Sari P Kukkonen

University of Kuopio, Kuopio, Eastern Finland Province, Finland

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Publications (11)23 Total impact

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    ABSTRACT: Baculoviruses can express transgenes under mammalian promoters in a wide range of vertebrate cells. However, the success of transgene expression is dependent on both the appropriate cell type and culture conditions. We studied the mechanism behind the substantial effect of the cell culture medium on efficiency of the baculovirus transduction in different cell lines. We tested six cell culture mediums; the highest transduction efficiency was detected in the presence of RPMI 1640 medium. Vimentin, a major component of type III intermediate filaments, was reorganized in the optimized medium, which associated with enhanced nuclear entry of baculoviruses. Accordingly, the phosphorylation pattern of vimentin was changed in the studied cell lines. These results suggest that vimentin has an important role in baculovirus entry into vertebrate cells. Enhanced gene delivery in the optimized medium was observed also with adenoviruses and lentiviruses. The results highlight the general importance of the culture medium in the assembly of the cytoskeleton network and in viral gene delivery.
    No preview · Article · Nov 2009 · Journal of Biotechnology
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    Dataset: Figure S3
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    ABSTRACT: Localication and entry of baculovirus after expression of various endocytic membrane traffic regulators. (A,B) Baculovirus (wt, MOI 200, red) localization with Flotillin-1 MAb at 15 min p.t. (A) or GPI-EGFP at 30 min p.t. (B) in 293 cells (green). Baculovirus (wt, MOI 200) entry into 293 cells transfected with the wt, CA and DN mutants of Cdc42-EGFP at 6 h p.t. (C–E), as well as DN and CA mutants of Rac1-EGFP at 2 h p.t. (F,G) (green). (H) 293 cells were transfected with the Ntb-domain of IL2-receptor visualizing the localization of internalized IL2-receptor after baculovirus (wt, MOI 200, red) transduction for 30 min. Ntb-detecting antibody conjugate (NtB(IL-2):Cy3-561; green) was added on the plasma membrane before baculovirus addition. (I–O) Baculovirus (p24mCherry, MOI 200) entry after 6 h p.t. in 293 cells transfected with the wt and DN mutant of CtBP1/BARS (BARS; I–J), CA and DN mutants of Pak1 (K,L) as well as wt, DN and CA mutants of Rab34 (M–O; green). In images A,B and F–H, baculovirus was labeled with capsid labeling antibodies vp39 MAb or BV Ab together with Alexa-555 secondary antibody (red). In images C-E and I-O, p24mCherry construct was used. In all images, scale bars 10 µm. (9.89 MB TIF)
    Preview · Dataset · Apr 2009
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    Dataset: Figure S2
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    ABSTRACT: Baculovirus induces ruffle formation. (A) Cellular ruffle formation in live baculovirus transduced HepG2 cells. Baculovirus (MOI 400) was directly fed onto cells in the confocal microscope, and details of the cell surface protrusions were followed immediately during 0–15 min p.t. DIC images reveal protrusions growing from the cell surface. Baculovirus is not visualized for clarity. (B) Baculovirus was internalized for 15 min into 293 cells and, after PFA fixation, baculovirus capsid and actin were labeled by antibodies (Ab, Alexa-488, green) and TRITC-phalloidin (red), respectively. Scale bars, 10 µm. (1.30 MB TIF)
    Preview · Dataset · Apr 2009
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    Dataset: Figure S5
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    ABSTRACT: Putative effects on baculovirus entry due to expression of various endocytosis regulating WT constructs. Various plasmid constructs were transfected into 293 cells for 48 hours prior to baculovirus (p24mCherry, 200 MOI) uptake for 30 min. The plasma membrane-bound virus was extensively washed before the PFA fixation of the. Transfected cells (n = 300–400 cells) were scanned by confocal microscopy from three separate samples and analyzed for their p24mCherry fluorescence intensity. The results are shown as mean values ± SE. Untransfected cells (control) and cells expressing plain EGFP (GFP) were as controls. (1.64 MB TIF)
    Preview · Dataset · Apr 2009
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    Dataset: Figure S4
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    ABSTRACT: The effect of EIPA on fluid-phase entry. The efficacy of EIPA was tested by internalizing TRITC-De for 30 min in untreated (A) or EIPA-treated (0.1 mM) 293 cells (B). A representative group of cells are shown with (right) or without (left) DIC image merged with TRITC-De. Scale bars, 10 µm. (2.63 MB TIF)
    Preview · Dataset · Apr 2009
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    Dataset: Video S1
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    ABSTRACT: Baculovirus internalization into living 293 cells was observed by confocal microscopy. In the imaging, fluorescent (p24mCherry, MOI 400) virus was fed onto cells and the attachment and internalization of the viruses were monitored thereafter. Differential contrast image (DIC), virus (red) and selected time frames (0–400 s) are shown. (3.92 MB MOV)
    Preview · Dataset · Apr 2009
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    Dataset: Figure S1
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    ABSTRACT: Clathrin-mediated endocytosis is not involved in baculovirus early uptake. (A,B) Effect of baculovirus transduction on clathrin localization pattern in 293 cells. By live confocal microscopy, the expressed, fluorescent light chain clathrin (clathrin light chain tomato) showed similar distribution after baculovirus transduction (C; wt, 1000 MOI, 60 min p.t.) as untransduced, transfected control cells (B). Pseudocolor images are presented for better visualization of the expressed clathrin. (C,D) The expression of wt (C) or DN (D) clathrin coat assembly regulator protein Eps15-EGFP (green) and internalization of baculovirus capsid (wt, vp39 MAb, 200 MOI; red) after 2 h p.t. in 293 cells. (E,F) Baculovirus (p24mCherry, 200 MOI; red) was internalized in untreated (E) or dynasore-treated (F) 293 cells for 30 min together with CME marker transferrin (A546-TF; red). (G) Baculovirus (p24mCherry, 200 MOI, red) localization in 293 cells with caveolin-1 MAb (green). Alexa-555 and -488 were used as secondary antibodies. In all images, scale bars 10 µm. (4.90 MB TIF)
    Preview · Dataset · Apr 2009
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    ABSTRACT: The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants of RhoA, and of Arf6, a regulator of clathrin-independent entry. Furthermore, the entry of baculovirus induced ruffle formation and triggered the uptake of fluorescent E. coli bioparticles. To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bacterial uptake. This study increases our understanding of virus entry strategies and gives new insight into baculovirus-mediated gene delivery in human cells.
    Full-text · Article · Feb 2009 · PLoS ONE
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    ABSTRACT: Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), a potent virus for mammalian cell gene delivery, possesses an ability to transduce mammalian cells without viral replication. We examined the role of the cellular cytoskeleton in the cytoplasmic trafficking of viral particles toward the nucleus in human hepatic cells. Microscopic studies showed that capsids were found in the nucleus after either viral inoculation or cytoplasmic microinjection of nucleocapsids. The presence of microtubule (MT) depolymerizing agents caused the amount of nuclear capsids to increase. Overexpression of p50/dynamitin, an inhibitor of dynein-dependent endocytic trafficking from peripheral endosomes along MTs toward late endosomes, did not significantly affect the amount of nuclear accumulation of nucleocapsids in the inoculated cells, suggesting that viral nucleocapsids are released into the cytosol during the early stages of the endocytic pathway. Moreover, studies with recombinant viruses containing the nuclear-targeted expression β-galactosidase gene (β-gal) showed a markedly increased level in the cellular expression of β-galactosidase in the presence of MT-disintegrating drugs. The maximal increase in expression at 10 h postinoculation was observed in the presence of 80 μM nocodazole or 10 μM vinblastine. Together, these data suggest that the intact MTs constitute a barrier to baculovirus transport toward the nucleus.
    Full-text · Article · Apr 2005 · Journal of Virology
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    ABSTRACT: Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced green fluorescent protein (EGFP)-displaying virus. Our confocal and electron microscopy results suggest that the transduction block in mammalian cells is not in the endosomal escape, as previously proposed, but rather in the cytoplasmic transport or nuclear entry of the virus capsid. Our results also suggest that the EGFP-tagged virus can be used for visualization of the virus biodistribution in vivo. Furthermore, capsid-modified baculoviruses hold great promise for the nuclear and subcellular targeting of transgenes and as a novel peptide display system for a variety of eukaryotic applications.
    Full-text · Article · Dec 2003 · Molecular Therapy
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    Kukkonen SP
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    ABSTRACT: Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced green fluorescent protein (EGFP)-displaying virus. Our confocal and electron microscopy results suggest that the transduction block in mammalian cells is not in the endosomal escape, as previously proposed, but rather in the cytoplasmic transport or nuclear entry of the virus capsid. Our results also suggest that the EGFP-tagged virus can be used for visualization of the virus biodistribution in vivo. Furthermore, capsid-modified baculoviruses hold great promise for the nuclear and subcellular targeting of transgenes and as a novel peptide display system for a variety of eukaryotic applications.
    Preview · Article · Nov 2003 · Molecular Therapy