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
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 (). 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; Lehiy, Blair, and Passarelli, unpublished results].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. (); 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
Fig. 1 Construction of two recombinant baculoviruses expressing a hemagglutin (HA) -tagged vfgf. A) Using an AcMNPV bacmid template lacking a functional vfgf gene, an HA-tagged vfgf under the control a 207-base pair native promoter element was inserted into (more ...)
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 ().
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 ( and results not shown). Despite numerous attempts, vFGF could not be detected in the supernatant of AcBAC-vfgfHARep-infected cells (). 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 (). vFGF was also detected in the supernatant of AcBAC-HSP70vfgfHA-infected cells, suggesting vFGF was efficiently secreted and released from cells (). 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.
Fig. 2 Production of vFGF during virus infection. Intracellular (A) and extracellular (B) production of vFGF from TN-368 cells infected with AcBAC-vfgfHA (vFGFHA) or AcBAC–HSP70vfgfHA (HSP70 VFGFHA) at an MOI of 10 PFU/cell. Cells were lysed at the times (more ...)
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. 3 Presence of vFGF on the surface of cells and budded virions. (A) SF-21 cells were infected with AcBAC-HSP70vfgfHA at a MOI of 5 PFU/cell. At 24 hours post infection, cells were fixed and immunolabeled with anti-HA.11 primary antibody and a gold-conjugated (more ...)
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 (). Only background levels of antibody were bound to the cell surfaces of AcBAC-vfgfKO-infected cells at all time points.
Fig. 4 TN-368 cells were infected at a MOI of 5 PFU/cell with either AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA. At 24 and 48 hours post infection (h p.i.), the cells were harvested and resuspended in PBS, pH 6.2. The surface of cells was labeled with (more ...)
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 (); however, these were absent in AcBAC-vfgfKO virions, indicating specificity (). 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 (). 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 ().
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 (). 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.
Fig. 5 Binding of budded virions to heparin-Sepharose beads. Purified virions from either AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA -infected SF-21 cells were incubated with heparin-Sepharose beads. The beads were washed with a salt-containing solution (more ...)
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 (). 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 (). This suggests a possible function for the virion-associated vFGF, but this function needs to be confirmed during infection.
Fig. 6 Virus induced cell migration. (A) SF-21 cells (2 × 104) were placed in the upper chamber of a migration chamber with an 8 μM pore size and purified infectious virions from AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA, were placed (more ...)
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 35
S-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 (). 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 (). 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. 7 Virus attachment and entry into SF-21 and TN-368 cells. 35S-methionine radiolabeled AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA virions (MOI of 1 PFU/cell) were allowed to attach to SF-21 (A, C) or TN-368 (B) cells at either 4 °C (A, B) (more ...)
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 (). 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 (, 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.