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Journal of Virology, December 2005, p. 15452-15459, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15452-15459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Baculovirus Entry into Human Hepatoma Cells

Heli Matilainen,{dagger} Johanna Rinne,{dagger} Leona Gilbert, Varpu Marjomäki, Hilkka Reunanen, and Christian Oker-Blom*

NanoScience Center, Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland

Received 31 July 2005/ Accepted 26 September 2005

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype member of the Baculoviridae family, has gained increasing interest as a potential vector candidate for mammalian gene delivery applications. AcMNPV is known to enter both dividing and nondividing mammalian cell lines in vitro, but the mode and kinetics of entry as well as the intracellular transport of the virus in mammalian cells is poorly understood. The general objective of this study was to characterize the entry steps of AcMNPV- and green fluorescent protein-displaying recombinant baculoviruses in human hepatoma cells. The viruses were found to bind and transduce the cell line efficiently, and electron microscopy studies revealed that virions were located on the cell surface in pits with an electron-dense coating resembling clathrin. In addition, virus particles were found in larger noncoated plasma membrane invaginations and in intracellular vesicles resembling macropinosomes. In double-labeling experiments, virus particles were detected by confocal microscopy in early endosomes at 30 min and in late endosomes starting at 45 min posttransduction. Viruses were also seen in structures specific for early endosomal as well as late endosomal/lysosomal markers by nanogold preembedding immunoelectron microscopy. No indication of viral entry into recycling endosomes or the Golgi complex was observed by confocal microscopy. In conclusion, these results suggest that AcMNPV enters mammalian cells via clathrin-mediated endocytosis and possibly via macropinocytosis. Thus, the data presented here should enable future design of baculovirus vectors suitable for more specific and enhanced delivery of genetic material into mammalian cells.

* Corresponding author. Mailing address: University of Jyväskylä, Nano Science Center, Department of Biological and Environmental Science, PO Box 35, FIN-40351 Jyväskylä, Finland. Phone: 358 14 260 2285. Fax: 358 14 260 2221. E-mail: christian.oker-blom{at}jyu.fi.

{dagger} H. M. and J. R. made equal contributions to this report.

Journal of Virology, December 2005, p. 15452-15459, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15452-15459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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