Silencing structural and nonstructural genes in baculovirus by RNA interference.
We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.
Flores-Jasso
C Fabian
CF
Departamento de Biología Celular, Instituto de Fisiología Celular, UNAM, Ciudad Universitaria, Mexico, D.F. 04510, Mexico.
Valdes
Victor Julian
VJ
Sampieri
Alicia
A
Valadez-Graham
Viviana
V
Recillas-Targa
Felix
F
Vaca
Luis
L
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000737:chemistry; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D034741:RNA, Small Interfering / Q000032:analysis / Q000235:genetics; D018411:Spodoptera; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics*
D034741:RNA, Small Interfering; D015678:Viral Structural Proteins