|Home | About | Journals | Submit | Contact Us | Français|
The Autographa californica M nucleopolyhedrovirus (AcMNPV) viral fibroblast growth factor (vFGF) has functional parallels to cellular FGFs. Deletion of the AcMNPV vfgf has no obvious phenotype in cell culture but delays the time of insect death. Here, we determined vFGF production during virus infection. vFGF was detected at 24 hours post infection and through the reminder of the infection cycle. Since vFGF is thought to be a secreted membrane-binding protein and virions acquire an envelope derived from the cell membrane, we examined virions for the presence of vFGF using microscopy, flow cytometry, and affinity chromatography. We found that vFGF associated with virions. Furthermore, budded virus carrying vFGF had more affinity to heparin than vFGF-deficient budded virus, consistent with the affinity of FGFs for heparan sulfate proteoglycans. Although the function of virion-associated vFGF is not clear, we found that virion-associated vFGF stimulated cell motility and affected virus attachment.
Fibroblast growth factors (FGFs) are a family of growth factors that play diverse roles in both embryonic development and homeostatic maintenance (Ornitz and Itoh, 2001). Members of this family include 22 vertebrate homologs and several well-characterized invertebrate orthologs, including Drosophila branchless, pyramus, and thisbe FGFs (Samakovlis et al.,1996; Stathopoulos et al., 2004). FGFs are typically secreted from the cell using the endoplasmic reticulum-Golgi pathway. On the surface of cells, FGFs bind to heparan sulfate proteoglycans through ionic interactions between the highly sulfated oligosaccharides and the basic amino acids of FGFs (Faham et al., 1996; Kreuger et al., 2005; Lin et al., 1999; Ornitz 2000). The binding of the growth factor to heparin sulfate serves to both protect FGF in the extracellular environment from degradation and mediate downstream specificity with potential cellular receptors though differences in the sugar sulfation patterns (Harmer 2006; Raman et al., 2002). Release of the heparan sulfate-FGF signaling complex from the cell is mediated by factors such as heparanase or matrix metalloproteinase 2 which cleave the oligosaccharide chain from its membrane bound protein anchor (Plotnikov et al., 1999; Szebenyi and Fallon, 1999; Vlodavsky and Friedmann, 2001; Tholozan et al., 2007). Once released, heparan-FGF complexes can bind tyrosine kinase fibroblast growth factor receptors (FGFR) through interactions between amino acids in the beta trefoil domain of FGF and the D2 extracellular domain of the receptor (Mohammadi et al., 2005a; Mohammadi et al., 2005b, Ornitz, 2000). Binding of a ligand with the extracellular domain of FGFR induces a conformational change in the transmembrane region of the receptor, allowing receptor homodimerization (Peng et al., 2009). Dimerization of the FGFRs leads to transphosphorylation of intracellular kinase domains and activation of the Ras-mitogen activated protein kinase signal cascade pathway (Ornitz and Itoh, 2001; Pye and Gallagher, 1999; Sutherland et al., 1996). The final outcome of this activation varies greatly, depending upon the ligand, receptor, and context of the signal, but can include induction of motility, cell proliferation, or differentiation (Ornitz and Itoh, 2001).
In addition to vertebrate and invertebrate FGFs, most baculoviruses also encode FGF orthologs with sequence identity to cellular FGFs (Detvisitsakun et al., 2005; Katsuma et al., 2004; Sutherland et al., 1996). The best characterized baculovirus, Autographa californica M nucleopolyhedrovirus (AcMNPV), encodes a single fgf that predicts a 181-amino acid protein with a defined and functional signal sequence (Detvisitsakun et al., 2005; Katsuma et al., 2004). AcMNPV vfgf expressed from a plasmid vector was secreted from cells and had strong affinity to heparin-Sepharose (Detvisitsakun et al., 2005). The specific role of vFGF in viral pathogenesis has not been defined, but evidence suggests that it may aid in the trafficking of the virus from midgut cells, the primary site of infection, to the tissues in the hemocoel (Detvisitsakun et al., 2007). vFGF may also be involved in hemocyte-mediated infection spread within insect tissues (Detvisitsakun et al., 2005; Katsuma et al., 2008).
Previous studies using Northern blot analysis and RT-PCR detected vfgf transcripts between 3 and 72 hours (h) post infection (p.i.) (Detvisitsakun et al., 2005; Katsuma et al., 2004); however, the production of vFGF during virus replication has not been determined. This study shows timing of vFGF production during infection, its secretion fate, and in vitro assays that suggest a function during virus infection of susceptible hosts.
Transcription of vfgf has been previously examined; vfgf was classified as an early gene since transcription was evident at early times post infection (p.i.) and in the presence of cycloheximide (Detvisitsakun et al., 2005; Katsuma, et al., 2004). However, protein production kinetics have not been reported during the infection cycle. To examine vFGF accumulation during infection, we constructed a bacmid expressing vfgf with a hemagglutinin (HA) tag at the C terminus to facilitate immunodetection. Briefly, 448 base pairs of vfgf (~82% from the N terminus) were replaced via homologous recombination with the zeocin resistance gene using the commercially available bacmid bMON14272. The enhanced green fluorescent protein gene (egfp) under the Drosophila heat shock protein (hsp) 70 promoter control, a high-level inducible promoter in insect cells, and the polyhedrin (polh) gene under polh promoter control were introduced into the polh locus of bMON14272, resulting in AcBAC–vfgfKO (Detvisitsakun et al., 2006). To construct AcBAC-vfgfHARep virus, egfp and polh cassettes, and the HA-tagged vfgf under the control of its own promoter was inserted in AcBAC-vfgfKO, so that only one copy of the gene was present (Fig. 1A). The vfgf promoter, a 207 base pair fragment, has been previously shown to support vfgf expression [Detvisitsakun et al., 2006; Lehiy, Blair, and Passarelli, unpublished results]. To construct a virus carrying vfgf driven by the Drosophila hsp70 promoter, AcBAC-HSP70vfgfHA, the 207 base pair vfgf promoter fragment was replaced with the Drosophila hsp70 promoter. The presence of vfgf and its correct insertion location within the viral genome was confirmed using PCR (results not shown). RT-PCR was used to ensure that vfgf was expressed in AcBAC-vfgfHARep. We detected vfgf transcripts at 6, 12, 24 and 48 h p.i. (Fig. 1B); AcBAC-vfgfRep virus, expressing untagged vfgf from the same promoter, also expressed vfgf transcripts (Detvisitsakun et al., 2006). Although it is possible that vfgf is better expressed at its native locus, this 207 base pair vfgf promoter region was sufficient to repair phenotypic defects in AcBAC-vfgfKO (Detvisitsakun et al., 2006).
To evaluate whether insertion of vfgf under either native or hsp70 promoter control in AcBAC-vfgfHARep and AcBAC-HSP70vfgfHA, respectively, affected virus replication, we performed single-step growth curve analyses. SF-21 cells were infected with either AcBAC-vfgfHARep, AcBAC-HSP70vfgfHA or AcBAC, a virus in which vfgf has not been perturbed, at a multiplicity of infection (MOI) of 5 plaque forming units (PFU)/cell, supernatant was collected at several times p.i., and the titer determined by TCID50. We did not detect any significant growth differences between the viruses throughout the time courses of infections (Fig. 1C).
We determined accumulation of vFGF driven by its native promoter by infecting TN-368 cultured cells at an MOI of 5 PFU/cell with either AcBAC-vfgfHARep or AcBAC-HSP70vfgfHA and immunodetection. vFGF steady-state levels were observed in immunoblots with whole cell lysates prepared from AcBAC-vfgfHARep-infected cells at 24 and 48 h p.i. but not at 12 h p.i. or earlier (Fig. 2A and results not shown). Despite numerous attempts, vFGF could not be detected in the supernatant of AcBAC-vfgfHARep-infected cells (Fig. 2B). AcBAC-HSP70vfgfHA-infected cells, as expected, exhibited much higher levels of vFGF accumulation starting at 12 h p.i., peaking at 24 h p.i. and continuing at 48 h p.i., even though 25-fold less total protein was loaded per lane than in the AcBAC-vfgfHARep cell lysate (Fig. 2A). vFGF was also detected in the supernatant of AcBAC-HSP70vfgfHA-infected cells, suggesting vFGF was efficiently secreted and released from cells (Fig. 2B). It is possible that the inability to detect vFGF in the medium of AcBAC-vfgfHARep-infected cells, where vfgf expression is lower than from AcBAC-HSP70-vfgfHA-infected cells, was due to protein detection sensitivity levels. As a signaling molecule, the necessary levels of vFGF for efficient function do not need to be high.
We detected vFGF in whole cell lysates and in cell culture media when expressed at high enough levels. When expressed at lower levels, vFGF secretion may be hard to detect. In addition, the presence of vFGF in whole cell lysates could be due to secreted vFGF tethered to the surface of cells by heparan sulfate proteoglycan interactions or its presence intracellularly. To validate that vFGF was cell surface-bound, SF-21 cells were infected with AcBAC-HSP70vfgfHA and harvested 24 h p.i. Infected cells were fixed and immunolabeled with anti-HA antibody followed by a gold-labeled secondary antibody prior to embedding in resin. Dense pockets of gold-labeled antibodies, corresponding to cell surface-bound vFGF, were evident on cells, confirming that vFGF was indeed secreted and retained on the surface of cells (Fig. 3A).
To further strengthen the observation that vFGF binds to cell membranes and have a quantitative comparison of cell bound-vFGF from each virus construct, TN-368 cells were infected with AcBAC-HSP70vfgfHA, AcBAC-vfgfHARep, or AcBAC-vfgfKO and harvested at 12, 24 and 48 h p.i. After harvesting, cell surfaces were immunolabeled with anti-HA primary antibody and an allophycocyanin (APC)-conjugated secondary antibody, and labeled cells were analyzed using fluorescence-based flow cytometry. The number of vFGF positive cells was considerably higher in AcBAC-HSP70vfgfHA-than AcBAC-vfgfHARep-infected cells at all time points examined although with both AcBAC-HSP70vfgfHA and AcBAC-vfgfHARep infections, the number of vFGF positive cells increased approximately 5.4 to 6-fold between 12 and 24 h p.i., respectively (Fig. 4). Only background levels of antibody were bound to the cell surfaces of AcBAC-vfgfKO-infected cells at all time points.
We predicted that if vFGF was secreted from cells and bound to heparan sulfate proteoglycans on cell membranes, virions budding from cells would acquire heparan sulfate-bound vFGF on their envelopes. To examine if budded virions incorporated vFGF on the virus particle, budded virions from AcBAC-vfgfKO-, AcBAC-vfgfHARep-, or AcBAC-HSP70vfgfHA-infected SF-21 cells were purified by gradient centrifugation, fixed onto Nickel Formvar/Carbon 200 mesh grids, and vFGF was detected using immunoelectron microscopy. Gold-labeled particles specific to HA-tagged vFGF were detected on AcBAC-vfgfHARep virions (Fig. 3B); however, these were absent in AcBAC-vfgfKO virions, indicating specificity (Fig. 3D). In the majority of experiments, we could only detect three or less gold-labeled particles on individual vFGF-HA carrying virions. In contrast, there were over 10-fold more gold-labeled particles conjugated to anti-GP64 that associated with GP64, an envelope associated viral protein essential for virus entry (Fig. 3C). Interestingly, the gold-labeled vFGF-HA particles were usually localized at one end of the virion. Virions derived from the AcBAC-HSP70vfgfHA-infected cells showed increased gold-labeled particles also at the bulbous end of the virion, reaffirming the polarization of virions and vFGF (Fig. 3E).
Since FGFs, including vFGF, have high affinity to heparin-Sepharose, we next compared the affinity of AcBAC-vfgfHARep, AcBAC-vfgfKO and AcBAC-HSP70vfgfHA purified virions to heparin-Sepharose and determined whether the presence or absence of vFGF on the virus surface affected this property. Equal numbers of infectious virions, calculated prior to use by TCID50, were incubated with heparin-Sepharose beads, sulfated and carboxylated glucosaminoglycans on Sepharose that yield an overall negative charge and serve as a cation exchangers. To reduce non-specific interactions, the beads were washed with 125 mM NaCl to disrupt ionic interactions prior to eluting the bound proteins with a 1.25 M NaCl solution. All fractions were dialyzed against TC-100 incomplete media and virus solutions titered. We found that dialysis was necessary since high salt concentrations used during washes and elution interfered with infections (Lehiy and Passarelli, unpublished results). AcBAC-vfgfKO virions had less affinity to heparin-Sepharose than either AcBAC-vfgfHARep or AcBAC-HSP70vfgfHA virions, with the majority of virions eluting in the unbound fraction, flow through, and wash fractions. In contrast, virions with vFGF on the surface had higher affinity to heparin beads, eluting mainly in the presence of high salt concentrations (Fig. 5). Non-specific virus binding to heparin-Sepharose can be attributed to either cellular FGFs incorporated on the viral envelopes, FGFs from the serum containing media, or other ionic interactions.
It has previously been demonstrated that heparin-Sepharose purified vFGF stimulates motility of insect cells by stimulating an FGFR and ensuing a subsequent signaling cascade (Detvisitsakun et al., 2005; Katsuma et al., 2006); thus, we asked whether virions containing vFGF could stimulate cell motility. Using transwells with polycarbonate membrane inserts, 2 × 104 SF-21 cells were placed in the upper chamber while 1 × 105 to 1 × 107 infectious purified AcBAC-HSP70vfgfHA or AcBAC-vfgfKO virions were placed in the lower chamber. After four hours, the transwell inserts were removed and cells that migrated to the lower chamber were quantified using CellTiter-Glo luminescent substrate that measures ATP production and is indicative of live cells. Cell migration increased proportionally to virus titer with 1 × 107 virions yielding statistically significant differences between the three viruses (Fig. 6A). To address whether vFGF cleaved from the virion induced motility, we treated 1 × 107 purified AcBAC-HSP70vfgfHA virions with 1IU of heparinase III, an enzyme known to cleave heparan sulfate proteoglycans from cellular surfaces. After treatment, the virion and supernatant fractions were used in cell motility experiments. Cell motility stimulated by heparinase III-treated virions decreased compared to untreated virions, while the supernatant fraction containing cleaved vFGF stimulated cell motility (Fig. 6B). This suggests a possible function for the virion-associated vFGF, but this function needs to be confirmed during infection.
vFGFs is a membrane-associated protein that interacts with other membrane proteins, including heparan sulfate proteoglycans and the FGFR. In addition, a number of viruses belonging to several virus families, use heparan sulfate molecules as their receptors (Flint, et al., 2004). Thus, we were interested in evaluating whether the presence or absence of vFGF on the surface of the virions affected virus attachment to and/or entry into permissive insect cells. To this end, virions from AcBAC-vfgfHARep, AcBAC-vfgfKO, and AcBAC-HSP70vfgfHA were radiolabed with 35S-methionine and partially purified to remove unbound radioactivity. Radiolabeled AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA was added at an MOI of 1 PFU/cell to chilled SF-21 or TN-368 cells. At specific times post attachment, the virus supernatant (i.e., unattached virions) was removed and the cells washed three times with cold PBS. After washing, the cells were lysed and the radioactivity determined in a scintillation counter. SF-21 cells treated with AcBAC-vfgfKO bound less radioactive particles than AcBAC-HSP70vfgfHA and AcBAC-vfgfHARep at every time point measured (Fig. 7A). Similar defects were observed using TN-368, although the binding defects of AcBAC-vfgfKO compared to viruses encoding vfgf were not as marked as those in SF-21 cells, and differences were not significant 30 minute post attachment (Fig. 7B). We repeated the binding assay at 25 °C using SF-21 cells and also observed a defect in attachment at this temperature, which was more prominent with shorter attachment times (Fig. 7C).
To determine whether the presence of vFGF on the budded virus envelope affected entry, we inhibited endosomal acidification, a step required for endosomal membrane fusion, with ammonium chloride at several time points after AcBAC-vfgfRep or AcBAC-vfgfKO attachment to SF-21 cells (Hefferon et al., 1999). At early times post attachment (0 through 20 minutes), treatment of cells with ammonium chloride resulted in an entry defect ranging between 24.6 and 34.5% for AcBAC-vfgfKO-compared to AcBAC-vfgfHARep-infected cells (Fig. 7D). At 30 and 60 minutes post attachment, treatment of AcBAC-vfgfKO-infected cells with ammonium chloride resulted in an entry defect of 15.9 and 14.1%, respectively, compared to AcBAC-vfgfHARep. AcBAC-vfgfRep- and AcBAC-vfgfKO-infected but ammonium chloride-untreated cells showed no significant differences (Fig. 7D, control column). Although we observed entry differences between AcBAC-vfgfHARep and AcBAC-vfgfKO, they were too small to infer any significance during the normal virus attachment and entry phase.
The role of secreted vFGF during virus infection has been hypothesized; it may stimulate tracheoblast motility during secondary infections or hemocyte chemotaxis during systemic infection (Detvisitsakun et al., 2005; Katsuma et al., 2008). Since its specific role has not been defined in vivo, it is important to determine at what time during the replication cycle is vFGF produced as well as the destination of the secreted product. This information will provide insight into functions of vFGF during the infection cycle in vitro and in vivo.
In this study, we observed that AcBAC-vfgfHA-infected TN-368 cells produced HA-tagged vFGF at 24 and 48 h p.i. This is consistent with vfgf transcripts previously detected starting at 3 h p.i. and in the presence of cycloheximide, and at late times, as part of multicistronic messages (Detvisitsakun et al., 2005). The inability to detect vFGF earlier or in the supernatant of infected cells is not surprising, since as a signaling ligand, vFGF would be required in concentrations under 5 ng/ml to induce activation of target cells (Detvistsakun, Wu, and Passarelli, unpublished results; Katsuma et al., 2006a). Since vFGF is secreted during virus infection of cells and probably binds cell surface heparan sulfated proteoglycans in vivo, we considered that as the budded virus exited from infected cells, its envelope would acquire vFGF-heparan sulfate proteoglycan complexes along with other cellular and viral membrane proteins (Fig. 8). Therefore, we investigated this possibility and found that vFGF was present on the cell surface of infected cells and that budded virus carried vFGF. Furthermore, the presence of vFGF affected the affinity of virions to bind heparin-Sepharose in vitro. Our studies were performed using two insect cell lines, SF-21 and TN-368, since viruses lacking vfgf have effects on both cell types and the species from which the cells were derived (Detvistsakun et al., 2007; Katsuma et al., 2006b).
Potential roles for vFGF on the surface of the virion are open to speculation, although this study provides some evidence suggesting chemotaxis of target cells may be involved. Motility of cells towards the virus would facilitate virus spread. This is consistent with viruses carrying vfgf, accelerating host mortality (Detvisitsakun et al., 2007; Katsuma et al., 2006b). Alternatively, the bonafide properties of FGF secretion may causally localize vFGF on the surface of cells and thus, the virus envelope. Studies addressing the function of vFGF on the membrane of virus particles during infection of the host may clarify its in vivo role.
Other viruses use cell surface heparan sulfate proteoglycans for entry into cells. Proteins on the surface of the human herpes virus 8 interact with sulfated heparan prior as a tether prior to glycoprotein-mediated cell adhesion and entry (Akula et al., 2001; Spear, 2004; Wang et al., 2001). In addition, herpes simplex 1 virus uses heparan sulfate to bind to the basal laminae of muco-cutaneous tissues prior to entry (Yura et al., 1992). We showed that the presence of vFGF on the surface of the virion facilitates virus attachment more efficiently possibly via interaction with surface heparan sulfate proteoglycans or FGFRs, however, it is not clear if these effects are significant during in vivo infections. Interestingly, transduction of mammalian cells with baculoviruses also shows a robust dependency on virus-heparin interactions but not on other negatively charged polyanions such as chondroitin sulfate or dermatan sulfate (Duisit et al., 1999). It is tempting to speculate that the affinity of vFGF and heparin enhances the electrostatic interactions between the virus and the cell surface, allowing more efficient virus attachment, but the significance of this effect during infection is not clear.
The cell line IPLB-SF-21 (SF-21) (Vaughn et al., 1977) derived from the fall armyworm, Spodoptera frugiperda, and TN-368 cells (Hink, 1970) derived from the cabbage looper, Trichoplusia ni, were maintained in TC-100 medium (Invitrogen) supplemented with 10% fetal bovine serum (Atlanta Biological) and 0.26% tryptose broth as described previously (O’Reilly et al., 1994).
We constructed a transfer vector to generate a recombinant of AcMNPV expressing vfgf under native promoter control. First, the oligonucleotides (Sac I-F 5′-CTTAAGTCT GCAGTTTTAC-3′ and Sac 1-R 5′-ATAAAAATGTTTTTATTGTAAAATACAC-3′) were used to amplify an 863-base pair fragment from a construct containing HA-tagged vfgf and flanking regions. DNA containing HA-tagged vfgf and 207-base pairs upstream of vfgf that serve as promoter region was ligated into the transplacement vector pFastBac–polh+gfp+ (Detvisitsakun et al., 2006) at Sac I sites to generate pFastBac–polh+-vFGFHA-gfp+. The correct amplified sequence was verified by nucleotide sequencing analysis. To create the transplacement vector with vfgf under Drosophila heat shock protein (HSP) 70 promoter control, the 207 base pair native promoter element was removed and the 739 base pair HSP 70 promoter element inserted. The correct insertion was also verified by nucleotide sequencing analysis.
AcBAC-vfgfHA Rep and AcBAC-HSP70 vfgfHA were generated by Tn7-mediated transposition by transforming MAX DH10Bac Efficiency competent Escherichia coli (Invitrogen) with pFastBac–polh+-vFGFHA-gfp+ or pFastBac–polh+-HSP70 vFGFHA-gfp+. Bacterial cells were incubated for 4 hours in SOC media (Invitrogen) at 37 °C and then plated on the appropriate selection media and incubated for an additional 18 hours according to the Bac-to-Bac Baculovirus Expression System Manual (Invitrogen). White colonies resistant to kanamycin and gentamicin were selected and the transposition event and flanking regions were verified by PCR analysis. Budded virus was produced by transfection of AcBAC-vfgfHARep or AcBAC-HSP70vfgfHA DNA into SF-21 cells by liposome-mediated transfection as previously described (Crouch and Passarelli, 2002).
SF-21 cells were infected at an MOI of 5 PFU/cell with AcBAC-vfgfHARep, AcBAC-HSP70vfgfHA or AcBAC. Budded virus was collected at different times p.i. and titers were determined by TCID50 (O’Reilly et al., 1994). The graph represents 3 independent experiments for each virus. Cells from the AcBAC-vfgfHARep-infections were harvested from plates, lysed with Trizol reagent (Invitrogen), and stored at −80 °C until the entire time course was complete. Using the standard protocol, total RNA from infected cells was extracted and quantified. Aliquots (4 μg) from each time point were treated with 2 units of DNAse I for 2 hours and then heat inactivated at 72 °C for 15 minutes. After DNAse I treatment, the Access RT PCR system (Promega) was used to amplify vfgf specific transcripts with 1μg of treated RNA as a template and oligonucleotides (F5′-GGAGCTGTTTACG GAACCATTG-3′ and R5′-CAGTGCCACATACGTCAACTTG-3′) as primers. To control for DNA contamination, parallel samples lacking AMV Reverse Transcriptase were tested. PCR conditions were carried out at 45 °C for 1 hour, followed by 40 cycles of 95 °C for 2 minutes, 45 °C for 30 seconds, and 72 °C for 1 minute.
TN-368 cells (2 × 106) were infected at an MOI of 10 PFU/cell with AcBAC–vfgfHARep or AcBAC-vfgfKO virus. At several times p.i., cells were collected in 100 μl of Laemeli loading buffer. Proteins were resolved in a sodium dodecyl sulfate–12 % polyacrylamide gel, transferred to a PVDF membrane, and detected using 1:3000 dilution of anti-HA.11 antibody (Covance), 1:5000 dilution of goat anti-mouse IgG–horseradish peroxidase (Bio-Rad). The SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) was used to detect cross-reactive proteins. Densitometry Integrated Density Values (IDV) were calculated using AlphaImager 2200 software.
SF-21 cells (2 × 108) were infected with AcBAC–vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA at an MOI of 0.1 PFU/cell. At 72 h p.i., cell supernatant was collected and budded virus isolated using density gradient centrifugation (O’Reilly et al., 1994). Purified virions were removed from sucrose or Nycoprep™ Universal (Axis Shield) gradients and resuspended in PBS, pH 6.2 (Potter and Miller, 1980). Virions (1:100 dilution) were bound to Nickel Formvar/Carbon 200 mesh grids (Ted Pella, Inc.) and immunolabeled with a 1:1500 dilution of anti-HA.11 antibody (Covance) or 1:1500 dilution of anti-GP64 (AcV1) antibody (Santa Cruz Biotechnology) and 1:100 dilution of anti-mouse IgG (whole molecule)–gold antibody (Sigma). Virions were visualized with a Philips MC-100 transmission electron microscope.
TN-368 cells (0.5 × 106) were infected with AcBAC–vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA at an MOI of 5 PFU/cell. At 24 and 48 h p.i., cells were harvested, washed twice with cold PBS (pH 6.2) and then resuspended in 100 μl of 1:1000 dilution of anti-HA.11 antibody (Covance). After 1 h, the antibody solution was removed and cells were washed once with PBS. Cells were then resuspended in 100 μl of 1:2000 dilution of APC-conjugated anti-mouse IgG antibody (BD Biosciences) for 1 h in the dark. Unbound antibody was removed and the cells were washed 3 times in PBS before fixing in 100 μl of 4% paraformaldehyde-1% glutaraldehyde in 0.1 M PBS. Cells were analyzed using a FACSCalibur flow cytometer.
SF-21 cells (2 × 108) were infected with AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA and virions were isolated and titered as described above (O’Reilly et al., 1994). Heparin Sepharose™ 6 Fast Flow beads (Amersham Biosciences) were washed with and resuspended in PBS pH 6.2. Aliquots (100 μl) from a 50% bead slurry were mixed with 5 × 107 infectious virions and then diluted to a final volume of 1 ml. The virus-heparin-Sepharose mixture was incubated overnight at 4 °C with steady rotation. After binding, the flow-through was collected and the beads were washed with 1 ml of TC-100 incomplete media. Bound vFGF was eluted with 1.25 M NaCl solution (1 ml). The input, flow-through, wash, and elution fractions were dialyzed in TC-100 incomplete media for a minimum of four hours at 4 °C to remove excess salt and maintain comparable salt concentrations in the different samples. After dialysis, the amount of infectious virions in each sample was determined by TCID50. Results are reported as a percentage of the total viral infectious units present in each sample treatment.
SF-21 cell migration was assessed using 8 μM pore size Costar transwells with polycarbonate membrane inserts. Approximately 2 × 104 cells were loaded onto transwell inserts and allowed to settle for 30 minutes. The transwell inserts were then transferred to 24-well plates containing virus and incubated for 4 h at 27 °C. Purified infectious virions (1 × 105 to 1 × 107) from AcBAC-vFGFHARep-, AcBAC-HSP70vfgfHA-or AcBAC-vfgfKO-infected cells were placed in the lower transwell chamber along with 500 μl of PBS, pH 6.2. After incubation, the transwell inserts were removed and cells that had migrated downward were quantified using CellTiter-Glo luminescent substrate to measure ATP, according to the protocol provided by the manufacturer (Promega). The level of luminescence was determined with the Wallac Victor3 1420 Multilabel counter (Perkin-Elmer). To remove virion-bound vFGF, 3 × 107 AcBAC-HSP70vFGFHA virions in 3 ml of buffer (20 mM Tris-HCl, pH 7, 0.1mg/ml BSA, and 4 mM CaCl2) were treated with 1IU of heparinase III from Flavobacterium heparinum (Sigma Aldrich) at 27 °C for 4 h. After treatment, virions were purified by centrifugation at 24,000 × g through a 25% Nycoprep cushion. The supernatant and cushion were carefully removed and the pelleted virus reconstituted with 1ml of PBS, pH 6.2. In the migration assay, 500 μl of clarified supernatant and 1 × 106 AcBAC-HSP70vfgfHA virions were used in cell motility assays.
SF-21 cells (8 × 107) were infected at an MOI of 0.1 PFU/cell with AcBAC-vfgfHARep, AcBAC-vfgfKO or AcBAC-HSP70vfgfHA. The supernatant was removed 29 h p.i. and replaced with Grace’s Insect Medium lacking L-methionine and unsupplemented (Invitrogen). After 1 h, EasyTag™ EXPRESS 35S Protein Labeling Mix (Perkin Elmer) was added to a final concentration of 10 μCi/ml and cells were incubated at 27 °C for 10 h. Cells were then supplemented with 10 mM unlabeled methionine and 10% FBS and incubated for an additional 48 h. After incubation, the supernatant was removed and centrifuged at 1000 × g for 5 minutes to remove any debris present. The cleared supernatant was centrifuged at 80,000 × g for 75 minutes at 4 °C through a 25% sucrose cushion to pellet the virus and then resuspended in 10 ml of TC-100 complete media supplemented with 10% FBS. The radiolabeled virus was titered using TCID50 end point dilution methods for a total of 3 times and radioactivity in the virus suspension measured by mixing 10 μl of virus with 1.5 ml of NET lysis buffer (20 mM Tris, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40 1 mM EDTA, pH 7.5) and 3.5 ml of EcoLUME ™ Scintillation fluid (MP Biomedicals). Cells (1 × 106) were plated on a 35-mM tissue culture dish and allowed to attach for 1 h at 27 °C before placing at a 4 °C for 3 h. After chilling, the cells were placed on ice blocks and the supernatant removed.
Chilled virus was then added to each well (MOI of 1 PFU/cell, 500 μl total volume) and removed at specific time intervals (0, 5, 10, 20–30 and 60 minutes). Immediately after, the cells were washed with 3 ml of ice-cold PBS, pH 6.2 and then lysed with 1.5 ml of NET buffer. The lysate was transferred to scintillation vials along with 3.5 ml of EcoLume ™ Liquid Scintillation fluid. Radioactivity was determined for 1 minute using a Beckman-Coulter LS6500 Scintillation counter. To normalize all samples, the CPM counts on all samples were adjusted relative to initial CPM counts.
SF-21 cells (1 × 106) were allowed to attach for 1 h at 27 °C prior to incubation at 4 °C for 2 h and infected at an MOI of 1 PFU/cell for 1 h at 4 °C with pre-chilled AcBAC-vfgfHARep or AcBAC-HSP70vfgfHA. After 1 h, the virus supernatant was removed and cells washed 3 times with pre-chilled PBS, pH 6.2. After washing, the cells were treated with warm TC-100 complete media and returned to a 27 °C incubator. At 0, 5, 10, 20, 30 and 60 minutes post attachment, ammonium chloride was added to the wells (final concentration of 25 mM) to inhibit endosome acidification. As a control, a well of infected cells was left untreated allowing endosomal acidification to continue normally. 24 h post attachment, the cells were removed from the plates, fixed with 100 μL of 4% paraformaldehyde-1% glutaraldehyde in 0.1 M PBS. Cells were analyzed using a FACSCalibur flow cytometer and the results of two independent experiments were shown here.
Analysis of standard deviations and statistical significance was done using Graphpad Prism® software.
We thank Dr. Dan Boyle for help with sectioning and imaging samples for microscopy and Justin Trowbridge for cell passage. We would also like to thank Maria Da Cunha for technical assistance in constructing and characterizing AcBAC-HSP70vfgfHA and Justin Blair for his work on defining the vfgf promoter.
This work was supported in part by NIH grant 5R21AI63089 and the NRI Competitive Grants Program, USDA, under agreement number 2008-35302-18849. This is contribution number 09-220-J from the Kansas State University Agricultural Experiment Station.