FIG. 7.

Cosilencing VP1 and VP6 partially recovers the translation of viral proteins. Cells were transfected with a mixture of siRNAs against VP1 and either VP6 or VP2, as indicated, and 48 h later they were infected with RRV at an MOI of 3. At 7 hpi cells were radiolabeled for 60 min with 25 μCi/ml of Easy-tag Express-³⁵S and then lysed. Protein synthesis was visualized by autoradiography (A) or proteins were transferred to a nitrocellulose membrane for Western blot assays using MAb 3A8 to stain VP2 and polyclonal rabbit antibodies to VP1 and TLPs. As secondary antibodies, Alexa Fluor-conjugated antibodies were used. (B) The relative amount of each viral protein was calculated by densitometry of the fluorescent bands using ImageQuant TL software (Amersham Biosciences), and it is expressed as a percentage of the corresponding protein obtained when the irrelevant control siRNA(Irr) was used, which was taken as 100%. Data represent the arithmetic means ± standard deviation of at least three independent experiments.