Department of Chemical Engineering, The University of Queensland, Queensland 4072, Australia1
Author for correspondence: Linda Lua. Fax +61 7 3365 4199. e-mail lindal{at}cheque.uq.edu.au
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Abstract |
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Introduction |
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Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV), a wild-type baculovirus, has the potential for use as a biopesticide for effective control of heliothis. HaSNPV is specific and highly virulent to its host (Hughes et al., 1983 ; Teakle et al., 1985
). The development of in vitro production of HaSNPV is becoming increasingly important as the demand for effective heliothis control increases.
Various factors relating to the replication of HaSNPV, such as virus infection kinetics, optimal time of infection (TOI), nutrient consumption and passage effect, have been studied previously (Chakraborty et al., 1995 , 1996
, 1999
; Chakraborty & Reid, 1999
). These studies were aimed primarily at optimizing production processes for this virus. Despite all the reported studies on HaSNPV, little is known about HaSNPV morphogenesis in Helicoverpa zea serum-free suspension culture. In vitro production of high quality and biologically active HaSNPV necessitates an understanding of virus replication and assembly in this production system. Knowledge of the morphogenic events in this system will aid in optimizing production parameters such as the time of harvest for fully mature polyhedra, and in documenting any structural differences between this system and other reported baculoviruses.
Previous studies on Helicoverpa zea SNPV (Goodwin et al., 1973 ; Granados, 1978
, 1981
; Ignoffo et al., 1971
; Lenz et al., 1991
; McIntosh & Ignoffo, 1981
; Rice et al., 1989
; Yamada et al., 1982
) revealed very little about the morphogenesis of the virus. A brief sequence of morphogenesis was described by Granados et al. (1981)
. However, virion occlusion and polyhedron envelope morphogenesis was not reported in detail. Ultrastructural studies on replication of other NPVs have been performed extensively over the last two decades. Most of these studies were done using either infected larval tissues (Adams et al., 1977
; Granados & Lawler, 1981
; Harrap, 1972b
; Hess & Falcon, 1978
; Kawamoto et al., 1977
; Tewari & Datta, 1996
), or by using monolayers of cells grown in serum-containing media (Carstens et al., 1979
; Fraser, 1986
; Knudson & Harrap, 1976
; MacKinnon et al., 1974
; Young et al., 1993
). The most well documented virus morphogenesis process available is for Autographa californica NPV (AcMNPV), which was reviewed by Williams & Faulkner (1997)
.
NPVs have a biphasic replication cycle that is localized in the cell nucleus (Vlak, 1992 ). The first phase of replication generates nucleocapsids and budded virus. The second phase involves the development of large proteinaceous paracrystalline occlusion bodies, also known as polyhedra, which are composed mainly of a polyhedrin protein of about 30 kDa, in which numerous enveloped virions are embedded. Varying from 0·5 to 15 µm in diameter, the polyhedra stabilize virions, thus allowing them to remain viable for long periods in the environment (Bergold, 1963
). Each polyhedron has an electron-dense layer known as the polyhedron calyx or polyhedron envelope (PE), the function of which is unknown. Gross et al. (1994)
suggested that the PE may prevent virions from dislodging from the polyhedron matrix and, in general, helps seal the virus complex. The PE consists mainly of carbohydrates, with 60% hexose, 29% pentose and small amounts of uronic acids and hexosamines (Minion et al., 1979
), and at least one protein (Whitt & Manning, 1988
).
This paper documents the morphogenic sequence of HaSNPV in H. zea serum-free suspension cultures and further discusses prominent structural differences between the morphogenesis of HaSNPV and that of other baculoviruses. This study improves our understanding of HaSNPV replication in serum-free suspension cultures and will undoubtedly aid in the development of an optimized production process of HaSNPV as a commercial biopesticide.
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Methods |
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Virus.
HaSNPV (uncloned) passage one stock was established in cells grown in SF900II plus 10% foetal bovine serum (CSL, Australia) with haemolymph collected from larvae (obtained from the Department of Primary Industries, Long Pocket, Queensland). The haemolymph from 22 infected larvae was pooled together into a total volume of 100 ml of medium. It was frozen in liquid nitrogen and later thawed and filtered through a 0·2 µm filter before use. 1·8 ml was used to infect a 20 ml Helicoverpa zea suspension culture at 3x105 cells/ml. 80 ml fresh medium was added at 4 days p.i., to prevent over growth of cells and to provide fresh nutrients. Budded virus of passage one was harvested at 80% cell viability (8 days p.i.). The cell suspension was centrifuged at 1000 g for 5 min at room temperature. The cell pellet was discarded and the virus-containing supernatant was stored at -70 °C. Passage two stock was made from passage one virus in the following way, for use in this study. A culture with a 50 ml working volume was seeded at 3x105 cells/ml in SF900II medium and allowed to grow to 1x106 cells/ml. It was diluted back to 5x105 cells/ml with fresh medium and infected with an m.o.i. of 0·5 p.f.u. per cell. Passage two budded virus was harvested at 70% cell viability (5 days p.i.), stored at -70 °C and titred before use.
Quantification of budded virus.
The virus titres of passage one and two stocks were determined by a plaque assay using Helicoverpa zea cells in SF900II plus 10% foetal bovine serum cells. 60 mm Petri dishes (Corning) were seeded with 3 ml Helicoverpa zea cells at 3x105 cells/ml, and incubated overnight at 28 °C. The medium was removed from the cells and 0·5 ml of virus at an appropriate dilution was inoculated onto the plates and rocked gently for 4 h. Virus inoculum was removed before 4 ml of mediumagarose mix was overlaid onto each plate. The overlay consisted of an equal volume of 2x SF900II plus 10% foetal bovine serum and 2% low melting point SeaPlaque agarose (FMC BioProducts).
Virus infection.
Duplicate 50 ml cultures were seeded at 3x105 cells/ml in SF900II medium and allowed to grow to 1x106 cells/ml before being diluted to 5x105 cells/ml with 50 ml fresh medium. These cultures were infected at an m.o.i. of 2 p.f.u. per cell. Samples were collected for transmission electron microscopy (TEM) processing at 4 hourly intervals post-infection for the first 48 h p.i. and subsequently every 24 h p.i. Samples from mock-infected cultures were also collected and processed for TEM. Cell density and viability were determined daily in triplicate using the 0·1% trypan blue exclusion method (Nielsen et al., 1991 ). To determine polyhedra density, cells were lysed with 0·5% SDS for 1 h at 28 °C before triplicate counts were done in a haemocytometer counting chamber.
An infected Sf9 cell pellet was obtained from Matthew Rosinki (University of Queensland). In brief, Sf9 cells were infected with a recombinant AcMNPV expressing -galactosidase (pAc-360
-gal), at an m.o.i. of 0·001 and harvested at 4 days p.i. Another HaSNPV infection in SF900II supplemented with 10% serum was performed to determine that some of the structures observed in the serum-free medium were not a result of the lack of serum. Infections in the serum-supplemented cultures were carried out under conditions similar to those used for serum-free cultures and infected cell samples were collected at 2 and 3 days p.i. for TEM. Fourth instar Helicoverpa armigera larvae were also infected with in vivo- produced HaSNPV polyhedra, and the fat bodies of infected larvae were harvested at 4 days p.i. for TEM.
Transmission electron microscopy.
TEM processing was similar to previous reports in the literature but was modified for cell culture samples. The cells were harvested at different times post-infection, pelleted and fixed with 3% glutaraldehyde in 0·1 M phosphate buffer pH 7·2, for at least an hour at 4 °C. Cells were washed once with phosphate buffer pH 7·2 at 4 °C and resuspended in 2% SeaPlaque agarose. The cellagarose block was cut into 1 mm squares and washed twice again with phosphate buffer, before being fixed with 1% osmium tetroxide in 0·1 M cacodylate buffer, pH 6·8, for 1 h at 4 °C. The samples were then washed once with 0·1 M cacodylate buffer, pH 6·8, and once with water at room temperature. After washing, they were sequentially dehydrated once in 50, 70 and 90% acetone and twice in 100% acetone for 5 min at each step. Samples were infiltrated with 50% Spurrs low viscosity resin for 1 h, followed by 100% Spurrs resin for a further 1 h. After one change in 100% Spurrs resin, they were left in resin overnight on a rotator. Samples were embedded in fresh resin and polymerized at 60 °C for 3 days. Ultra-thin sections (6080 nm) were cut with a Leica Ultracut UCT microtome. The sections on copper grids were stained for 2 min in 5% uranyl acetate and 1 min in Reynolds lead stain before being viewed under a JEOL 1010 transmission electron microscope.
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Results |
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Mock-infected cells harvested at 24 and 48 h p.i. were also observed under TEM as a control to the infection studies (Fig. 2a, b
). Infection with an m.o.i. of 2 p.f.u. per cell was insufficient to observe early infection events (such as virus attachment and entry into host cells) in the first 4 h p.i. Infection with a higher m.o.i. of 10 or 100 p.f.u. per cell could not be performed due to the low virus stock titre (2x107 p.f.u./ml). Pictures were obtained from the secondary infections (at 24 h p.i.) to illustrate virus attachment and entry at the plasma membrane (Fig. 2c
). The first indication of virus infection was enlargement of the cell nucleus and formation of a loose network of granular material in the middle of the nucleus (Fig. 2d
). Development of this intranuclear virus replication centre, the virogenic stroma, was first detected at 12 h p.i. Clumps of heterochromatin, normally seen dispersed throughout the nucleoplasm of an uninfected cell (Fig. 2a
, b
), were reduced and displaced peripherally along the inner nuclear membrane by the forming stroma. Maturation of the virogenic stroma yielded a significant and morphologically distinct peristromal compartment of nucleoplasm, called the ring zone (Williams & Faulkner, 1997
). Examination of the 16 h p.i. sample revealed formation of short progeny nucleocapsids in the stromal network (Fig. 2e
). These nucleocapsids appeared to associate very closely with the stromal edge. Short membrane profiles were observed in the ring zone. These membrane profiles likely represent the source of virion envelopes. Some unidentified circular structures were also seen near the inner nuclear membrane.
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Discussion |
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In brief, the main morphogenic events are replication of progeny virus, virion envelopment, virion occlusion and PE formation. The first sign of infection is formation of the virogenic stroma, an electron-dense matte with electron-lucent intrastromal spaces. Nucleocapsids develop adjacent to the stroma and inside the intrastromal spaces, near the edge of the stromal matte. The virogenic stroma contains discrete DNA- and RNA-containing structures that are related to replication and packaging of the progeny virus (Young et al., 1993 ). In vitro studies on AcMNPV-infected Sf21 cells have shown that the electron-dense matte is extremely sensitive to RNase whereas areas adjacent to the intrastromal spaces are sensitive to DNase.
Nucleocapsids made in the virogenic stroma are subsequently enveloped by one of two processes. They are either enveloped during budding through the cell plasma membrane, acquiring a loosely fitting envelope (budded virus), or enveloped in the nucleus through de novo envelope morphogenesis to form preoccluded virions. In this study, both morphogenic events occurred at about the same time post-infection. These two forms of virus differ morphologically and biochemically. Braunagel & Summers (1994) have demonstrated that the protein and lipid composition of the envelopes of budded virus and preoccluded virions is significantly different.
The de novo intranuclear nucleocapsid envelopment process is clearly observed in this study, and is similar to such processes reported for other baculoviruses (Fraser, 1986 ; Stoltz et al., 1973
). At 16 h p.i., short membrane profiles synthesized de novo in the nuclei of infected cells accumulate in areas of the ring zone, and nucleocapsids that migrate out to the ring zone, form preoccluded enveloped virions. These intranuclear membrane profiles were not observed in mock-infected cultures.
The initial stages of polyhedron formation can be detected by deposition of polyhedrin protein between enveloped virions, forming a typical lattice structure between the virions. Naked, unenveloped nucleocapsids are not occluded. The virion envelope may have a possible role as a site for polyhedrin protein polymerization (Wood, 1980 ). The envelope of occluded virions in the polyhedrin matrix fits snugly around the nucleocapsid, unlike the budded virus envelope. Enlargement of the polyhedra were observed over time, between 24 and 36 h p.i. It is unclear what factors determine the size of polyhedra or why the deposition of polyhedrin protein ceases at a certain stage. Harrap (1972a
) suggested that the available pool of polyhedrin protein monomer may be exhausted at this time.
Early PE morphogenesis in HaSNPV appears to be marked by the appearance of calyx precursor structures in the virogenic stroma. These structures appear to move progressively to the ring zone to interact with maturing polyhedra. Ultrastructurally, they appear similar to the calyx precursors that interact with fibrillar structures of AcMNPV (van Oers & Vlak, 1997 ; Williams & Faulkner, 1997
; Williams et al., 1989
) and other baculoviruses (Knudson & Harrap, 1976
; MacKinnon et al., 1974
). However, in HaSNPV, these structures appeared to emerge and develop randomly in the nuclei, especially in the virogenic stroma, and to have no definite form. The calyx precursor structures often encircle the polyhedra during acquisition of the PE. These calyx precursor structures are most likely the PE precursors, as they appear to be involved in the application of the PE to the surface of the polyhedra. Their appearance coincided with polyhedron formation and they were not observed in the nuclei of infected cells after completion of PE formation, indicating that they have a role in PE morphogenesis.
Fibrillar structures enriched in P10 proteins are commonly found in the nuclei and cytoplasm of AcMNPV-infected cells (Lee et al., 1996 ; Rohrmann, 1992
, Williams & Faulkner, 1997
). In AcMNPV-infected cell nuclei, these fibrillar structures usually associate with maturing polyhedra during PE morphogenesis. This phenomenon is frequently observed and well documented for other baculovirus infections such as TnMNPV (MacKinnon et al., 1974
), SfMNPV (Knudson & Harrap, 1976
) and Orgyia pseudotsugata (Op)MNPV (Gross et al., 1994
). However, such a prominent fibrillar structure was not observed in the nuclei of HaSNPV-infected cells. Instead, fibrillar structures (presumptive homologues of AcMNPV fibrillar structures), resembling to some extent the AcMNPV fibrillar structures, were clearly detected only in the cytoplasm of HaSNPV-infected cells. Mock-infected cells showed no fibrillar structures, indicating that these cytoplasmic fibrillar structures are associated with HaSNPV infection events. These cytoplasmic fibrillar structures were first detected at around 2832 h p.i., just after initial virion occlusion events in the nuclei. Occasionally, calyx precursor-like structures were seen interacting with these cytoplasmic fibrillar structures. It cannot be ruled out that the absence of fibrillar structure in the nuclei of infected cells and their location only in the cytoplasm is an artefact of serum-free cultures. However, HaSNPV infections in serum-supplemented medium produced similar results (appearance of fibrillar structures in the cytoplasm of infected cells but not in the nuclei). This is further confirmed by the observation that Helicoverpa armigera larva fat cells infected with HaSNPV also have cytoplasmic fibrillar structures only.
The P10 protein is known to be a structural component of the fibrillar structures, calyx precursors and PE in AcMNPV-infected cells (Lee et al., 1996 ). P10 is a poorly conserved protein that is hyperexpressed very late in infection (Rohrmann, 1992
; van Oers & Vlak, 1997
) and is involved in disintegration of the nucleus and release of polyhedra late in infection (van Oers et al., 1993
). Recently, the first p10 gene from an SNPV, Buzura suppressaria (Busu)NPV, was identified (van Oers et al., 1998
). An AcMNPV recombinant expressing the BusuNPV p10 gene (using a p10-negative AcMNPV virus) formed fibrillar structures in the cytoplasm of infected Spodoptera frugiperda cells. The cytoplasmic fibrillar structures resemble those fibrillar structures found in AcMNPV-infected cells. Thus, it may be possible that P10 proteins of SNPV are located differently to that seen in MNPV, such that in SNPV fibrillar structures may be exclusively located in the cytoplasm. P10 proteins could be present in the nuclei of HaSNPV-infected cells but remain undetected, as they do not form fibrillar structures in the nucleus.
NPV replication and assembly in the cell nucleus evidently involves a complex but elegantly controlled synthetic process. This paper clearly documents the unique morphogenesis of calyx precursor structures and cytoplasmic fibrillar structures during the HaSNPV PE morphogenic process, which has not been reported previously in the literature. Calyx precursor structures and the exclusive cytoplasmic location of fibrillar structures could be unique to HaSNPV morphogenesis since they are observed in both serum and serum-free cultures and also in the in vivo system. This ultrastructural study provides an outline of a general mode of HaSNPV replication in Helicoverpa zea cells, with time-scales for several morphogenic events, which will facilitate further investigations on production of viral proteins specific to PE formation, such as P10 and PE protein. Research to identify the nucleotide sequences and level of expression of these critical viral proteins is under way.
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Acknowledgments |
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We would like to thank Dr Robert E. Teakle (Queensland Department of Primary Industry) for the generous gift of HaSNPV, Dr Shukla Chakraborty (UQ) for her efforts in collection of the original virus haemolymph, Dr Dall (CSIRO) for the gift of the Helicoverpa zea cell line and Matthew Rosinki (UQ) for his kind contribution of the rAcMNPV-infected Sf9 cells. Dr Teakles assistance in the larvae EM work is also gratefully acknowledged. We also acknowledge the valuable technical advice provided by the Centre for Microscopy and Microanalysis (UQ).
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References |
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Received 30 March 2000;
accepted 26 June 2000.