Functional analysis of Aaeg Dcr2 PAZ domain mutants. (A) Antiviral activity of Aaeg Dcr2. AF319 cells were transfected with expression plasmids encoding myc-tagged WT Dcr2 or Dcr2 PAZ domain mutants M1–M4 or eGFP (negative control). These cells were infected with SFV-FFLuc (MOI = 0.1) 24 h posttransfection (hpt). FFLuc levels were measured at 48 h postinfection (hpi) and are shown as mean ± SEM relative light units compared to eGFP control set to 1 from three independent repeats, performed in technical triplicate with (*) P < 0.05 according to Student's t-test. (B) Silencing activity of mutant Aaeg Dcr2. AF319 cells were cotransfected with (i) myc-tagged pPUb plasmids expressing WT Dcr2 or Dcr2 PAZ domain mutants M1–M4 (with eGFP as control), (ii) FFLuc and RLuc (internal control) reporter plasmids, and (iii) dsRNA targeting FFLuc (FFLuc dsRNA, blue) or eGFP (eGFP dsRNA; nonsilencing control, gray). At 24 hpt, FFLuc and RLuc levels were measured and are shown as mean ± SEM relative light units (FFLuc/RLuc) normalized to dseGFP from three independent repeats, performed in technical triplicate, with (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 versus controls according to two-way ANOVA.
