Suppression of apoptosis in insect cells stably transfected with baculovirus p35: dominant interference by N-terminal sequences p35(1-76).
Expression of p35 from the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) suppresses virus-induced apoptosis and promotes virus replication in Spodoptera frugiperda (SF21) cells. To examine the molecular mechanism by which p35 prevents apoptosis in insects, SF21 cells were stably transfected with p35. Neomycin-resistant cell lines that synthesized protein P35 were identified. Stable transfection with p35 protected SF21 cells from apoptosis induced by actinomycin D concentrations that caused apoptotic death of untransfected cells. Cellular expression of p35 also blocked apoptosis induced by infection with p35 null mutants and restored mutant replication to levels comparable to those of wild-type virus. In contrast, stable expression of the mammalian death suppressor bcl-2 failed to block actinomycin D- or AcMNPV-induced apoptosis. Thus, p35 was sufficient to prevent apoptosis, whereas bcl-2 was not, suggesting that the activities of the two nonhomologous death regulators are functionally distinct. Stable expression of the truncation mutant p35(1-76), containing the N terminus of p35, failed to block apoptosis. However, p35(1-76) interfered with p35 antiapoptotic activity, since stably transfected cells underwent apoptosis upon infection with wild-type AcMNPV. Despite normal levels of viral p35 transcription, P35 levels were selectively reduced during infection. Thus, p35(1-76) acted as a dominant inhibitor by directly or indirectly affecting the synthesis or stability of viral P35. These results suggested that the N terminus of P35 constitutes a functional domain which is required to interact with other proteins, possibly host invertebrate death regulators or P35 itself.
Institute for Molecular Virology, Graduate School, University of Wisconsin-Madison 53706.
J L
JL
Cartier
P A
PA
Hershberger
P D
PD
Friesen
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D005816:Genetic Complementation Test; D015151:Immunoblotting; D051034:Inhibitor of Apoptosis Proteins; D007700:Kinetics; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2; D012367:RNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D018411:Spodoptera; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis*; D001616:beta-Galactosidase / Q000096:biosynthesis
D051034:Inhibitor of Apoptosis Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012367:RNA, Viral; D014764:Viral Proteins; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus; D003609:Dactinomycin; D004247:DNA; D011505:Protein-Tyrosine Kinases; D001616:beta-Galactosidase