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. 2013 Sep;87(17):9822-35.
doi: 10.1128/JVI.01004-13. Epub 2013 Jul 3.

Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization

Paula Turkki  1 , Kaisa-Emilia Makkonen, Moona Huttunen, Johanna P Laakkonen, Seppo Ylä-Herttuala, Kari J Airenne, Varpu Marjomäki
Affiliations

Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization

Paula Turkki et al. J Virol. 2013 Sep.

Abstract

Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.

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Figures

Fig 1
Fig 1
A block in infection/transduction is not due to lack of receptor or deficient binding of virus. (A) Cells were incubated with baculovirus (MOI, 800) and analyzed at 48 h p.t. with a confocal microscope. The percentages of cells expressing the fluorescent reporter gene are shown. (B) Echovirus infection (8 × 107 PFU/ml) was analyzed at 6 h p.i. Cells were immunolabeled with antibody against EV1, and the percentages of cells showing cytoplasmic expression of newly synthesized virus particles are shown. (C to F) Cell lines showed no statistical difference in virus binding or numbers of viral receptors. The amounts of bound viruses, syndecan 1, and α2β1-integrin were determined by immunolabeling with antibodies against the virus, syndecan 1, and α2-integrin. Cells were imaged by confocal microscopy, and the relative amounts of viruses and their receptors are shown as total intensity of antibody per nucleus volume. Analysis was performed using BioImageXD. The error bars indicate SEM.
Fig 2
Fig 2
BV aggregates syndecan 1 on the plasma membrane in Ea.hy-926 and MG-63 cells. EV-1 internalization is not blocked. (A and B) With differential labeling before and after permeabilization, we were able to differentiate antigens on the cell surface from the intracellular antigens. After baculovirus (MOI, 400) internalization (5 h p.t), cells were immunolabeled and imaged by confocal microscopy. (C and D) Baculovirus internalization was studied in Ea.hy926 cells. The cells were treated with baculovirus (MOI, 500), fixed at different time points, immunolabeled with BV and sdc-1 antibodies, and imaged with a confocal microscope. Colocalization and particle analysis were performed with BioImageXD. (E) Internalization of clustered α2β1-integrin. α2β1-integrin was clustered with antibodies in the presence of fluorescence-conjugated secondary antibodies (Alexa 555). After 2 h of internalization, α2β1-integrin remaining on the cell surface was labeled with secondary antibody conjugated with a different fluorescent dye (Alexa 488), showing clustered intracellular α2β1-integrin in red and extracellular α2β1-integrin in green. (F) Quantification of images with BioImageXD showed no statistical difference between the cell lines. Scale bars, 20 μm. The error bars indicate SEM. **, P < 0.01; ***, P < 0.001.
Fig 3
Fig 3
Nonpermissive and permissive cell lines show differences in expression patterns of F-actin, syntenin, vimentin, and pPKCα. (A) Immunolabeling of syntenin and vimentin in different cell types. Actin was labeled using TRITC-conjugated phalloidin. (B) pPKCα immunolabeled from untreated (control [ctrl]) or virus-treated (virus 2 h p.i.) Ea.hy926 and HepG2 cells. Scale bars, 20 μm.
Fig 4
Fig 4
Vimentin, syntenin, and pPKCα expression levels are changed in optimal medium. (A) Baculovirus transduction (MOI, 1,000 in nonpermissive cells and 400 in permissive cells) or echovirus 1 infection (a proportional amount of cells highly expressing viral capsid proteins after 6 h p.i.; 8 × 107 PFU/ml in nonpermissive cells and 3.2 × 106 PFU/ml in permissive cells) determined from cells cultured in DMEM or RPMI medium. (B) Confocal images of cells immunolabeled with antibodies against vimentin, syntenin, or pPKCα and, in the case of F-actin, fluorescence-conjugated phalloidin. (C) Western blot showing the effect of syntenin siRNA transfection on syntenin levels in cells. (D) Syntenin knockdown could not rescue EV1 infection or BV transduction in nonpermissive Ea.hy926 cells. (E) Western blots showing the level of pPKCα in nonpermissive cells cultured in different media. Scale bars, 20 μm. The error bars indicate SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 5
Fig 5
pPKCε is associated with BV and syndecan 1 aggregate on the plasma membrane in nonpermissive cells cultured in DMEM, with downregulation of pPKCε by RPMI medium and EV1 internalization. (A) Amounts of pPKCε in cells cultured in different media. pPKCε was immunolabeled with anti-pPKCε antibody, and the amount of pPKCε in cells was determined from confocal images and analyzed with BioImageXD. (B) Confocal images of pPKCε associated with baculovirus aggregate 5 h p.i. Scale bars, 20 μm. (C) pPKCε labeled in Ea.hy926 cells after SDS-PAGE and blotting after BV or EV1 treatments for 15 min to 2 h. The error bars indicate SEM. **, P < 0.01.
Fig 6
Fig 6
PMA treatment induces EV-1 infection and BV transduction in permissive cells, but not in nonpermissive cells. (A) Baculovirus transduction efficiency (MOI, 1,000 in nonpermissive cells and 400 in permissive cells) after transduction was determined from similar samples with fluorescence-activated cell sorter (FACS) analysis by monitoring reporter gene expression. (B) Echovirus 1 (8 × 107 PFU/ml in nonpermissive cells and 3.2 × 106 PFU/ml in permissive cells) infection efficiencies were determined in different media after PMA (30-min treatment) by immunolabeling and confocal microscopy. (C) Syntenin-targeted siRNA treatment did not have an effect on pPKCα in Ea.hy926 cells. The error bars indicate SEM. *, P < 0.05; **, P < 0.01.
Fig 7
Fig 7
PMA treatment modulates cellular distribution of vimentin to a more restrictive phenotype in Ea.hy926 cells. (A) Vimentin was immunolabeled from Ea.hy926 after PMA treatment (30 min) and imaged with a confocal microscope. Cell outlines are shown. (B) Vimentin in HepG2 cells after similar treatments. (C) EV1 infection (8 × 107 PFU/ml) and BV transduction (MOI, 500) were determined after siRNA transfection and subsequent PMA treatment (30 min). The error bars indicate SEM. (D) Vimentin knockdown efficiency after siRNA transfection (120 h) in Ea.hy926 cells was evaluated by immunolabeling. Scale bars, 20 μm. The error bars indicate SEM. *, P < 0.05; **, P < 0.01.
Fig 8
Fig 8
Factors associated with permissive and nonpermissive cell phenotypes affecting BV transduction and EV1 infection efficiency.
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