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1 Department of Microbiology and Immunology, Dalhousie University, Halifax, NS B3H 4H7, Canada
2 Laurentian Forestry Centre, Canadian Forest Service, Natural Resources Canada, Quebec City, QC G1V 4C7, Canada
Correspondence
Don Stoltz
dstoltz{at}dal.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Atlantic Forestry Centre, Canadian Forest Service, Natural Resources Canada, Fredericton, New Brunswick E3B 5P7, Canada. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Webb, 1998). Following release into the oviduct in the form of virus-containing ‘calyx fluid’, virions are injected into host larvae during oviposition; here, they infect a number of tissues, but infection is abortive in the conventional sense, since no progeny virus particles are produced. Nonetheless, viral DNA is maintained and expressed; viral gene products are believed to create an environment conducive to the survival and successful development of parasitoid eggs and/or larvae. Survival of the parasitoid is in turn necessary for the continued transmission of the chromosomally inherited polydnavirus genome. Thus, infection of host (typically lepidopteran) tissues, coupled with virus-specific gene expression, is of critical importance. Yet, little is known about how polydnaviruses recognize, enter and subsequently colonize host tissues.
In recent years, investigators have begun to develop in vitro systems with which to examine some aspects of polydnavirus infection using established lepidopteran cell lines (Béliveau et al., 2003; Kim et al., 1996; Stoltz et al., 1988; Volkoff et al., 1999, 2001), assuming such systems to mirror, at least in part, events that occur in vivo. These efforts have met with some degree of success; for example, patterns of polydnavirus gene expression in vitro have in some cases been shown to be very similar to those observed in vivo (Béliveau et al., 2003; Stoltz et al., 1988; Volkoff et al., 1999, 2001). The availability of in vitro systems also provides an opportunity to investigate early stages in the infection process. In vivo, polydnavirus nucleocapsids enter the cytosol following a putative membrane fusion event involving the (sole) envelope in the case of bracoviruses (Stoltz & Vinson, 1977) and the inner membrane (of two envelopes) in the case of ichnoviruses (Stoltz & Vinson, 1979). Here, focusing on the latter, we describe an in vitro system for studying early events in the infection of Sf-21 cells by ichnoviruses from three different ichneumonid parasitoids. Since the plant glycoside digitonin has been shown to crack the ichnovirus outer membrane (Deng et al., 2000), thus exposing the inner one, we reasoned that treatment with this chemical might lead to enhanced infectivity in vitro. Here we show that treatment of virions with digitonin does indeed lead to substantially increased binding and infectivity. In addition, we report that digitonin-treated ichnovirus particles are fusogenic from without.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Stoltz & Makkay, 2000), although we have more recently been using Spodoptera exigua as host for H. exiguae. It should be pointed out that our H. fugitivus colony collapsed during the winter of 2005/2006, and was replaced with field-collected wasps in May of 2006. Tranosema rostrale was obtained from spruce budworm (Choristoneura fumiferana) larvae parasitized in the field (Cusson et al., 2002; Doucet & Cusson, 1996) in 2005 and 2006; despite numerous attempts, we have not as yet been able to establish a laboratory colony of this parasitoid. Ichnoviruses carried by these parasitoids are designated HfIV, HeIV and TrIV, respectively. It should be noted that, in the studies reported here, viruses were used in the form of calyx fluid (CF), which in addition to a predominant particulate fraction (virions) also contains a number of soluble polypeptides of unknown function; in preliminary work, we have learned that virions pelleted out from CF are indeed, as expected, fusogenic (unpublished observations). In our experiments, quantities of virus-containing inocula are expressed in terms of calyx fluid equivalents (CFE); 1 CFE, then, is the amount of calyx fluid that can be extracted from the ovaries of a single parasitoid, which in the case of H. fugitivus amounts to approximately 9 µg in terms of protein concentration. In typical experiments, aliquots were taken from CF pooled following extraction from the ovaries of several different females, since the amount of CF per female varies considerably. In experiments using HfIV, 0.5 CFE in SF-900 II medium (Invitrogen) containing 3 % fetal bovine serum (FBS) was routinely used. Relative particle count comparisons carried out on negatively stained virus preparations revealed that a single H. fugitivus female produces, on average, at least four times as much CF as a T. rostrale female, and twice as much as an H. exiguae female (data not shown); accordingly, in the case of TrIV and HeIV, 2.0 and 1.0 CFE were used per well, respectively.
The cell line used for most of the work reported here was Sf-21, grown in Sf-900 II medium (Invitrogen) containing 3 % FBS. This cell line was chosen simply because preliminary experiments revealed it to be the most susceptible to virus-induced cell fusion.
Binding.
Binding of HfIV was assessed using immunofluorescence microscopy. Briefly, Sf-21 cells were seeded on 22 mm glass coverslips in 6-well tissue culture dishes, at a density of 106 cells per well. The cells were either mock-infected, or infected with either virus (in the form of CF) or digitonin-treated virus (see below) in 300 µl Sf-900 II medium per well. Attachment was typically allowed to proceed for 1 h at 4 °C, after which time the cells were placed at room temperature, and 2 ml of fresh medium was added to each well. After 1–2 h incubation at room temperature, the cells were washed with PBS, fixed in 0.4 % paraformaldehyde for 30 min and permeabilized for 30 min at room temperature using 0.1 % Triton X-100 in PBS. Cells were blocked overnight at 4 °C in 5 % normal goat serum in PBST (PBS containing 0.1 % Tween 20). The primary antibody was a mouse polyclonal monospecific serum raised against an HfIV 43 kDa major nucleocapsid polypeptide. The HfIV polypeptide co-migrated with a major H. exiguae polypeptide known to be associated with the nucleocapsid (see lane 4 of Fig. 4a in Cook & Stoltz, 1983), and so was used to generate an antiserum at a time when our H. exiguae colony was in poor condition. The 43 kDa HfIV polypeptide was electro-eluted from SDS-PAGE gels, and injected subcutaneously into BALB/c mice, as described previously (Harlow & Lane, 1988). Application of the primary antibody was followed by incubation in the secondary antibody, a goat anti-mouse Alexa-488 conjugate (Molecular Probes). Both antibodies were used at concentrations of 1 : 1000 in blocking solution, and applied for 1 h at room temperature. Counterstaining was performed using TO-PRO-3 (Molecular Probes) according to the manufacturer's protocol. Coverslips were affixed to glass slides with Prolong Gold anti-fade mounting medium (Molecular Probes) prior to observation with a Zeiss Axiovert 200 fluorescence microscope.
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Electron microscopy.
Relative infectivity rates (for HfIV only) were assessed primarily using transmission electron microscopy (TEM). For TEM, CF from three female parasitoids was dissected out into 0.5 ml of medium and applied to a 25 cm2 flask of Sf-21 cells grown to approximately 80 % confluency. After 1.5 h, cells were processed for electron microscopy. We defined infectivity as the release of viral nucleocapsids into the cytosol; thus, each nucleocapsid observed free in the cytosol or present in the nucleus was considered to represent a successful infection event. Fixation was initially for 1 h at room temperature in 3 % glutaraldehyde in 0.05 M sodium cacodylate buffer containing 0.25 M sucrose. Post-fixation was for 2 h in 2 % OsO4 in the same buffer, followed by overnight en bloc staining in 0.1 % aqueous uranyl acetate. Following dehydration in ethanol, cell pellets were embedded in TAAB resin. Micrographs were taken using a Philips EM 300 operating at 80 kV.
Statistical analysis.
Syncytial indices (y) estimated for control and virus-treated Sf-21 cells were submitted to an ANOVA, using the GLM procedure of SAS, version 9.1 (SAS Institute). Data were first transformed (z=log[(y+0.25)/(100–y–0.25)]) to stabilize the variance and meet the analysis' assumption of normality. The model included the main effects of three factors: (i) a factor labelled ‘category’ at two levels: presence (1) or absence (0) of virus, (ii) virus species (TrIV, HfIV or HeIV) when category=1, and (iii) treatment (mock or mock+digitonin when category=0, and CF, CF+digitonin or –80 °C-treated CF when category=1). It also included an effect of the interaction between treatment and virus when category=1. The special structure of the ANOVA model reflects the embedding of the virus and treatment effects within the levels of category. The number of replicates per category, treatment and virus combination varied from 3 to 15. Means and their 95 % confidence intervals were computed on the scale of z and back-transformed for presentation in Fig. 5. Since the virus–treatment interaction was not significant (P = 0.051), differences between treatments were assessed from means for the three viruses combined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), would enhance binding of virus to the cell surface. At 1–1.5 h post-infection (p.i.), few untreated HfIV virions were present on the surface of Sf-21 cells (Fig. 2a). On the other hand, digitonin-treated virions coated cell surfaces in significant numbers (Fig. 2b). Fluorescent signals were not observed on the surfaces of mock-infected cells. While previous studies had established that ichnoviruses are infectious in vitro (Béliveau et al., 2003; Kim et al., 1996; Volkoff et al., 1999), nothing was known about the early stages of viral entry, nor was any information available concerning the frequency or efficiency of the entry process. Accordingly, given that digitonin treatment enhanced binding of virus particles to Sf-21 cell surfaces, we wished to determine whether this also led to enhanced infectivity, here defined as nucleocapsid entry into the cytosol and/or nucleus, as assessed using either TEM or confocal fluorescence imaging (Fig. 3). Our observations indicate that digitonin treatment of CF enhances HfIV infectivity (Table 1).
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–6). Initial events led to the formation of binucleate and trinucleate cells linked by putative fusion ‘bridges’, which were often joined by neighbouring cells to form large polygonal syncytia (Fig. 4a, b); recruitment of additional cells appeared to cease at a maximum of approximately 15–20 nuclei/syncytium. Preliminary anecdotal observations suggested that digitonin-treated virus particles preferably bound to the aforementioned fusion bridges (Fig. 4c), but it remains as yet unclear whether this actually constitutes an intermediate event in the fusion process. It should be noted that, while Sf-21 cells often do not appear to be close to each other, and are not obviously motile, they nonetheless possess long microvilli, which would allow cell-to-cell contact over distances of 10–20 µm (unpublished observations). Left overnight, syncytia tended to lose their irregular outlines, assuming a somewhat more rounded configuration (not shown). The fusogenicity of all three ichnoviruses examined (TrIV, HfIV, and HeIV) was significantly increased by exposure to digitonin (CF vs CF/D, P < 0.001; Fig. 5), which alone had only a marginal (mock vs mock/D, P = 0.048) effect on the background incidence of syncytium formation. In comparison, syncytial indices estimated following inoculation of Cf-21 cells with untreated CF were significantly greater than those of control cells (mock vs CF, P < 0.001), but similar to those of digitonin-treated cells (mock/D vs CF, P = 0.059). Initially using TrIV, it was later discovered that a single freeze–thaw cycle (–80 °C for at least 30 min) of either wasps or extracted CF was sufficient for the latter to induce significant fusogenicity; freezing was subsequently found to be effective in the case of both H. fugitivus and H. exiguae ichnoviruses (CF vs CF/–80, P < 0.001; Fig. 5). Interestingly, however, freezing had no obvious effect on the morphology of negatively stained virus particles (not shown). Data concerning the frequency of syncytia having different numbers of nuclei at 1 h p.i. are presented in Fig. 6. These data reveal that, while the majority of cells involved in syncytium formation in this particular experiment were binucleate, substantial numbers of syncytia had more than two nuclei per cell (Fig. 6). In additional experiments, we observed that a majority of syncytia on occasion contained three or more nuclei; conditions affecting the size of syncytia remain to be elucidated.
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Given that cell fusion was typically observed within 30 min p.i., it appeared likely that fusion from without (FFWO) was occurring (fusion from within, by contrast, is defined as requiring de novo expression of viral gene products, which in other systems does not become apparent until several hours p.i.). In order to confirm that FFWO was the case, two complementary approaches were pursued. In one, psoralen/long-wave UV treatment was used exactly as described (Guzo & Stoltz, 1985) to block any potential transcriptional activity. In the other, cycloheximide was used as described (Mori et al., 2002) to inhibit protein synthesis. Neither treatment had any effect on the fusogenicity of virus preparations (data not shown), supporting a putative FFWO scenario.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), we were particularly interested in determining whether we could find a means to enhance this arguably necessary event in vitro. Since exposure of the virion inner membrane in vivo appeared to be a prerequisite for entry into certain cell types (Stoltz & Vinson, 1979), we reasoned that enhanced binding and/or infectivity might be achieved by treatment of virus preparations with digitonin, which is known to crack open the outer membrane (Deng et al., 2000). As we show here, this assumption would appear to be correct. Specifically, both binding and infection were increased by exposure of virions to digitonin. This observation should facilitate future studies on the entry of nucleocapsids initially into the cytosol and subsequently into the nucleus. Serendipitously, we have also found that at least some ichnoviruses, thus treated, are fusogenic; to the best of our knowledge, this property has not previously been associated with polydnavirus infection. We believe that our observations are best explained in terms of a fusion from without scenario, for the following reasons: (i) fusion begins to occur rapidly after the addition of virus to cells; (ii) polydnaviral structural polypeptides, including the most likely candidate fusogens, are unlikely to be synthesized in host lepidopteran cells/tissues, given that genes encoding these appear not to be packaged (Deng et al., 2000; Espagne et al., 2004; Tanaka et al. 2007; Webb et al. 2006); and (iii) exposing virus preparations to psoralen/long-wave UV irradiation did not inhibit fusogenicity, suggesting that transcriptional activity is not required. It is important to note that during the course of this study, two different populations of both H. fugitivus and T. rostrale parasitoids were used; with respect to fusogenicity, results were identical, as far as we could determine, such that in preparing data for presentation we have not made any distinction between these. Therefore, in spite of the fact that polydnavirus genomes are polymorphic (Stoltz & Xu, 1990), it seems likely that fusogenicity as a biological activity has been conserved.
FFWO has been associated with infection in a variety of enveloped viruses (Blissard & Wenz, 1992; Clavel & Charneau, 1994; Falke et al., 1985; Knutton, 1979; Milne et al., 1998; Moss, 2006; Pedersen et al., 2006; Saharkhiz-Langroodi & Holland, 1997; Siess et al., 1996; Terry-Allison et al., 1998) and rotaviruses (Falconer et al., 1995). Two scenarios have been suggested: in one, virus particles themselves serve as bridges linking the surfaces of neighbouring cells (Knutton, 1979; Knutton et al., 1977). Alternatively, virion envelope polypeptides inserted into the plasma membrane during entry might mediate fusion with adjacent cells (Clavel & Charneau, 1994; Moss, 2006). These two possibilities are of course difficult to distinguish in practice, and are not mutually exclusive. In either case, however, FFWO can be viewed as a direct consequence of virus binding and entry occurring at the cell surface. FFWO can also in some cases be artificially induced by adding virus to cells under low pH conditions, thus duplicating at the surface of the cell the endosomal environment into which virions would normally be segregated; examples of this include the baculoviruses (Blissard & Wenz, 1992) and, possibly, vaccinia virus (Moss, 2006), for which the protein(s) responsible for cell fusion are also required for infectivity (Ojeda et al., 2006; Senkevich et al., 2004; Townsley et al., 2005). Interestingly, like the ichnoviruses, extracellular enveloped vaccinia virus particles possess a second envelope, or ‘wrapper’ membrane, which must first be removed in order for both cell fusion and infection to occur (Ichihashi, 1996; Moss, 2006; Vanderplasschen et al., 1998). It was recently shown that the outer membrane of the extracellular enveloped vaccinia particle can be disrupted by polyanionic substances such as glycosaminoglycans (Law et al., 2006), known to be present in basement membranes. This is a provocative observation, since the outer membrane of at least one ichnovirus appears to be disrupted or shed upon contact with basement membranes (Stoltz & Vinson, 1979).
With regard to the ichnoviruses, there is at present no compelling evidence to suggest that the initial infection event occurs at any location other than the cell surface (Stoltz & Vinson, 1979, and unpublished observations), with haemocytes representing the possible sole exception (see below). For example, careful observation of body fat and muscle tissues from newly parasitized host larvae indicates that virions are taken up by neither endocytosis nor phagocytosis. In addition, the inner membrane is exposed, during passage of virions through basement membranes, prior to uptake into these target tissues (Stoltz & Vinson, 1979). Accordingly, we have suggested that the inner membrane mediates a membrane fusion event required for nucleocapsid entry, and now further propose that FFWO is also mediated by this membrane, or protein(s) associated with it. It is of course formally possible that a fusogen resides between the two viral envelopes. That said, no discernible electron density has ever been observed between these membranes; in addition, in preliminary experiments we have been unable to find evidence of a fusogenic activity associated with a putative viral tegument, or with outer membrane fragments released from digitonin-treated virions (unpublished observations).
In parasitized host larvae, there are two categories of potentially susceptible targets for infection by the ichnovirus particles injected during oviposition: tissues covered by basement membranes (e.g. body fat and muscle), and circulating haemocytes. A question therefore arises as to why cell fusion has never been observed in newly parasitized larvae, since haemocytes in particular represent an early and obvious target tissue. However, since haemocytes are not surrounded by basement membranes, they are most likely to come into contact only with intact virions, which may be unable to initiate a productive infection at the cell surface. In fact, work in progress indicates that haemocytes (particularly granulocytes), unlike other potential target tissues, efficiently phagocytose ichnovirus particles. In addition, we have preliminary evidence to the effect that the outer membrane becomes vesiculated once inside phagosomes (unpublished observations). Uptake of intact ichnovirions into phagocytic vesicles would readily explain the observed absence of haemocytic syncytia in vivo.
Our work, taken together with previous observations (Stoltz & Vinson, 1979), raises the issue of defining a functional role for the ichnovirus outer membrane. In that regard, it could reasonably be argued that the acquisition of an additional unit membrane envelope by budding would provide a non-lytic avenue for virion release from calyx epithelial cells. The outer membrane, once acquired, might play a somewhat ancillary role, perhaps in contributing to virion stability in extracellular environments, but there are other possibilities: for example, the outer membrane might have an affinity for basement membranes, as is indeed suggested by previously published electron micrographs (Stoltz & Vinson, 1979); this, if true, would bring virions into a closer proximity with a number of potential target tissues, and it should be noted that TrIV appears to preferentially target tissues that are surrounded by basement membranes (Béliveau et al., 2000; Cusson et al., 1998; Volkoff et al., 2002).
Much additional work will be required in order to resolve a number of questions raised by our study. For example, the degree to which infectivity in vitro accurately reflects events that occur in vivo remains to be determined. In this regard, Spodoptera frugiperda, from which Sf cell lines are derived, is not recorded as a host for any of the parasitoid species used in this study (Krombein et al., 1979). Interestingly, preliminary studies have revealed that some cell lines, including Sf-9, are refractory to virus-induced cell fusion, and possibly to virus infection as well. In keeping with this, in other systems cellular factors appear able to differentiate between entry and fusion activities (e.g. Siess et al., 1996). It will also be of interest to identify the putative fusogenic polypeptide(s) that are assumed to be associated with the virion inner membrane, and to examine the contribution, if any, of CF supernatant proteins to the cell fusion phenomenon. The question of whether digitonin simply exposes a fusogenic viral inner membrane, or in addition potentiates a fusogenic activity, needs to be considered. Digitonin is likely to complex with cholesterol and/or phytosterols present in exposed membranes (Gogelein & Huby, 1984), with consequences yet to be understood. On the other hand, fusogenicity is enhanced by freeze–thawing as well, so there is no apparent requirement for digitonin. The fact that freezing had no obvious effect on virus structure is at present enigmatic; we suggest that perhaps freezing renders the outer membrane more fragile, such that it is somewhat more prone to disruption upon contact with cell surfaces. Finally, work now in progress will attempt to address the question of whether the inner membrane protrusion often revealed following treatment of virions with digitonin (see Fig. 1) plays any role in entry; previously described for both HeIV and CsIV (Stoltz & Vinson, 1979), this is the first report of an inner membrane protrusion being observed in association with HfIV virions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 26 April 2007;
accepted 10 July 2007.
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