CSIRO Entomology, GPO Box 1700, Canberra ACT 2601, Australia1
Author for correspondence: Julie Olszewski. Present address: Department of Biological Sciences, Imperial College of Science, Technology, and Medicine, Imperial College Road, London SW7 2AZ, UK. Fax +44 207 584 2056. e-mail j.olszewski{at}ic.ac.uk
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Abstract |
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Introduction |
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While all members of the Poxviridae possess a series of conserved fundamental characteristics such as genome structure and virion morphology, EPVs distinctively and characteristically incorporate their mature intracellular virions into large proteinaceous occlusion bodies known as spheroids (Arif, 1995 ). These structures are believed to reduce the impact of environmental stresses on the occluded virus particles following their release after death of the host, and are thus assumed to function in a manner analogous to counterparts termed polyhedra, produced by insect-infecting members of the baculovirus and reovirus families (Miller, 1996
).
The most abundant component of the EPV spheroid is a high molecular mass (100120 kDa) matrix protein known as spheroidin (SPH), which is apparently uniquely associated with this group of viruses (King et al., 1998 ). SPH from HaEPV has a deduced relative molecular mass of 115438, and is relatively closely related to homologues from other lepidopteran-associated EPVs, showing amino acid identities of 82 and 79% to SPH proteins from Amsacta moorei EPV (AmEPV) and Choristoneura fumiferana EPV (Sriskantha et al., 1997
). SPH is abundantly expressed by HaEPV in both in vivo and in vitro infections (Dall et al., 1993
; J. A. Olszewski & D. J. Dall, unpublished). Similarly, studies of AmEPV in vitro have shown that SPH is the most abundantly expressed viral protein in infected cells (Winter et al., 1995
) and, in addition, that its synthesis is not essential to virus replication (Palmer et al., 1995
).
Recognition of these characteristics led to suggestions that the AmEPV spheroidin locus (sph) might present a valuable site to develop a system for expression of heterologous proteins (Palmer et al., 1995 ; Li et al., 1998
), in a manner similar to that previously developed for the prototypic poxvirus and baculovirus, vaccinia virus and Autographa californica nucleopolyhedrovirus (AcNPV) (Mackett et al., 1982
; OReilly et al., 1992
). In the case of HaEPV, this possibility was further enhanced by observations that the virus could replicate in a non-lytic manner in serum-free cultures of lepidopteran cells (D. J. Dall, unpublished), potentially simplifying the purification of foreign proteins secreted into the culture medium. Additionally, HaEPV replicates in cells which have been widely used for baculovirus expression systems, such as Spodoptera frugiperda (Sf9) and Trichoplusia ni Hi-5 cells (Invitrogen).
Initial experiments on the AmEPV sph locus suggested that it may be less productive than originally envisaged. Palmer et al. (1995) reported that expression of chloramphenicol acetyltransferase from the AmEPV sph locus in Lymantria dispar cells occurred at levels of only 510% of those observed when the same protein was expressed in a baculovirus system under the polyhedrin (polh) promoter. Subsequently, however, it was reported that the activity of the promoter could be substantially improved by strict conservation of the native TAAATG consensus nucleotide sequence at the translation initiation codon (King et al., 1998
). Further to this observation, Li et al. (1998)
demonstrated that high-level expression of
-galactosidase from the AmEPV sph promoter was dependent on retention of native sph coding sequence from the 5' region of the gene, although the fact that expression was from a heterologous genomic locus makes the general validity of this observation more difficult to assess. Taken together, these findings provide only limited support for further development of AmEPV as a protein expression system; nevertheless, the possibility that sph loci of other EPV isolates might possess more favourable capabilities remains to be examined.
Furthermore, AmEPV is unusual among lepidopteran EPVs in lacking the locus that encodes the viral fusolin (FUS) protein. This polypeptide is the primary constituent of EPV-associated spindle bodies (Dall et al., 1993 ; Lai-Fook & Dall, 2000
), and is the most abundant protein in preparations of HaEPV originating from in vivo infections. Homologues of this protein are also found in many baculoviruses, and are generally referred to as gp37 (Phanis et al., 1999
). Gp37 synthesis has recently been inactivated in AcNPV and thus been shown to be non-essential for baculovirus replication (Cheng et al., 2001
). The observation that neither of two fully sequenced EPVs possess copies of the fusolin (fus) gene (Afonso et al., 1999
; Bawden et al., 2000
) suggested that the protein is also unlikely to be essential for replication of HaEPV. On this basis we considered that the HaEPV fus locus might provide another strong viral promoter from which heterologous gene products could be expressed.
In this paper we report the engineering of HaEPV, using a seamless cloning strategy, to exactly replace either the sph or fus gene with nucleotide sequence encoding the green fluorescent protein (GFP; Chalfie et al., 1994 ). We examine the growth kinetics of the recombinant HaEPVs in Sf9 insect cells in culture, report kinetics of GFP production by the recombinants, and compare these with those of a recombinant AcNPV baculovirus, in which production is controlled by the polh promoter.
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Methods |
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Plasmid constructs.
About 0·5 µg or 10 ng of viral genomic or plasmid DNA, respectively, was used as template for PCR amplifications using Taq DNA polymerase (GibcoBRL). Reaction cycles used a template denaturation step of 5 min at 94 °C, followed by 36 cycles of 94 °C for 1 min, 45 to 55 °C for 1 min, and 72 °C for 1 min. PCR products were cloned and their sequence verified using automated DNA sequencing.
A seamless cloning strategy was employed to make the transfer vector pTV497, subsequently used for replacement of the HaEPV sph gene (GenBank accession no. AF019224) with a modified version (see below) of the Aequorea victoria gfp gene. An HaEPV region upstream of the sph gene (-1 to -797) was PCR amplified from wt HaEPV genomic DNA with primers TV497A and TV497B (Table 1). These primers added an EcoRI restriction site at the 5' end of the amplified fragment, and BsmBI, XhoI and BglII restriction sites at the 3' end (Fig. 1A
). After co-digestion with EcoRI and BglII this fragment was cloned into EcoRI/BamHI-linearized pTZ19R vector plasmid (AmershamPharmacia) to create pTV497-UI2. A 1400 bp region downstream of the spheroidin translation termination codon was amplified from wt HaEPV genomic DNA with primers TV497C and TV497D (Table 1
), adding XbaI and HindIII sites at the 5' and 3' ends, respectively, of the product. After digestion this product was cloned into XbaI/HindIII-digested pTV497-UI2 to create pTV497-I.
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A corresponding strategy was employed to insert the same gfp(RS) gene under control of the HaEPV fus promoter. Construction of the transfer vector pTV698, used to replace the HaEPV fus gene (GenBank accession no. L08077) with the gfp(RS) gene, entailed PCR amplification of the region upstream of the fus gene, using wt HaEPV genomic DNA and oligos TV2962A and TV697.1 (Table 1). This procedure introduced BsmBI and XhoI sites at the 3' end of the resulting 1·08 kb product. The product was purified (GeneClean; Bio101), end-filled and kinased using DNA Pol I and T4 polynucleotide kinase (GibcoBRL), following manufacturers protocols. The resultant fragment was cloned into SmaI-digested pTZ19R, and inserted products were screened for correct orientation. A clone with the 3' end of the inserted sequence proximal to the XbaI site of the parental vector was selected, digested with XhoI and XbaI, and a XhoIXbaI-derived gfp(RS) fragment, as described above, was inserted, creating plasmid pFusPR-GFP. The region downstream of the HaEPV fus locus was amplified from wt HaEPV genomic DNA using oligos TV2962C and TV2962D (Table 1
), introducing XbaI and HindIII sites at the 5' and 3' ends, respectively, of the 1·25 kb fragment. This XbaIHindIII product was cloned into similarly digested pFusPR-GFP, and joining of the fus promoter and the GFP(RS)-encoding sequence was achieved as described above, producing the vector pTV698 (Fig. 1F
).
The transfer vector BacPAK8/GFP(RS), expressing gfp(RS) under control of the AcNPV polh promoter was constructed by inserting the XhoIXbaI gfp(RS) fragment into the BacPAK8 vector (Clontech).
Isolation and purification of recombinant viruses.
Recombinant HaEPVs were generated by transfecting transfer vector plasmid DNAs into Sf9 cells using DOTAP reagent (Roche) and standard protocols (OReilly et al., 1992 ) and then, after 18 h, infecting the same cells with wt HaEPV. Media was collected from the transfection/infections 5 days later and used to infect other Sf9 cells. Cells infected with recombinant HaEPVs were selected using fluorescence activated cell sorting (FACS) and introduced into six-well plates that had been lightly seeded with uninfected Sf9 cells. Several rounds of alternating FACS and plaque purification or end-point dilution (OReilly et al., 1992
) were required before a stock free of contaminating parental wt HaEPV, as judged by PCR analysis (see below), was obtained for each recombinant virus.
The BacPAK-GFP(RS) virus was produced by co-transfecting the BacPAK8/GFP(RS) transfer vector with BacPAK6 viral DNA, previously digested with Bsu36I, according to the manufacturers protocols (Clontech). Plaques which contained fluorescent cells were picked, amplified, and subjected to a second round of plaque purification. Verification of the recombinant junction at the polh locus was carried out by PCR analysis using primers Bac1 and Bac2 (Clontech).
PCR screening of virus isolates.
PCR was used to screen for the presence of parental and recombinant HaEPV genomes following amplification in cell culture. Culture medium (25 µl) of was digested with 25 µl Proteinase K (400 µg/ml in 20 mM Tris, pH 8·0, 10 mM EDTA and 0·5% SDS) at 37 °C. Samples were boiled for 10 min, and then diluted with 200 µl dH2O. PCR amplifications used 5 µl of this material as template. Stocks of recombinant viruses were analysed with two primer pairs in order to confirm the presence of the expected recombinant, and absence of parental virus (Fig. 2A, B
).
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Time-course of extracellular virus production.
Sf9 cells (7500 per well) were seeded in 96-well tissue culture-treated microtitre plates (Falcon) and allowed to attach. Infections were established, as described above, for wt HaEPV, HaEPV(fus-) and HaEPV(sph-), at m.o.i. values of 0·1, 0·5 and 2·0 infectious particles per cell. Inocula were carefully removed after 2 h and replaced with fresh medium. At various time-points (time zero being the time at which the infectious inoculum was added) cells and media were collected from duplicate wells for each viral inoculum per m.o.i. Cells were pelleted by microcentrifugation (2000 r.p.m., 5 min) and supernatants were transferred to clean tubes and stored at -80 °C until all samples had been collected. The amount of infectious virus present in each sample was quantified by TCID50 analysis.
Analysis of GFP production by fluorimetry.
Sf9 cells (5000 per well) were seeded in alternating wells of 96-well plates and allowed to attach. Cells were mock-infected or infected with HaEPV(sph-), HaEPV(fus-) or BacPAK-GFP(RS) at m.o.i. values of 0·1, 0·5 or 2·0, in quadruplicate, as described above. GFP fluorescence was either measured directly from these infected plates, or media and cells were collected, then diluted and measured in parallel in microtitre plates. To estimate the quantity of GFP produced by each infection on the basis of its measured fluorescence intensity, parallel measurements were made on a dilution series (50 to 1500 ng per well) of purified recombinant red-shifted EGFP (Clontech) prepared in medium containing uninfected cells. Fluorescence was measured with a FLUOstar fluorimeter (BMG LabTechnologies) using a standard fluorescence reading head, an excitation filter of 485 nm and an emission filter of 520535 nm, 10 flashes per well with the gain set against the greatest quantity EGFP standard.
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Results |
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A PCR strategy was also used to screen isolates of HaEPV putatively engineered at the fus locus. Thus, primer EPSP13 (Table 1), which anneals to sequence upstream of the fus locus, was used in conjunction with primer GFX2 to amplify a product of 850 bp from both the pTV698 transfer vector plasmid and the recombinant virus isolate HaEPV(fus-) (Fig. 2B
, upper, and 2D
, left panel). The corresponding DNA fragment was not amplified from media collected from wt HaEPV-infected cell cultures or from uninfected cells, or from water only negative control reactions (Fig. 2D
, left panel). In contrast, primer pair EPSP13/EPSP16 (Table 1
) identified the presence of virus carrying the parental fus locus in media from wt-infected cells (Fig. 2B
, bottom, and 2D
, right panel). Amplification of this 950 bp product was not observed in reactions where media from HaEPV(fus-)-infected cells were used as template, or in other control reactions (Fig. 2D
). These data show that a recombinant form of HaEPV, in which the fus coding sequence was replaced by that of gfp(RS), had been successfully isolated. This is the first demonstration of deletion of an endogenous fus coding sequence from an EPV, and shows that this gene is non-essential for growth of HaEPV in cell culture.
Kinetics of extracellular virus production by recombinant forms of HaEPV
The kinetics of extracellular virus (ECV) production of HaEPV in cell culture have not previously been described. In work reported here we assayed temporal and quantitative aspects of the process by determining the infectious titre of media harvested from cultures of Sf9 cells infected with either wt or recombinant forms of the virus. As shown in Fig. 3(A), ECV progeny of wt HaEPV from infections established at an m.o.i. of 0·1 were first detectable 3 days post-infection (p.i.), and had increased substantially in concentration at 5 days p.i., when the recorded titre was 1·58x104 TCID50 units/ml. ECV continued to accumulate, albeit more slowly, between 5 and 9 days p.i., and ultimately achieved a titre of 1·26x105 TCID50 units/ml. The experiment was concluded at 9 days p.i., on the basis of data from pilot experiments (not shown) that suggested that significantly higher ECV titres are not achieved past this time.
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Kinetics of ECV production were also assessed for infections established at the higher m.o.i. of 2·0. The most apparent consequence of this 20-fold increase in inoculum concentration was the presence of higher titres of progeny ECV at early times of infection, an effect that was particularly evident for the wild-type parental form. Assays at later stages of infection (59 days p.i.) showed similar levels of progeny production for all three forms of the virus, with observed maxima between 1·95 and 2·69x105 TCID50 units/ml (Fig. 3B). These results indicate that replacement of either of the HaEPV sph or fus genes with one encoding GFP does not result in substantive alteration in the kinetics or amount of ECV production, and by extrapolation, that it is also unlikely to affect any fundamental parameters of virus replication in cell culture.
Production of GFP by recombinant forms of HaEPV
In order to compare the sph and fus promoters with each other, and with the polh promoter driving expression of the same gene in the context of baculovirus infection, we established parallel infections of Sf9 cells with three recombinant insect viruses: HaEPV(fus-), HaEPV(sph-) and BacPAK-GFP(RS). Infections were established at three m.o.i. values 2·0, 0·5 and 0·1 (Fig. 4A, B
, C
, respectively) and total GFP produced was measured from 0·5 to 13 days p.i., using a fluorescent plate reader. At an m.o.i. of 2·0, GFP expression from BacPAK-GFP(RS) could first be detected at 2 days p.i., and levels of GFP rose rapidly between 2 and 5 days p.i. to a maximum output of 2·27 µg GFP per well (Fig. 4A
, solid bars). GFP amounts did not increase after 5 days p.i., and remained essentially constant until a slight decrease was noted at 13 days p.i. Therefore, from 5 to 13 days p.i. either little de novo synthesis of GFP occurred, or the rate of production closely matched the proteins turnover rate (Fig. 4A
). In contrast, GFP produced by HaEPV(sph-) infections at an m.o.i. of 2·0 accumulated more gradually, with a maximum of 0·68 µg GFP per well being measured at 11 days p.i. (Fig. 4A
, striped bars). Under these experimental conditions, the HaEPV sph promoter thus produced about 30% as much foreign protein as the polh promoter. Surprisingly, GFP expression from HaEPV(fus-)-infected cells was observed to be much lower than that from cells infected with either of the other two recombinants at the same m.o.i. Under these experimental conditions maximum GFP production was recorded at 0·05 µg GFP per well, and was attained at 1113 days p.i. (Figs 4A
and 5
, open bars). The amount of GFP produced by activity of the fus promoter in this system was thus about 7% of that produced by the sph promoter.
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Closer inspection of the kinetics of GFP production from HaEPV(fus-)-infected Sf9 cells (Fig. 5) showed that accumulation essentially plateaued after 4 days p.i. for infections initiated at an m.o.i. of 0·5 (Fig. 5
, grey bars), but continued to increase until about 11 days p.i. for infections initiated at an m.o.i. of 0·1 (Fig. 5
, black bars). In these assays maximum accumulation was measured as being 0·06 and 0·08 µg GFP per well, respectively. Once again, these levels of production were substantially below those of either of the other two recombinants assayed under the same conditions, representing about 2% and 35% of the corresponding amounts of GFP produced by BacPAK6-GFP(RS) and HaEPV(sph-), respectively. Nevertheless, like both other recombinants, HaEPV(fus-) produced the most GFP from infections initiated at an m.o.i. of 0·1, and took correspondingly longer to reach this maximum. On the basis of these observations, and the ECV studies reported above, we consider that the observed low level of accumulation of GFP reflects HaEPV fus promoter activity in this system, rather than some interference in viral replicative process(es).
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Discussion |
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In order to assess whether HaEPV might be as useful for heterologous protein expression as established baculovirus-based systems, we used Sf9 cells to make direct comparisons of production of GFP by the two recombinant HaEPVs and a commercial BacPAK-based baculovirus, which expressed the same protein from the polh promoter. In all instances we observed that the kinetics and amounts of protein produced were dependent on the m.o.i. used to establish infection. Thus, for all three m.o.i. values tested, GFP production by BacPAK-GFP(RS) occurred in a rapid burst between 2 and 5 days p.i. (Fig. 4); at m.o.i. values of 2·0 and 0·5, total GFP did not increase after 5 days p.i., while when an m.o.i. of 0·1 was used, maximum accumulation was reached at 78 days p.i. In contrast, GFP production by HaEPV(sph-) was always more gradual, and protein continued to accumulate over a period of 311 days p.i. (Fig. 4
). Under optimal conditions for GFP production (m.o.i. of 0·1), the HaEPV(sph-) recombinant produced about 60% of the quantity synthesized by the BacPAK-GFP(RS) recombinant; under less favourable conditions (e.g. at an m.o.i. of 2·0), its comparative production was about 30%.
While our experiments show that HaEPV(sph-) virus produces less GFP than BacPAK-GFP(RS) in Sf9 cells, it is conceivable that these production kinetics may be favourable in some bioproduction situations. For example, an accepted difficulty of use of baculovirus systems for expression of some heterologous proteins, especially those which require extensive post-translational processing and/or secretion, is that the rapid course of baculovirus infection frequently compromises host cellular functions required for these purposes (Jarvis, 1997 ). Stably transformed insect cell lines have been developed which utilize early baculovirus or non-viral promoters to try to circumvent this problem (Jarvis, 1997
; Hegedus et al., 1998
). However, use of the strong sph promoter in the context of viable HaEPV-infected cells potentially presents another means by which this issue might be addressed, without the requirement for production of stable cell lines.
We consider the next major test of the utility of HaEPV as an expression system to be the production of protein(s) whose functionality requires post-translational modification and/or secretion; it will here be of interest to determine whether the slower kinetics of HaEPV infection result in production of better quality products. It is also of considerable interest to assess the possibility of utilizing non-lytic recombinants of HaEPV for continuous production of protein, as opposed to the current batch infections used for large scale recombinant protein production with baculovirus.
An unexpected result of this study was the observation that expression of GFP from the fus locus of HaEPV produced a considerably lower quantity of protein (37%) than expression from the sph locus. Since fusolin is the major constituent protein of HaEPV spindle bodies (SB), and more abundant than spheroidin in viral preparations from in vivo infections (Lai-Fook & Dall, 2000 ; Dall et al., 1993
) we expected that both loci would produce comparable levels of protein. Given that three pure isolates of HaEPV(fus-) showed similar levels of GFP expression (data not shown), we do not believe that the results reported here reflect some deficiency in the virus used in these studies. We do consider it likely that the HaEPV fus promoter is more active in some cell types than others, an hypothesis based in part on observed distributions of SB in tissue preparations from infected caterpillars (J. Lai-Fook & D. J. Dall, unpublished), and which is now amenable to testing via in vivo infection with these recombinants.
Although isolation of the recombinants described here proved to be a lengthy process, we have demonstrated that expression of GFP can be used to identify and select cells infected with recombinant forms of the virus. Other work in this laboratory (data not shown) has demonstrated that intergenic sequences of HaEPV can be used as sites for insertion and expression of heterologous coding sequences placed under control of HaEPV-derived promoters, and also that more than one such sequence can be expressed simultaneously. These latter findings offer a potential means to link effective expression of a desired product, as described here through use of the sph locus, with minimal expression of a selectable marker driven by another HaEPV promoter from an intergenic locus. Alternatively, a dual expression vector could be constructed to target the spheroidin locus, with the spheroidin promoter driving foreign gene expression in an opposite orientation with another promoter driving marker gene expression, as has been done with baculovirus expression systems at its polyhedrin locus (OReilly et al., 1992 ). Further development of transfer vectors and engineered parental viruses should provide more versatility and convenience, and lead to the refinement of a selectable HaEPV-based protein expression system.
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Acknowledgments |
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References |
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Received 7 August 2001;
accepted 12 October 2001.
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