Improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes.
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.
Salminen
Mirka
M
Department of Biological and Environmental Science, P.O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Jyväskylä, Finland.
Airenne
Kari J
KJ
Rinnankoski
Rita
R
Reimari
Jenni
J
Välilehto
Outi
O
Rinne
Johanna
J
Suikkanen
Sanna
S
Kukkonen
Sari
S
Ylä-Herttuala
Seppo
S
Kulomaa
Markku S
MS
Vihinen-Ranta
Maija
M
D021581:Active Transport, Cell Nucleus; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism / Q000821:virology; D000072159:Dynactin Complex; D015870:Gene Expression; D005822:Genetic Vectors; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure / Q000821:virology*; D006801:Humans; D007763:Lac Operon; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D019251:Nucleocapsid / Q000378:metabolism / Q000648:ultrastructure; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C000606361:DCTN2 protein, human; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase