Divergences in Protein Activity and Cellular Localization within the Campoletis sonorensis Ichnovirus Vankyrin Family

Insects and cell lines. C. sonorensis parasitoids and their host, H. virescens, were reared at 27°C with a photoperiod of 16 h of light and 8 h of dark, as described previously (27). Ovarian tissue-derived Sf9 (Invitrogen, Carlsbad, CA), Sl2 (derived from SPC-SI 52 cells described in Mialhe et al. [41] and kindly provided by Nor Chejanovsky, The Volcani Center, Bet Dagan, Israel), and High Five (Invitrogen, Carlsbad, CA) insect cell lines were maintained in the laboratory in TNM-FH-10% fetal bovine serum (FBS) or TC-100-5% FBS. Drosophila S2 cells were maintained in Schneider's Drosophila medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS.

Antibody production.The production of polyclonal anti-P-vank-1 and anti-P-vank-4 antibodies in rabbits against recombinant P-vank-1 and P-vank-4 polypeptide sequences was described previously (28).

Recombinant baculovirus construction and vankyrin protein expression.The Invitrogen (Carlsbad, CA) Bac-N-Blue baculovirus protein expression system was used for introducing and expressing the CsIV vankyrin proteins from recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV). PCR products generated for all CsIV-specific vankyrin genes were ligated to the pUni/V5-His-TOPO (Invitrogen, Carlsbad, CA) vector, producing C-terminal fusions of CsIV vankyrin proteins with the V5 epitope and a six-histidine tag. vankyrin inserts were sequenced on an ABI Prism 310 Bioanalyzer to verify the integrity of the ORFs. TOPO vectors were recombined with the pBlueBac (Invitrogen, Carlsbad, CA) transfer vector and with linearized Bac-N-Blue baculovirus DNA (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol, to produce recombinant AcMNPV-expressing vankyrin proteins under the very late polyhedrin promoter. The recombinant baculoviruses were transformed into cultured Sf9 cells for virus amplification and protein production. Recombinant plaques were purified and amplified to generate P3 viral stocks of all recombinants. For baculovirus infection assays, cultured cells were infected with P3 virus stocks of the respective recombinant vankyrin-expressing AcMNPV at a multiplicity of infection (MOI) of 5. Recombinant proteins were visualized at 3 days postinfection (p.i.) by immunofluorescence assays and by Western blotting, as previously described using an anti-V5 antibody (Ab) (Sigma, St. Louis, MO). Specificities of anti-P-vank-1 and anti-P-vank-4 antibodies for the recombinant baculovirus-raised proteins were also determined via Western blotting as described.

Western blots.Crude protein extracts were prepared from 3-day recombinant AcMNPV-infected Sf9 cells, 3-day plasmid-transfected Sf9 cells, and 3-day plasmid-transfected Drosophila S2 cells. Cells were dislodged from T-75 tissue culture flasks by pipetting and centrifuged at 500 × g for 5 min at 4°C. The pellet was washed three times with 1× phosphate-buffered saline (PBS) prior to protein extraction. Washed Sf9 cells were resuspended in 500 μl of lysis buffer (10 mM Na₂HPO₄, 30 mM NaCl, 0.25% Tween 20, 10 mM EDTA, 10 mM EGTA, pH 7.0) and sonicated (three times at 15-s intervals) to release proteins. Samples were centrifuged at 16,000 × g for 10 min, and supernatants were analyzed for protein content by Bradford assay. Proteins (5 to 20 μg) were denatured by boiling for 5 min in 1× sample buffer (50 mM Tris-Cl, pH 6.8, 100 mM dithiothreitol, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol). Denatured samples were loaded and separated on 15% SDS-polyacrylamide gel electrophoresis gels and transferred to Immobilon-P (Millipore) membranes using transfer buffer (39 mM glycine, 48 mM Tris, 0.037% SDS, 20% MeOH, pH 8.3) and a Bio-Rad Semidry transfer cell. Membranes were blocked in 5% BLOTTO (BLOT-Quickblocker; GENO-TECH, Inc.) and TTBS (100 mM Tris-HCl, 0.9% NaCl, 0.1% Tween 20, pH 7.5) for 1 h and then incubated overnight at 4°C in a 1:5,000 dilution of rabbit polyclonal anti-V5 antibody (Sigma, St. Louis, MO), a 1:1,000 dilution of anti-P-vank-1 polyclonal Ab, or a 1:1,000 dilution of anti-P-vank-4 polyclonal Ab. Membranes were washed (three times for 10 min in TTBS) and incubated for 45 min in a 1:5,000 dilution (in BLOTTO) of alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G; Sigma, St. Louis, MO). Subsequently, blots were washed twice in TTBS for 10 min and twice in alkaline phosphatase substrate buffer (0.1 M NaCl, 5 mM MgCl₂, 100 mM Tris-HCl, pH 9.5) for 15 min prior to colorimetric assay. Antibody-protein complexes were then visualized by exposure to a chromogenic substrate of alkaline phosphatase (Nitro Blue Tetrazolium plus 5-bromo-4-chloro-3-indolyl phosphate) (Sigma, St. Louis, MO).

Time course infection assays.Time course infection assays in Sf9 and Sl2 cells were performed with the recombinant AcMNPV viruses expressing the CsIV vankyrin genes and proteins. Sf9 and Sl2 cells were seeded into six-well tissue culture plates and infected at 80% confluence with wild-type AcMNPV or individual recombinant vankyrin-expressing viruses at an MOI of 5. Cell morphology and survival were monitored at 24-h intervals over a 7-day time course of infection. Direct comparisons were made between cell stabilities in Sf9 and Sl2 cultures infected with the wild-type AcMNPV control infections and cultures infected with the recombinant AcMNPV variants expressing the seven CsIV vankyrin proteins. Time course infection assays were performed in triplicate to verify results.

Transient expression vector construction.The multiple cloning site (MCS) of the pJDsRed plasmid vector (kindly provided by P. Shirk and H. Bossin, U.S. Department of Agriculture, Gainesville, FL) was PCR amplified with primers flanked by HindIII restriction sites. The amplimer was digested and subcloned into the HindIII site of the pGEM-13Xf(+) plasmid (Promega, Madison, WI). The recombinant pGEM-13Zf(+)-MCS vector served as a platform for the design of vankyrin constructs having C-terminal V5-His₆ fusions and constitutive AcMNPV immediate early 1 (IE1) promoters. The AcMNPV IE1 promoter was PCR amplified from AcMNPV DNA preparations purified during the process of generating recombinant AcMNPVs described above. Forward and reverse IE1 primers were tagged with SalI and BglII sites, respectively, for cloning into the recombinant pGEM-13Zf(+)-MCS plasmid. The simian virus 40 polyadenylation signal (PA) was similarly amplified from plasmid vector DNA with oligonucleotide primers flanked with XhoI (forward) and AflII (reverse) sites. Resultant amplimers for the IE1 promoter and PA signal were digested with appropriate enzyme combinations and directionally inserted into the pGEM-13Zf(+)-MCS plasmid to generate a working pGEM-13Zf(+)-IE1-MCS-PA plasmid. The generation of CsIV vankyrin constructs with the C-terminal V5 epitope and polyhistidine fusions in Invitrogen's pUni-V5-His TOPO TA vector was described above. Vankyrin ORFs were amplified by PCR from recombinant TOPO vectors using gene-specific forward primers flanked with KpnI and a universal reverse primer flanked with NarI to recover the vankyrin-V5-His fusions intact. Resultant PCR products were digested with KpnI and NarI, band purified, and ligated into corresponding sites of the 13Zf(+)-IE1-MCS-PA vector to generate functional 13Zf(+)-IE1-vankyrin-PA expression constructs. Vankyrin ORFs were verified by sequencing as described previously.

Transfection of recombinants.Sf9 and High Five cells were plated at 75 to 80% confluence (1.6 × 10⁶ to 2.5 × 10⁶ cells/ml) and prewashed with Grace's insect medium (minus supplements and FBS). In vitro transfections of the recombinant 13Zf(+) vectors expressing the vankyrin gene constructs were performed according to Invitrogen's Bac-N-Blue transfection procedure with appropriate modifications where necessary. Briefly, 1 μg of recombinant 13Zf(+) vector was combined with 1 ml of Grace's insect medium and 20 μl of Cellfectin reagent (Invitrogen, Carlsbad, CA) in the absence of medium supplements and FBS. The mixture was incubated for 15 min at room temperature and overlaid onto prewashed High Five or Sf9 cells in six-well plates. Overlaid cells were incubated at room temperature and gently agitated for 4 h. Following the 4-h transfection, 1 ml of the appropriate complete growth medium (TNM-FH-10% FBS for Sf9 cells, TC-100-5% FBS for High Five cells, and Schneider's medium-10% FBS for Drosophila S2 cells) was added, and cells were incubated at 27°C for an additional 24 h. Cells were assayed for transfection efficiency via monitoring the IE1-YFP-PA control 13Zf(+) plasmid. Remaining vankyrin-expressing transfections were monitored for vankyrin expression by immunofluorescence assay (IFA) or Western blotting as described.

Immune challenge.To study protein localization and the effects of immune stimulation on vankyrin localization patterns, Sf9 and High Five lepidopteran cell lines were selected as they are derived from ovarian tissues of a semipermissive (Spodoptera frugiperda) and fully permissive (Trichoplusia ni) host of C. sonorensis wasps. These cells lines were also selected as they have been documented to exhibit inducible antimicrobial peptide responses and immunogenic properties closely related to hemocytes (2, 3, 27, 60; J. A. Kroemer and B. A. Webb, unpublished data). For immune stimulation experiments, recombinant plasmid-transfected Sf9 and High Five cells were exposed to 10 μg/ml Escherichia coli (111:B4) LPS (Sigma, St. Louis, MO) or 10 μg/ml laminarin (β-1,3-glucan with β-1,6 cross-linkages) (Sigma, St. Louis, MO) for 6 h prior to IFA procedures described below. Localization patterns of variants were also monitored after infection with recombinant AcMNPVs for 24 h at an MOI of 1 and after infection with recombinant baculoviruses for 48 h at an MOI of 5. These experiments were performed to visualize vankyrin localization in cells just at the onset of late expression (cell nuclei are small and cells are less compromised) and near the end of the lytic cycle (cell nuclei are swollen and cells are in a highly compromised state).

IFAs.Control, 3-day posttransfection, and 24- to 48-h baculovirus-infected Sf9 or High Five cells were plated via dropwise addition of resuspended cells to coverslips. Sf9 and High Five cells were allowed to spread on coverslips for 1 h in the appropriate complete growth medium. Adherent cells were washed briefly in ice-cold 1× PBS, fixed in 3.7% formaldehyde-PBS solution for 30 min, and permeabilized with PT (PBS-0.3% Triton X-100) detergent for 30 min. Following permeabilization, cells were blocked for 1 h in PT-0.1% bovine serum albumin, and incubated in a 1:100 dilution of primary antibody (anti-V5 polyclonal Ab; Sigma, St. Louis, MO) for 45 min at 37°C. Cells were then washed with PT solution (three times for 5 min each) and incubated with a 1:500 dilution of anti-rabbit immunoglobulin G-AlexaFluor 488-conjugated secondary antibody (Molecular Probes, Invitrogen, Eugene, OR) for 45 min at 37°C. Cells were washed as previously described and counter-stained for F-actin with a Texas red-phalloidin conjugate (Molecular Probes, Invitrogen, Eugene, OR) for 30 min, washed with PT, and analyzed for fluorescence signals using a Leica TCS-NT laser scanning confocal microscope. Cell counts were determined directly on slides to verify that transfections and/or expression of different vankyrin protein variants had no differential or adverse effects on cell adherence to coverslips. Baculovirus-infected cells showed no loss of adherence until 72 h postinfection. The majority of recombinant AcMNPV-infected Sf9 cells began to show significant signs of death and lysis by 72 h postinfection (see Fig. 2), resulting in substantial loss of cells on coverslips. For this reason, IFAs were performed on cells 24 to 48 h postinfection to minimize loss of living/adherent cells.

Fluorescent cell count assay.Sf9 cells were transfected independently with the seven recombinant 13Zf(+)-IE1-vankyrin-PA plasmids and analyzed for vankyrin protein expression by IFA as described. Twenty random images were collected from slides at a magnification of ×100, with an additional five images collected at a magnification of ×40 for each vankyrin protein variant. Numbers of positive fluorescent cells and total cell counts were collected for each image. Data from four ×100 images were pooled into a single trial, and ×40 images were analyzed as independent trials, for a total of 10 trials in each data set (five trials at ×100 [20 images/4 images per pool] plus five trials at ×40). Percentages of fluorescent cells per trial were calculated for each vankyrin variant and averaged for the 10 trials. Pairwise comparisons between all groups were made with one-tailed independent two-sample t tests (α = 0.01; P ≤ 0.001) assuming unequal variances between samples to determine significant differences in detected protein expression for each vankyrin variant. The analyses were performed on three independent transfection experiments to confirm results.

Absolute QRT-PCR.Plasmid DNA was purified by standard alkaline lysis miniprep from Sf9 cells 3 days posttransfection of recombinant 13Zf(+)-IE1-vankyrin-PA plasmids and resuspended in 15 μl of sterile H₂O. Purified plasmid DNA was diluted 1/10 and absolute quantitative real-time PCR (QRT-PCR) was performed according to Stagliano et al. (44a) to determine relative plasmid incorporation into Sf9 cells for each individual vankyrin plasmid variant. Briefly, universal forward (5′-GGAGTCGACGACCTCGTACTTTTGGCTTC-3′) and reverse (5′-GGAAGATCTTAGTCACTTGGTTGTTCACGA-3′) oligonucleotide primers were used for amplification of the IE1 promoter region present in all plasmid vectors. Restriction enzyme sites incorporated into forward (SalI) and reverse (BglII) primers for cloning purposes are in bold type. One microliter of the 1/10 diluted plasmid DNA from each transfection was subjected to 50 rounds of PCR in the presence of SYBR green dye (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Plasmid DNA standards were prepared from serial dilution (10-fold) of 0.001 μg of a purified 13Zf(+)-IE1-MCS-PA stock. The fluorescence intensity was monitored after each PCR cycle, with mean threshold cycles determined for all unknowns and standards. Reactions were performed in triplicate for standards and in quadruplicate for plasmid DNA from each unknown. Threshold PCR cycles of unknowns were calculated from the four independent runs and plotted for each transfection sample to determine relative DNA recovery between each independent transfection. Starting quantities for all unknown DNA samples were calculated on the basis of the linear standard equation formulated from starting quantities, and mean log threshold fluorescence values were obtained from standards. Reactions were performed on a Bio-Rad MyiQ single-color real-time PCR thermalcycler. The experiment was performed in triplicate to verify results.

Relative QRT-PCR.Total RNA was isolated from Sf9 cells 3 days posttransfection of the seven 13Zf(+)-IE1-vankyrin-PA plasmids using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Oligo(dT)-primed cDNA synthesis reactions were performed according to Hilgarth and Webb (19) using Superscript RNase H reverse transcriptase (Invitrogen, Carlsbad, CA) on each total RNA pool. Reactions were performed according to the manufacturer's protocol on 3 μg of total RNA from each transfection. RNA was treated with DNase I for 15 min prior to cDNA synthesis to remove contaminating plasmid DNA and verified for absence of contaminating DNA by PCR. For PCR amplification reactions, a universal reverse oligonucleotide primer (5′-GGAGGCGCCCAGTCGAGGCTGATAGCGAGC-3′) was used with the following forward oligonucleotide primers specific to the vankyrin genes: I²-vank-1, 5′-CCAGGTACCGAAATGAATATTGTTGGAAT-3′; I²-vank-2, 5′-CCAGGTACCGATGGAGATTTGTCAGATAAG-3′; I²-vank-3, 5′-CCAGGTACCGATGGAAAATTCTCAAATTGC-3′; P-vank-1, 5′-CCAGGTACCGACAATGGAGATTTCTCA-3′; P-vank-2, 5′-CCAGGTACCGATGGATATTTCTGGAATTCAA-3′; P-vank-3, 5′-CCAGGTACCGATGGACATTTCTGGAATTCG-3′; P-vank-4, 5′-CCAGGTACCGGGAACAATGAATATTGC-3′. Restriction enzyme sites incorporated into forward (KpnI) and reverse (NarI) primers for cloning purposes are in bold type. Relative QRT-PCRs on vankyrin genes were performed according to Stagliano et al. (44a). cDNA pools created for each transfection were diluted 1/30, and 1 μl of the diluted cDNA was subjected to 50 rounds of PCR in the presence of SYBR Green dye (Bio-Rad, Hercules, CA) according to manufacturer's instructions. The fluorescence intensity was monitored after each PCR cycle with mean threshold cycles determined for all unknowns and a serially diluted (fivefold) cDNA standard testing positive for amplification of each gene. Reactions were performed in triplicate for standards and in quadruplicate for diluted cDNA from each unknown. Mean threshold PCR cycles of unknowns were calculated from the four independent runs, normalized to 18S ribosomal rRNA quantities (amplified with the primers FW, [5′-CAGTGATGGGATGAGTGCTTTTATTAGAT-3′] and RV [5′-AGGCCCTCCGTCGATTGGTTTT-3′]), and plotted for each cDNA sample to visualize relative vankyrin RNA expression between each unknown. Starting quantities for all unknown cDNA samples were calculated on the basis of the linear standard equation formulated from starting quantities and mean log threshold fluorescence values obtained from standards. Reactions were performed on a Bio-Rad MyiQ single-color real-time PCR thermalcycler. The experiment was performed in triplicate to confirm results.

Recombinant vankyrin protein expression and antibody cross-reactivity.Recombinant vankyrin proteins exhibited cross-reactivity with the most closely related proteins of the same subclass (FB or HC specific) but do not react with other proteins of the vankyrin family in CsIV (Fig. 1). The anti-P-vank-1 Ab reacted strongly with recombinant P-vank-1 protein and weakly with recombinant I²-vank-3 protein, while the anti-P-vank-4 Ab reacted with both recombinant P-vank-2 and P-vank-4 proteins. All recombinant vankyrin proteins appeared to react equally well with the anti-V5 Ab. These data suggest that differences in protein structure and antigenicity exist within the CsIV vankyrin proteins.

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FIG. 1.

Cross-reactivity of antibodies raised to recombinant CsIV vankyrin proteins. (A) All CsIV vankyrin proteins were produced in a recombinant AcMNPV baculovirus, and crude supernatant extracts (10 μl) from Sf9 cells were assayed by Western blotting for reactivity with anti-V5, anti-P-vank-1, and anti-P-vank-4 Abs. The seven recombinant proteins reacted strongly with the V5 Ab. The anti-P-vank-1 Ab reacted strongly with recombinant P-vank-1 protein and weakly with recombinant I²-vank-3 protein. The anti-P-vank-4 Ab reacted strongly with recombinant P-vank-2 protein and weakly with recombinant P-vank-4 protein. (B) Direct comparisons of vankyrin protein sequences revealed that only the most closely related proteins (bold boxes) within the FB- and HC-specific vankyrin subclasses cross-reacted with the anti-P-vank-1 or anti-P-vank-4 Ab.

Vankyrin proteins cause differential phenotypic effects when expressed from recombinant baculoviruses in vitro.In vitro infection studies with two insect cell lines (Sf9 and Sl2) indicate that vankyrin proteins, expressed from recombinant AcMNPV baculoviruses, exhibit different pathological effects (Fig. 2). Viruses expressing recombinant FB-specific P-vank-1 and I²-vank-3 proteins exhibit enhanced longevity and delayed cell lysis compared to wild-type AcMNPV and viruses expressing other vankyrin family variants. Enhanced cell survival in P-vank-1- and I²-vank-3-infected cells is apparent starting 3 days postinfection and can be seen up to 7 days following infection of both cell lines. Figure 2 represents images taken at day 4 postinfection when the effects are most pronounced. These findings suggest that differential activities occur between vankyrin protein variants.

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FIG. 2.

Vankyrin effects on recombinant AcMNPV infection at 4 days postinfection. Sf9 (A) and Sl2 (B) cells exhibit differential morphological effects when exposed to recombinant AcMNPVs expressing CsIV vankyrin proteins under the very late polyhedrin promoter. Cells infected with recombinant, polyhedrin-negative viruses expressing FB-specific P-vank-1 and I²-vank-3 proteins (asterisks) are more stable and resemble noninfected cells at 4 days postinfection (p.i.). Cells exposed to recombinant viruses expressing the remaining CsIV vankyrin proteins undergo lysis by 4 days p.i. and resemble cells infected with wild-type, polyhedrin-positive AcMNPV. Viruses expressing recombinant P-vank-3 also cause Sf9 cells to assume a lenticular/sickle shape (arrow) late in infection. Magnification, ×40.

Vankyrin proteins show divergences in subcellular localization and response to immune stimulation in vitro.The different vankyrin proteins of CsIV respond differentially to immune stimulation and virus infection when expressed transiently from plasmids or from recombinant AcMNPV in Sf9 cells (Fig. 3). The FB-specific P-vank-1 and I²-vank-3 proteins showed both nuclear and cytoplasmic localization in unstimulated Sf9 cells but a strong bias toward nuclear localization in unstimulated High Five cells. The HC-specific P-vank-2, P-vank-4, and I²-vank-1 proteins were exclusively nuclear in transiently transfected Sf9 and High Five cells while the remaining two variants localized predominantly to large (P-vank-3; HC) or small (I²-vank-2; FB) cytoplasmic granules (Fig. 3A). When transfected Sf9 cells were exposed to LPS or laminarin for 6 h prior to IFAs, some vankyrin variants exhibited a shift in localization patterns. The P-vank-1 and I²-vank-3 proteins adopted a cytoplasmic bias in localization with many I²-vank-3-expressing cells showing a uniform distribution of protein throughout the cell (Fig. 3B). The P-vank-2 protein shifted from exclusive nuclear localization to a more uniform distribution in cells. I²-vank-1, I²-vank-2, P-vank-3, and P-vank-4 proteins did not show an apparent shift in localization in response to LPS or laminarin stimulation, although the granulated cytoplasmic appearance of I²-vank-2 in unstimulated cells adopted a smoother and more uniform appearance in stimulated cells (Fig. 3B). When cells were infected with recombinant AcMNPV expressing the vankyrin variants under the very late polyhedrin promoter, additional phenomena were observed. At the onset of very late expression (24 h p.i.; MOI of 1), all seven vankyrins appeared in the nuclei of infected Sf9 cells. At a higher MOI of 5 and by 48 h p.i., vankyrin proteins show localization patterns resembling more closely those exhibited by LPS- or laminarin-stimulated cells (Fig. 3C). Reciprocal infection studies (MOI of 5 at 24 h and MOI of 1 at 48 h) revealed similar trends (data not shown), although slides in these cases exhibited a small distribution of cells with opposing signals. We suspect this result was likely due to asynchronous progression of virus infection over 48 h at the lower MOI of 1, resulting in small populations of cells at different stages of infection. Also, the higher levels of initial virus exposure at the MOI of 5 may have promoted a hyperimmuno-sensitized state in some cells, resulting in the small number of cytoplasmic signals observed within 24 h of infection at this MOI.

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FIG. 3.

Immune stimulation and vankyrin localization. In vitro cellular localization patterns of recombinant CsIV vankyrin proteins in the presence and absence of immune stimuli. (A) V5 epitope-tagged CsIV vankyrins were expressed transiently from recombinant plasmids under the AcMNPV IE1 promoter and assayed for their cellular localization with the V5 antibody (green stain) 3 days posttransfection in High Five and Sf9 cells in the absence of immune stimulation. Two of the seven CsIV vankyrins (P-vank-3 and I²-vank-2; asterisks) showed localization to cytoplasmic granules (arrows), while the remaining five variants localized predominantly to cell nuclei (arrows). P-vank-1 and I²-vank-3 proteins were also seen in the cytoplasm of some cells (not shown). Magnification: ×150; scale bar, 20 μm. (B) IFAs were performed on Sf9 cells transiently transfected with vankyrin plasmids 6 h poststimulation with LPS (top) and laminarin (bottom). P-vank-1 and I²-vank-3 proteins (asterisk) shifted toward a cytoplasmic bias in staining in response to the immune stimuli with almost no cells exhibiting a strong nuclear stain. The P-vank-2 protein (boxed asterisk) shifted from an exclusively nuclear signal toward a more uniform distribution of protein within the cell. Differences in cellular localization were not observed between induced versus uninduced cells for the I²-vank-1, I²-vank-2, P-vank-3, and P-vank-4 proteins. (C) The CsIV vankyrin proteins were produced in recombinant AcMNPV baculoviruses and expressed very late in infection under the polyhedrin promoter. Proteins were assayed for cellular localization in Sf9 cells following 24 h of infection (MOI = 1) and 48 h of infection (MOI = 5). Cytoplasmic (in terms of uninduced cells, as in panel A) vankyrins shifted to the nucleus (circled asterisks) in response to the physiological conditions of earlier infection (arrows). All other vankyrins remained exclusively in the nucleus as in transient transfections (arrows). Vankyrins adopted localization patterns similar to LPS- and laminarin-stimulated cells (asterisks and boxed asterisk, respectively) by 48 h p.i. Noninfected Sf9 and wild-type (WT) AcMNPV-infected cells showed no specific nuclear or cytoplasmic staining. Cells were counterstained with Texas red-phalloidin to visualize actin and the cellular cytoskeleton. Cell nuclei of wild-type AcMNPV-infected Sf9 cells were detected by propidium iodide staining. Magnification: ×100; scale bar, 50 μm.

Vankyrin proteins are produced at different levels in transfected insect cells.Sf9 and Drosophila S2 cells transfected with nearly identical recombinant 13Zf(+) plasmids (differing only in the vankyrin gene cassettes) show apparent differences in protein expression but no difference in plasmid transfection efficiency or RNA expression. Absolute real-time PCR against purified DNA from transfected Sf9 cells indicated that DNA incorporation into cells remained relatively equivalent between transfections (Fig. 4A). Relative real-time PCR analysis showed that RNA transcription of the seven vankyrin cassettes also occurs at nearly equivalent levels posttransfection of recombinant 13Zf(+)-IE1-vankyrin-PA plasmids (Fig. 4B). In contrast, all vankyrin proteins were detected in IFAs, but cell numbers expressing visually detectable levels of recombinant P-vank-2, P-vank-4, and I²-vank-2 were significantly and consistently lower than those expressing P-vank-1, P-vank-3, I²-vank-1, and I²-vank-3 variants (Fig. 4C). Western blots against cell extracts from vankyrin plasmid-transfected cells confirmed the protein expression differences observed in IFAs with only P-vank-1, P-vank-3, I²-vank-1, and I²-vank-3 proteins detected in transfected Sf9 and Drosophila S2 cell extracts (Fig. 4D). Together, these data strongly suggest that different vankyrin proteins are subject to different levels of translational and/or proteolytic control in insect cells.

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FIG. 4.

Transient transfection and expression of CsIV vankyrins. (A) Absolute quantitative real-time PCR of recombinant 13Zf(+)-IE1-vankyrin-PA plasmids purified 3 days posttransfection of Sf9 cells. Recombinant plasmid recovery from transfected cells was equivalent between transfections. Error bars represent ±1 standard deviation from the mean. (B) Relative quantitative real-time PCR of vankyrin RNA expressed from Sf9 cells 3 days posttransfection of recombinant plasmids. Vankyrin RNA expression was similar across transfections for all vankyrin variants. Error bars represent ±1 standard deviation from the mean. (C) IFA analyses suggest that differences in regulation of protein expression occur between individual vankyrin variants. Percentages of transfected cells expressing visually detectable levels of recombinant P-vank-1, P-vank-3, I²-vank-1, and I²-vank-3 proteins were significantly higher (P < 0.001) than those of other vankyrin variants. Error bars represent ±1 standard deviation from the mean. (D) Western blotting with the anti-V5 Ab was performed on Sf9 and S2 cell extracts 3 days posttransfection of recombinant 13Zf(+)-IE1-vankyrin-PA plasmids. As seen in IFAs, the seven recombinant vankyrin proteins exhibited different levels of protein production in transfected cells. The P-vank-1 and I²-vank-3 proteins were expressed highly and detected readily in cell extracts. Recombinant P-vank-3 and I²-vank-1 proteins were also detected on Western blots but at lower levels. The P-vank-2, P-vank-4, and I²-vank-2 proteins were expressed at levels too low to be detected on Western blots using a colorimetric assay.

Protein localization patterns correlate with phylogenetic relationships of vankyrin proteins.The protein localization patterns and activities of vankyrin variants correlate closely with phylogenetic relationships (Fig. 5). The most ancestral and divergent HC- and FB-specific vankyrins (P-vank-3 and I²-vank-2) are those that appear in the cytoplasm of unstimulated cells and show a cytoplasm-to-nuclear shift in earlier stages of recombinant AcMNPV infection. The remaining three HC vankyrins (P-vank-2, P-vank-4, and I²-vank-1) show exclusive nuclear localization in unstimulated cells, with only a moderate cytoplasmic shift seen in the P-vank-2 protein in response to LPS, laminarin, and very late virus infection. The remaining two FB vankyrins (P-vank-1 and I²-vank-3) show a bias toward nuclear localization in unstimulated cells and a shift toward predominant cytoplasmic localization in LPS- or laminarin-stimulated cells, as well as very late virus infection. The P-vank-1 and I²-vank-3 proteins are also those that show cell stabilizing effects when expressed from recombinant AcMNPVs (Fig. 2).

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FIG. 5.

Relationships between vankyrin phylogeny and subcellular localization. (A) The vankyrin phylogenetic tree is rooted based upon the assumption of the biological clock. Alignment parameters are as follows: gap penalty, 15; gap length penalty, 6.66; DNA weight matrix, International Union of Biochemistry. The tree was assembled with MEGALIGN software (DNASTAR, Madison, WI) and constructed via the Clustal W method of DNA alignment. (B) Localization patterns of vankyrin proteins in immune-stimulated and unstimulated cells correlate closely with phylogenetic relationships. N, nucleus; C, cytoplasm; N+C, nuclear and cytoplasmic distribution; RAcMNPV, recombinant AcMNPV.