Department of Chemical Engineering, The University of Queensland, Queensland 4072, Australia1
Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia2
Author for correspondence: Linda Lua. Fax +617 3365 4199. e-mail lindal{at}cheque.uq.edu.au
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Abstract |
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Introduction |
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Frequent mutations have been identified within a specific region (map units 35·037·0) in the FP mutants of AcMNPV (Beames & Summers, 1988 , 1989
; Fraser et al., 1983
) and GmMNPV (Fraser et al., 1985
, 1983
). This region contains the 25K FP gene, which encodes a 25 kDa protein that is essential for virion occlusion and polyhedron formation (Beames & Summers, 1988
, 1989
; Wang et al., 1989
). Most reports on FP mutations were correlated to large insertions of host DNA or deletions of the viral genome (0·12·8 kb), which were detectable by restriction endonuclease (REN) digestion analysis (Beames & Summers, 1988
, 1989
; Fraser et al., 1985
, 1983
; Wang et al., 1989
). The FP mutations of LdMNPV were exceptions (Bischoff & Slavicek, 1997
; Slavicek et al., 1995
). These LdMNPV FP mutants exhibit FP characteristic traits; however, REN digestion analysis did not correlate the FP phenotypes observed to any DNA insertions or deletions of detectable lengths. Bischoff & Slavicek (1997)
later reported 1 bp insertions or small deletions (8 or 24 bp) in the 25K FP gene of LdMNPV FP mutants, suggesting that the mechanism of FP mutation in LdMNPV could be different from that of AcMNPV.
In recent years, studies have been done to determine the role of the 25 kDa protein in the infection cycle of baculoviruses. However, the analysis of the function of the 25 kDa protein is not conclusive (Beniya et al., 1998 ; Braunagel et al., 1999
; Harrison & Summers, 1995a
, b
). Studies showed that the 25K FP protein is a structural protein in the nucleocapsids of both BV and occlusion derived virus (ODV) but a large fraction of the 25K FP proteins remains cytoplasmic throughout infection (Braunagel et al., 1999
; Harrison & Summers, 1995a
). The relationship between the location of the 25K FP gene product and the phenotype caused by the gene mutation is still unclear. Considering the varied effects following mutation of this gene, it is possible that the gene has more than one function during the invasion and infection process. Studies also suggest that the 25K FP protein could be required for efficient protein accumulation and trafficking by regulating transcription, mRNA stability, translation or altered protein stability of one or many viral proteins, which directly or indirectly affect occlusion morphogenesis (Beniya et al., 1998
; Braunagel et al., 1999
).
Reduced occlusion production and decreased virulence as a result of FP mutations would greatly hinder the commercialization of baculoviruses produced by in vitro cell culture, for use as biopesticides. As large volumes of virus inocula are needed for large-scale production of baculoviruses (Rhodes, 1996 ), it may be impossible to generate enough virus inocula at low passage numbers for such production, as emergence of FP mutants appears to be inevitable during the scale-up of virus inocula used for cell culture systems. Understanding the nature of FP mutations and how they are selected may help in either developing a process for isolating virus isolates with a stable MP phenotype or to minimize the selective advantage of FP mutants. Research on FP mutation not only deserves further attention from the commercial perspective but also for its intrinsic interest.
Chakraborty & Reid (1999) reported the effect of serial passaging on HaSNPV in cell culture; however, no insights were given on the phenotype or genotype of HaSNPV FP mutants. The current paper reports detailed phenotypic and genotypic studies of HaSNPV FP mutants. HaSNPV was serially passaged in Helicoverpa zea serum-free suspension cultures and the phenotypic changes that occurred during the transition of an MP dominated population to a FP one was investigated using transmission electron microscopy (TEM). The ultrastructural differences between the MP and FP infected cells during progeny virus production and assembly were documented. REN digestion profiles of genomic viral DNA during serial passaging were determined, the wild-type HaSNPV 25K FP gene was identified and sequenced, and a FP mutant that carries a mutation in the 25K FP gene was isolated.
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Methods |
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Serial passaging of HaSNPV in vitro.
For all passages, duplicate 100 ml working volumes of Helicoverpa zea cell cultures at 5x105 cells/ml were infected at an m.o.i. of 0·5 p.f.u. per cell. All cultures were seeded at 3x105 cells/ml (50 ml volumes), and allowed to grow to 1x106 cells/ml, before being diluted back to 5x105 cells/ml with 50 ml fresh medium (100 ml final volume). To obtain infectious BV for subsequent passage, 50 ml of the cell suspension was harvested at 70% viability, typically 4 or 5 days post-infection (p.i.), leaving the other 50 ml cell suspension for polyhedra production. All virus stocks were stored in 1·8 ml or 4 ml aliquots at -70 °C. The virus titre of each passage was determined using a plaque assay (Lua & Reid, 2000 ), before use as inoculum for the next passage. Cell densities and cell viabilities of all cultures were determined daily in triplicate using the 0·1% trypan blue exclusion cell count method (Nielsen et al., 1991
). Final polyhedra yields were determined at 10 days p.i. Cells were first lysed with 0·5% SDS for 1 h at 28 °C before triplicate counts of the polyhedra were determined using a haemocytometer counting chamber.
Plaque purification of HaSNPV FP mutant.
A second HaSNPV population (uncloned) was established in cell culture with collected haemolymph under similar conditions to those described above. The virus was serially passaged out to passage 6. BV at passage 6 was diluted out onto culture plates as in a plaque assay and an FP plaque was picked on the basis of its phenotypic appearance under light microscopy (Hink & Vail, 1973 ). The FP mutant (designated as FP8AS) was plaque purified three times before being scaled up to obtain BV DNA for PCR amplification and sequencing. The FP phenotype of the plaque purified mutant was confirmed using TEM.
TEM and percent of FP infected cells.
At each passage, infected cell pellets were harvested at 6 days p.i. for determination of percentage of FP/MP using TEM. Cell suspension (1 ml) was centrifuged at 12000 r.p.m. for 10 min. The cell pellets were resuspended in 3% glutaraldehyde fixative solution and kept at 4 °C. The protocol for TEM processing is documented in Lua & Reid (2000) . The% FP infected cells was determined by assessing m infected cells under TEM and scoring each cell as either a FP infected cell or an MP infected cell. If we assume that each observation is independent, the number of FP infected cells observed, x, will be binomially distributed with the probability p equal to the% FP in the population (Taylor, 1997
). The confidence interval for p is given by:
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Bioassay.
The bioassay used to estimate the potency of the polyhedra produced against Helicoverpa armigera larvae measured the concentration of polyhedra required to kill a larva. The potency is usually expressed as LC50 (median lethal concentration), which is the concentration of polyhedra required to kill 50% of the population. Infected cells with polyhedra at 10 days p.i. were harvested and kept frozen at -20 °C for bioassay. Polyhedra were extracted and bioassays were performed by Peter Christian (CSIRO Entomology, Canberra, Australia). The potency of the polyhedra to be tested was compared to a standard virus, GemStar® [Helicoverpa zea (Hz)SNPV produced in vivo], by simultaneously testing both.
REN digestion analysis.
BV DNA from passage 2 to passage 6 was purified for REN digestion analysis. To purify DNA from BV, cells were first removed by low speed centrifugation (1000 g, 10 min). The BV was pelleted by ultracentrifugation of the supernatant at 10000 g for 45 min at 4 °C, using an ultracentrifuge with an SW28 rotor (Centrikon T-2070, Kontron Instruments) and 38·5 ml Ultra-Clear centrifuge tubes (Beckman). Each BV pellet was resuspended with 200 µl cold PBS. DNA was purified from the BV using the QIAamp DNA Blood Mini Kit (Qiagen) according to the protocol provided by the manufacturer. The viral DNA was digested separately with EcoRI, HindIII and BamHI, at 37 °C for 4 h. Each digestion consisted of 25 µl viral DNA, 2 µl enzymes and the appropriate buffer for specific restriction enzyme requirements. The fragments were separated by electrophoresis on a 0·7% agarose gel at 60 W for 2 to 3 h and stained with ethidium bromide. Digital images of the agarose gels were obtained using a Kodak EDAS 120 digital camera with software provided (Kodak Digital Science 1D, version 3.0.0).
PCR amplification and sequencing of the 25K FP gene.
This work was performed before the entire genome of HaSNPV was published (Chen et al., 2001 ). A set of upstream and downstream degenerated oligonucleotides was first designed from multiple alignment of the 25K FP gene of different baculoviruses and used to identify the 25K FP gene of HaSNPV. They were oligonucleotides 5' KGYAGYGTNGARATFTAYGG 3' (Primer 1) and 5' NATYTTNACNGGNCCRTCRTA 3' (Primer 2). The reaction buffer supplied by the manufacturer of the Taq DNA polymerase (Boehringer Mannheim) was used with dNTPs (Pharmacia) at a final concentration of 200 µM. The amplification cycle consisted of 3 min denaturation at 94 °C, annealing for 1 min at 45 °C and extension at 72 °C for 1 min. The amplification cycle was repeated 30 times. Specific oligonucleotides, 5' CGGTATTCACGATAGAAA 3' (Primer 3) and 5' CGTAGTCTATGTCTAGAT 3' (Primer 4), were designed to confirm the nucleotide sequence of the first identified fragment of the 25K FP gene. Primers 3 and 4 were also used for primer walking sequencing, using the purified genomic viral DNA as template. A 40 µl sequencing reaction consisting of 16 µl BigDye premix, 13 pM primer and 200300 ng viral genomic DNA was set up: 99 cycles consisting of 30 s at 95 °C for denaturation of template, 20 s at 50 °C for annealing of primer and 4 min at 60 °C for extension of nucleotides were operated. The final products were cleaned up with the Centrispin-20 columns (Geneworks), according to the protocol provided by the manufacturer, before being sequenced. Oligonucleotides 5' GTACGCACACATATACAC 3' (Primer 5) and 5' GCTAGTCAAATGAGTCGC 3' (Primer 6) were used to amplify and sequence the entire 25K FP gene. Oligonucleotides 5' ACGGACTGGATGAGCTTC 3' (Primer 7) and 5' CGGTACTCGGTAAATCTG 3' (Primer 8) were used to amplify and sequence the entire 25K FP gene including upstream and downstream regions of the gene. The annealing temperature used in the PCR amplification cycles for Primer 3 to Primer 8 was 50 °C. The nucleotide sequence of amplified DNA was determined directly. Sequencing was performed using the PRISM (Applied Biosystems) terminator chemistry method according to the manufacturers instructions: 3090 ng DNA and 3·2 pM primer in a 16 µl reaction was prepared for sequencing, whilst the other reagents for the reaction were supplied in the PRISM kit. Reaction conditions (including amplification cycle) and the removal of unincorporated reaction components were performed according to the manufacturers recommendations. Sequence analysis were done with SEQUENCHER (Genecodes Corp.). All nucleotide sequences were determined on both strands from at least two independently amplified templates and consensus sequences were obtained from multiple determinations to avoid errors arising from the use of Taq polymerase.
Cloning PCR products.
Amplicons of the 25K FP gene of passage 6 BV were cloned into pGEM-T Vector (Promega) and transformed into E. coli JM109 competent cells. After transformation, the positive clones were picked and DNA templates for sequencing were isolated using the Qiagen Plasmid Mini Kit.
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Results |
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Nucleocapsid envelopment and virion occlusion
In a wild-type MP infected cell, nucleocapsids are produced in the virogenic stroma, and migrate to the ring zone of the nucleus to acquire their envelopes through a de novo intranuclear membrane synthesis process (Fig. 2A). The enveloped virions then gather in the ring zone, near the inner nuclear membrane, ready for occlusion into the polyhedrin protein matrix. Abnormalities in morphogenesis became apparent at the fourth passage. A majority of the nucleocapsids in the ring zone of the FP infected cells did not acquire envelopes (Fig. 2B
). The intranuclear membrane profiles believed to be involved in the nucleocapsid envelopment process were clearly observed in these FP infected cells. However, only infrequently were the nucleocapsids enveloped in the FP infected cell cases, indicating an impaired nucleocapsid envelopment process in FP infected cells.
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Obvious signs of infection, such as presence of virogenic stroma, nucleocapsids and intranuclear membrane profiles, were evident in the FP infected cells. Despite the signs of infection, some FP infected cells do not form polyhedra (Fig. 3A). Another phenotypic characteristic of FP infected cells was accumulation of intranuclear membrane profiles in the ring zone of infected nuclei (Fig. 3B
). In some FP mutant cases, the intranuclear membranes became elongated, angular and unusual in shape, forming massive swirls in the ring zone of the nuclei. In addition, some FP infected cells also produced polyhedra with morphologically aberrant virions occluded (Fig. 3C
) or associated with the edge of an occlusion (Fig. 3D
), or polyhedra with very few virions occluded (Fig. 3E
).
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Discussion |
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The number of polyhedra produced by a HaSNPV FP infected cell was significantly lower (<10 polyhedra per section) than a HaSNPV MP infected cell ( 50 polyhedra per section) (see Table 1 and Fig. 2
). Harrison et al. (1996)
demonstrated that the rate of polyhedrin mRNA expression is significantly reduced in cells infected with AcMNPV FP mutants compared to that of an MP infection. In FP infected cells, polyhedrin localizes less efficiently to the nucleus during the early occlusion phase of infection (24 h post-infection), yet polyhedrin mRNA stability is similar in MP and FP mutant infected cells. The reduction in polyhedrin synthesis and nuclear localization could account for the reduced number of polyhedra observed with HaSNPV FP mutant infections and other baculoviruses by resulting in a significantly decreased concentration of polyhedrin available in the nucleus for occlusion assembly. Clearly, this requires further investigation of the effect of the 25K FP gene mutation on the expression and localization of polyhedrin in the HaSNPV infected cells.
An accumulation of a large number of naked nucleocapsids in the nuclei of FP infected cells, presumably a result of an impaired nucleocapsid envelopment, was a striking observation in this study. Although nucleocapsid assembly is apparently normal, there are few enveloped virions (the ODV) present in FP mutant infected cells. The appearance of intranuclear accumulations of short, open-ended, membrane profiles in the FP mutant infected cells also suggested that there is a specific failure in the envelopment process of nucleocapsids, not in the production of envelopes themselves. Altered intranuclear nucleocapsid envelopment due to FP mutation in AcMNPV was reported as early as 1974 (Ramoska & Hink, 1974 ). Naked nucleocapsids did not appear to be occluded. It is well documented that only enveloped virions are associated with polyhedrin protein, leading to occlusion of the virions (Harrap, 1972
). The virion envelope (or proteins embedded in it) may be necessary for an interaction of the virion with polyhedrin protein during the occlusion process (Wood, 1980
). Thus, an impaired intranuclear nucleocapsid envelopment process in HaSNPV FP mutant infected cells may directly or indirectly result in the production of FP polyhedra that have no virions occluded.
The rate of FP mutant proliferation relative to parental type in different cellvirus systems is variable. In this work, more than 80% of cells were infected by FP mutants at passage 4 during serial passaging of HaSNPV, and 100% FP mutant infected cells were obtained by passage 6. With TnMNPV, the virus population was 90% FP only after 10 passages (Potter et al., 1976 ) but mutation/selection was much faster for LdMNPV, which shows a 96% FP population after passage 2 (Slavicek et al., 1995
). OpSNPV FP mutation is affected by host cells (Sohi et al., 1984
), but formation of FP variants of GmMNPV is not a host-dependent phenomenon and their detection can be influenced by the overlay formulation used during plaque assays (Fraser & Hink, 1982
). This suggests that differences in media used to propagate cells and prepare overlays may contribute to inconsistencies in the quantitative detection of FP plaques. FP mutants are often distinguished from MP virus on the basis of a less refractive plaque morphology, which can be subjective. Therefore, in this study, TEM was used to score MP and FP mutant infected cells at each passage instead of conventional plaque assays.
A trait commonly observed in most baculovirus FP mutations is an increase in the production of BV. Higher BV titres were reported in FP mutants of AcMNPV (Harrison & Summers, 1995b ; Wood, 1980
), TnMNPV (Potter et al., 1976
, 1978
) and LdMNPV (Slavicek et al., 1995
). The BV titres of LdMNPV FP mutants increase significantly, as high as 150- to 250-fold (Slavicek et al., 1995
). However, an increase of BV production due to FP mutation was not observed for HaSNPV. A significant 3-fold increase in the BV titre was only detected at the fourth passage of the HaSNPV isolate, but decreased in the subsequent passages. Chakraborty & Reid (1999)
reported a steady increase in BV titres from passage 2 to passage 5 during serial passaging of HaSNPV in SF900II medium supplemented with 10% serum but titres decreased in the subsequent passages. Interestingly, declining BV titres during serial passaging of HzSNPV (Yamada et al., 1982
) and LdMNPV (Lynn, 1994
) were also reported. A decrease in BV production during serial passaging in cell culture could either be a trait specific to the Helicoverpa sp. or may be dependent on the cellvirus system. As HaSNPV FP mutations do not cause an increase in BV production, one possible reason for the FP mutants to out-complete MP virus could be their ability to bind and infect cells at a higher efficiency than MP virus.
The phenotypic observations on the HaSNPV FP mutants suggest genomic changes occurring in the 25K FP gene, which could be large insertions or deletions within the gene. However, the genomic digestion profiles of all passages generated separately with three restriction endonucleases were similar, indicating that no large insertions or deletions of the viral genome occurred during serial passaging. The consensus sequence data obtained from the 25K FP gene amplicon of both passage 2 and passage 6 material are identical. This result was unexpected since the HaSNPV FP mutants exhibit well-documented FP phenotypes that are correlated to mutations in the 25K FP gene of other baculoviruses. Speculations on the possibility that passage 6 comprised a mixture of FP mutants that carry very small insertions or deletions as a result of random mutations, thus resulting in a consensus sequence of the wild-type 25K FP gene, was confirmed when 25K FP gene sequences obtained from individual clones of passage 6 amplicons revealed a mixture of point mutations and/or point insertions within the gene. Two out of nine clones (C2 and C48) carried 1 bp insertions within a region of adenine repetitive sequences. Five other clones carried point mutations on the gene. Further investigation with a plaque-purified FP mutant (FP8AS) also revealed a 1 bp insertion in the same region of repetitive sequences as clone C2 within the 25K FP gene. Frameshift mutations as a result of point insertions in FP8AS, clones C2 and C48 would lead to truncated 25K FP proteins. The 25K FP gene mutations observed in HaSNPV are not specific to a particular region but rather they are randomly found throughout the gene. Hence, not all baculovirus FP mutants have similar genetic mutations within the 25K FP gene.
The FP mutation of HaSNPV is likely to arise through a different mechanism from AcMNPV and GmMNPV (Beames & Summers, 1988 ; Fraser et al., 1983
) but probably similar to that for LdMNPV (Bischoff & Slavicek, 1997
; Slavicek et al., 1995
). HaSNPV FP mutants are not generated through large DNA insertions or deletions that are of sufficient size to allow detection by genomic REN digestion analysis. Five nucleotide sites consisting of 5' TTAA 3', which are frequently associated with transposon insertions within the AcMNPV and GmMNPV 25K FP genes (Beames & Summers, 1988
; Fraser et al., 1985
; Wang et al., 1989
), were found in the HaSNPV 25K FP gene. However, 14 such sites were identified in the AcMNPV 25K FP gene (Wang et al., 1989
), which could lead to a higher rate of transposon insertions for AcMNPV. One bp insertions in repetitive sequences and point mutations observed for HaSNPV are more similar to the 25K FP gene mutations reported for LdMNPV (Bischoff & Slavicek, 1997
). As discussed by Bischoff & Slavicek (1997)
, 1 bp insertions within repetitive sequences (observed here with isolate FP8AS, clones C2 and C48) are most likely the result of DNA polymerase slippage on the newly synthesized strand.
The work has further confirmed the importance of the 25K FP gene during the production and assembly of progeny virus. From both a fundamental and applied science perspective, the effects of mutation in this gene deserve further study. Characterization of HaSNPV MP and FP virus is currently under way in our laboratory, in an effort to understand the selective advantage of HaSNPV FP mutants since they do not appear to gain their advantage simply through the production of higher BV titres. In particular, the cellvirus binding and uptake characteristics of MP and FP virus are under investigation.
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Acknowledgments |
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Footnotes |
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References |
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Received 6 November 2001;
accepted 20 December 2001.