Divergences in protein activity and cellular localization within the Campoletis sonorensis Ichnovirus Vankyrin family

Abstract

Ichnoviruses (IVs) occur in obligate symbiotic associations with endoparasitic ichneumonid wasps. IVs are injected with eggs during parasitization, where viral infection and gene expression alter host physiology to ensure endoparasitoid survival. The seven Campoletis sonorensis IV (CsIV) vankyrin genes encode proteins that possess ankyrin repeat domains resembling the inhibitory domains of NF-kappaB transcription factor inhibitors (IkappaBs). The CsIV vankyrins are divided into two subclasses: those expressed primarily in the host fat body (three genes) and those expressed in host hemocytes (four genes). CsIV vankyrin proteins showed limited antigenic similarity when analyzed by Western blotting. Cellular localization and expression patterns of recombinant vankyrin proteins in High Five and Sf9 insect cells differed within and between the subclasses and in cells exposed to lipopolysaccharide, laminarin, or viral immune challenge. In unstimulated Sf9 cells, five vankyrins were detected in cell nuclei. The remaining two proteins localized predominantly to cytoplasmic granules. Immune stimulation of cells resulted in a nuclear-to-cytoplasmic shift of three vankyrins but did not affect localization of other variants. When expressed from recombinant Autographa californica multiple nucleopolyhedroviruses (AcMNPVs), all vankyrins showed a nuclear localization during early stages of infection with patterns resembling those of immune-challenged cells as the infection progressed. Two fat body vankyrins also produced unique biological effects when expressed from recombinant AcMNPV. Insect cells infected with these viruses exhibited enhanced longevity compared to those infected with viruses expressing other vankyrins. Together, these data suggest that vankyrin proteins in CsIV have divergent physiological functions.

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.

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.

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.

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.

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.