title: Baculovirus entry into human hepatoma cells.
abstract: 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.
authors.affiliation: University of Jyväskylä, Nano Science Center, Department of Biological and Environmental Science, PO Box 35, FIN-40351 Jyväskylä, Finland.
authors.forename: Heli
authors.initials: H
authors.lastname: Matilainen
authors.forename: Johanna
authors.initials: J
authors.lastname: Rinne
authors.forename: Leona
authors.initials: L
authors.lastname: Gilbert
authors.forename: Varpu
authors.initials: V
authors.lastname: Marjomäki
authors.forename: Hilkka
authors.initials: H
authors.lastname: Reunanen
authors.forename: Christian
authors.initials: C
authors.lastname: Oker-Blom
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002460:Cell Line; D011992:Endosomes / Q000821:virology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000821:virology*; D006801:Humans; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000502:physiology*; D018411:Spodoptera / Q000821:virology*; D014771:Virion / Q000502:physiology*; D014779:Virus Replication
chemical_list: D049452:Green Fluorescent Proteins