Sari P Kukkonen

University of Kuopio, Kuopio, Eastern Finland Province, Finland

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Publications (4)17.49 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.
    Journal of Biotechnology 11/2009; 145(2):111-9. DOI:10.1016/j.jbiotec.2009.11.003 · 2.88 Impact Factor
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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.
    PLoS ONE 02/2009; 4(4):e5093. DOI:10.1371/journal.pone.0005093 · 3.53 Impact Factor
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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 beta-galactosidase gene (beta-gal) showed a markedly increased level in the cellular expression of beta-galactosidase in the presence of MT-disintegrating drugs. The maximal increase in expression at 10 h postinoculation was observed in the presence of 80 muM nocodazole or 10 muM vinblastine. Together, these data suggest that the intact MTs constitute a barrier to baculovirus transport toward the nucleus.
    Journal of Virology 04/2005; 79(5):2720-8. DOI:10.1128/JVI.79.5.2720-2728.2005 · 4.65 Impact Factor
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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.
    Molecular Therapy 12/2003; 8(5):853-62. · 6.43 Impact Factor