Figure 5.

CrPV-1A interferes with the function of pre-programmed holo-RISC in vitro and in vivo. (a) S2 extracts were programmed with siRNA for 45 min followed by addition of 1A [(0.35 µM CrPV-1A, lane 3), (0.35 µM DCV-1A, lane 4) or (BSA, lane 5)] for 10 min. RISC assay was initiated by incubating ³²P-mRNA substrate for additional 3hrs and 5′ cleaved product was analyzed using denaturing polyacrylamide gel electrophorsis (PAGE). (b) Silencing of firefly luciferase in stable S2 cells was programmed by co-transfecting firefly and renila reporter plasmid with specific FLuc dsRNA (dsLuc) or control eGFP dsRNA (dsGFP). 16 hrs post-transfection, luciferase expression was monitored either in presence (dsLuc+CrPV-1A, dsGFP+ CrPV-1A) or absence (dsLuc) of CrPV-1A for a period of 24 hrs. (c) Stable S2 cells were transfected with FLuc dsRNA for 48 hrs followed by induction of suppressor for additional 20 hrs. The post-nuclear cytoplasmic extract was centrifuged at 200,000×g to separate the ribosomal pellet (P200) from the supernatant (S200). (d) P200 and S200 fractions were western blotted using Drosophila Argonaute 2 (α-Ago2) polyclonal and anti-Flag (α-Flag) monoclonal antibodies. RNA extracted from P200 fraction was analyzed for Luc siRNA by northern blot. P200 ethidium bromide (Et. Br) represents RNA isolated from soluble high salt P200 extraction. (e) DCV-1A and FHV B2 either inhibits Dicer 2 processing of long dsRNA or siRNA incorporation in to RISC by virtue of their dsRNA or siRNA binding activity. Conversely, CrPV-1A acts at the level of holo-RISC by protein-protein interaction with Drosophila Argonaute 2.