title: RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome.
abstract: Double-stranded RNA (dsRNA) initiates cellular posttranscriptional responses that are collectively called RNA silencing in a number of different organisms, including plants, nematodes, and fruit flies. In plants, RNA silencing has been associated with protection from virus infection. In this study, we demonstrate that dsRNA-mediated interference also can act as a viral defense mechanism in mosquito cells. C6/36 (Aedes albopictus) cells were stably transformed with a plasmid designed to transcribe an inverted-repeat RNA (irRNA) derived from the genome of dengue virus type 2 (DEN-2) capable of forming dsRNA. Clonal cell lines were selected with an antibiotic resistance marker and challenged with DEN-2. The cell lines were classified as either susceptible or resistant to virus replication, based on the percentage of cells expressing DEN-2 envelope (E) antigen 7 days after challenge. Eight out of 18 (44%) cell lines designed to express irRNA were resistant to DEN-2 challenge, with more than 95% of the cells showing no DEN-2 antigen accumulation. One of the DEN-2-resistant cell lines, FB 9.1, was further characterized. DEN-2 genome RNA failed to accumulate in FB 9.1 cells after challenge. Northern blot hybridization detected transcripts containing transgene sequences of both sense and antisense polarity, suggesting that DEN-2-specific dsRNA was present in the cells. In addition, a class of small RNAs 21 to 25 nucleotides in length was detected that specifically hybridized to labeled sense or antisense DEN-2 RNA derived from the target region of the genome. These observations were consistent with RNA silencing as the mechanism of resistance to DEN-2 in transformed mosquito cells.
authors.affiliation: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Foothills Research Campus, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Zach N
authors.initials: ZN
authors.lastname: Adelman
authors.forename: Irma
authors.initials: I
authors.lastname: Sanchez-Vargas
authors.forename: Emily A
authors.initials: EA
authors.lastname: Travanty
authors.forename: Jon O
authors.initials: JO
authors.lastname: Carlson
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Beaty
authors.forename: Carol D
authors.initials: CD
authors.lastname: Blair
authors.forename: Ken E
authors.initials: KE
authors.lastname: Olson
mesh_terms: D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002461:Cell Line, Transformed; D009033:Culicidae / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics* / Q000502:physiology; D016679:Genome, Viral; D008969:Molecular Sequence Data; D010957:Plasmids; D034622:RNA Interference; D012367:RNA, Viral / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D012367:RNA, Viral