RNAi induced by dsPns10 inhibiting the assembly of Pns10 tubule and secondary infection of neighboring cells by RDV in VCMs.

<p>(<b>A</b>) Detection of siRNAs in VCMs transfected with synthesized dsPns10 or dsYFP at 72 h after transfection. Approximately 5 µg of total RNA was probed with DIG-labeled transfected RNA. Lower panel: detection of 5.8S rRNA as a control to confirm loading of equal amounts of RNA in each lane. (<b>B, C</b>) Intercellular spread of RDV was inhibited by RNAi induced by dsPns10 in VCMs. At 24 h after transfection with cellfection transfection reagent (control; images I), dsYFP (images II) or dsPns10 (images III), VCMs were inoculated with RDV at a low MOI (0.001) and cultured in the presence of virus-neutralizing antibodies, which prevent free virus infection. At 3 days p.i., cells were immunolabeled for RDV virions with virus-FITC (green) or for Pns10 tubules with Pns10-rhodamine (red), and then examined by confocal microscopy. Images in <b>B</b> show green fluorescence from virus antigens in widefield view. Images in C are composites of those for green fluorescence (virus antigens) and red fluorescence (Pns10 tubules). Insets in image III of panel <b>C</b> show green fluorescence (virus antigens) and red fluorescence (Pns10 antigens) of the merged images in the boxed area. Images are representative of multiple experiments with multiple preparations. Bars, 20 µm.