains were absent from the Acp34.8- viral genome.

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Fig. 2. PCR screening of putative recombinant AcUW1-LacZ. (A) Insertion of the gene encoding GFP. (B) Deletion of the gene encoding GFP and 531 bp from the p34.8 locus. (A) Lanes 110 are PCR products from different GFP+ plaques. Lanes 11, 12 and 13 are PCR controls on transfer vectors pAcPstH, pAcGFP and a mixture of both, respectively. (B) Lanes 1, 2, 3 and 4 are PCR products from AcUW1-LacZ , AcGFP, a GFP- plaque and pAcp34.8-, respectively. M represents 1 kb DNA ladder marker (BioLabs). Arrows point to PCR products from pure plaques. The high molecular mass bands in both A (lanes 11, 12 and 13) and B (lane 4) are the plasmid templates.
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Restriction endonuclease analysis of AcGFP and Acp34.8- substantiated the PCR screening data that a double-crossover event resulted in the formation of the latter virus. When the gene encoding GFP was inserted into the p34.8 locus in the AcUW1-LacZ 5·4 kb PstI-H fragment, two new PstI fragments of 4·1 and 2·3 kb were generated (Fig. 3A
). The 4·1 kb fragment contained the 0·92 kb GFP gene insert. In the deletion mutant, Acp34.8-, the PstI-H fragment had lost 0·531 kb from p34.8 giving rise to a new 4·9 kb PstI fragment (Fig. 3A
). The additional PstI site located after the GFP stop codon in the AcGFP genome was from the transfer vector pAcGFP derived from the multiple cloning site of pBlueBac4.5 (Fig. 1B
). The PstI restriction data were further confirmed by digestion of the viral genomes with EcoRI (data not shown).

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Fig. 3. REN (A) and Southern blot (B) analysis of AcUW1-LacZ , AcGFP and Acp34.8- viral DNAs. (A) Digestion with PstI and analysis on a 0·7% agarose gel. Arrows point to fragments that had changed mobility generated by rearrangement. (B) A Southern blot of DNA from panel (A) probed with 32P random primer-labelled NotIXbaI fragment containing the conserved domains sequence of p34.8 from pAcPstH.
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Viral DNAs digested with PstI (Fig. 3A
) were blotted onto a nylon membrane and probed with 32P-labelled 0·53 kb NotIXbaI fragment from the p34.8 ORF. The probe hybridized to the AcUW1-LacZ fragment PstI-H and to the AcGFP 2·3 kb fragment but not to any of the Acp34.8- DNA fragments (Fig. 3B
). These data clearly confirm that the conserved domains of p34.8 were deleted from the latter virus. Hybridization to EcoRI-digested viral genomes confirmed the above results (data not shown).
Northern blot analysis
Hybridization of 32P-labelled NotIXbaI fragment to Northern blots detected transcripts in SF-21cells infected with AcUW1-LacZ and AcGFP but not in cells infected with Acp34.8- (Fig. 4A
). The size of the transcripts from AcUW1-LacZ and AcGFP were 1 and 1·7 kb, respectively. The larger size of the transcripts in cells infected with AcGFP supports the evidence that the gene encoding GFP was inserted into the p34.8 ORF. The abundant transcription from this locus in AcGFP is attributed to the polyhedrin promoter, which is clearly much stronger than that of p34.8. Transcription from p34.8 promoter was obscured by the abundance of transcripts generated by the polyhedrin promoter. RTPCR analysis corroborated the data from Northern hybridization (Fig. 5
). Using a pair of primers (FP1 and RP1) from p34.8 sequence, a 1·9 kb RTPCR product was amplified. When the GFP forward primer (GFPF122) and p34.8 reverse primer (RP1) were used, an expected 1·4 kb RTPCR product was produced (Fig. 5
). Controls did not show any products indicating that the RTPCR products were from mRNA and not from viral DNA (Fig. 5
). When the total 32P-labelled p34.8 ORF was used as a probe in the Northern blots, the mRNAs from AcUW1-LacZ, AcGFP and Acp34.8- viruses were 1·0, 1·7 and 0·5 kb, respectively (Fig. 4B
). The lack of transcripts from Acp34.8- and the size of the transcripts from the three viruses confirmed that the NotIXbaI part of the ORF had been deleted successfully (Fig. 4A
).

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Fig. 4. Northern blot analysis of transcripts from AcUW1-LacZ, AcGFP and Acp34.8- virus-infected SF-21 cells. The numbers indicate time (h) post-inoculation. (A) Blot probed with 32P random primer-labelled NotIXbaI from pAcPstH. (B) Blot probed with 32P random primer-labelled PCR product of the p34.8 ORF.
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Fig. 5. RTPCR analysis. Total RNA isolated at 36 h post-infection was used as template by AMV reverse transcriptase. Lanes 1 and 2 are RTPCR products from a pair of primers in the p34.8 sequence (FP1 and RP1). Lanes 3 and 4 are products from a forward primer in the GFP ORF sequence (GFPF122) and a reverse primer in the p34.8 sequence (RP1). Lanes 1 and 3 are controls not containing AMV reverse transcriptase. M, 1 kb DNA marker (BioLabs).
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LD50 bioassays and viral kinetics
Since entomopoxvirus fusolin has been reported to have an enhancing effect on baculoviruses, we were interested to find out if its homologue P34.8 had a similar effect. Inactivation of p34.8 offers the possibility of investigating the effect on infectivity of OBs to larvae or on the kinetics of virus replication in tissue culture. The LD50 of purified Acp34.8- OBs was determined in third-instar T. ni larvae by using the diet plug method (Wang & McCathy, 1993
). Parental AcUW1-LacZ was used as a control. No significant difference in LD50 was observed between the deletion mutant and the parental viruses (Table 1
). It has been reported previously that AcMNPV is the only baculovirus identified so far where P34.8 was associated with the OBs (Gross et al., 1993
; Phanis et al., 1999
; Vialard et al., 1990
). The lack of difference in the infectivity of OBs from the two viruses could also be due to the fact that only very small amounts of the protein are associated with the OBs (Gross et al., 1993
; Vialard et al., 1990
). In the kinetics of replication analysis, no significant difference in the slopes of the growth curves between AcUW1-LacZ and Acp34.8- was detected (P=0·326). This indicated that inactivation of p34.8 had no effect on virus replication in SF-21 cells (Fig. 6
).

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Fig. 6. Growth of AcUW1-LacZ and Acp34.8- in SF-21 cells. SF-21 cells were infected with AcUW1-LacZ and Acp34.8- at a multiplicity of 1 p.f.u. per cell. Samples were taken from the infected cells at 12, 24, 36 and 48 h post-infection and assayed for budded viruses by the end-point dilution method. Differences in virus replication were analysed by regression analysis.
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Discussion
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This study was initially undertaken to determine if the EPV gene encoding fusolin could substitute for p34.8 or other homologues in baculoviruses. Unlike the fusolin gene, it was assumed that p34.8 was probably essential for baculovirus replication because p34.8-null mutants could not be isolated (Wu & Miller, 1989
). Since the codon usage of this gene favours A and T residues (Wu & Miller, 1989
), similar to most entomopoxvirus genes, it is likely that during evolution the gene was transferred from EPVs to baculoviruses. If this is true, then could the fusolin gene substitute for p34.8 in baculoviruses? Recently, this gene and its encoded product from CfDEFNPV was characterized and it was shown that the gene product accumulates as bipyramidal crystals in the cytoplasm of infected cells (Gross et al., 1993
; Li et al., 2000
). The question arose as to the essential function of a cytoplasmic protein in baculovirus replication. For this reason, we decided to ascertain the requirement for p34.8 in baculoviruses before attempting to swap it with an EPV fusolin gene.
We attempted to inactivate p34.8 in AcUW1-LacZ with an insertion of the gene encoding GFP (0·92 kb). PCR amplification of the p34.8 locus revealed that a recombinant virus with a GFP insert could be generated. We also deleted all the five conserved domains from the ORF (531 bp). PCR amplification unequivocally identified a virus with a double-crossover substitution that was free from contaminating parental or single-crossover genotypes (Fig. 2B
). No viable virus with inactivated p34.8 was isolated by previous investigators probably because their sample size (eight plaques) was too small and they did not use PCR to identify mutant viruses (Wu & Miller, 1989
). In the experiments reported here, PCR amplification was invaluable in identifying authentic mutants and their purity. Transcripts from the p34.8 deletion mutants had the expected size of 0·5 kb. The presence of viable virus with inactivated p34.8 was authenticated by PCR amplification, REN and Southern, Northern and RTPCR analyses. Also, sequencing of the PstI fragment generated by the deletion demonstrated that 531 bp were missing from the ORF (Fig. 1C
, D
).
Inactivation of p34.8 did not appear to have an effect on the viral LD50 in T. ni larvae or on growth kinetics in SF-21 cells (Table 1
, Fig. 6
). It could be that too little protein is associated with the OBs to have a demonstrable effect on LD50. The plentiful expression of the protein by CfDEFNPV (Li et al., 2000
) should help in determining the function of this protein in baculoviruses.
In conclusion, two independent criteria were used to show that p34.8 was not essential for virus replication. The gene was inactivated with an insert encoding GFP and by deleting the large portion of the ORF containing all the conserved domains. In both cases, viable mutants were isolated and authenticated. This locus in the viral genome could, therefore, potentially provide another site for the engineering of baculoviruses in cases where other non-essential loci, such as those of polyhedrin or p10, needed to remain in the virus.
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Acknowledgments
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We would like to thank Hilary Lauzon and Tim Ladd for assistance in DNA sequencing and Dr Richard Fleming for help in statistical analysis. This research was supported by Canadian NSERC strategic grant number STPO193108.
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Received 14 August 2000;
accepted 20 October 2000.