Silencing of the baculovirus Op-iap3 gene by RNA interference reveals that it is required for prevention of apoptosis during Orgyia pseudotsugata M nucleopolyhedrovirus infection of Ld652Y cells.
The Op-iap3 gene from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) inhibits apoptosis induced by a mutant of Autographa californica MNPV (AcMNPV) that lacks the antiapoptotic gene p35, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-iap3 during OpMNPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during OpMNPV infection, we used RNA interference (RNAi) to silence Op-iap3 expression during OpMNPV infection of Ld652Y cells. Infected cells treated with Op-iap3 double-stranded RNA (dsRNA) did not accumulate detectable Op-iap3 mRNA, confirming that the Op-iap3 gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in OpMNPV-infected cells treated with Op-iap3 dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-iap3 dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during OpMNPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function.
Means
John C
JC
Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.
Muro
Israel
I
Clem
Rollie J
RJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016367:Baculoviridae / Q000235:genetics / Q000472:pathogenicity; D002478:Cells, Cultured; D020868:Gene Silencing; D051034:Inhibitor of Apoptosis Proteins; D007915:Lepidoptera / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000472:pathogenicity*; D034622:RNA Interference; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D051034:Inhibitor of Apoptosis Proteins; D014764:Viral Proteins; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus