FIG 6.
Co-IP of TiLV RNA with Protein 4. TiLV-infected E-11 cells (A) or virions (B) were lysed in a nondenaturing buffer, and the extracts were immunoprecipitated with αProtein4 or αProtein5 antibodies, or with a preimmune serum. Co-IP of the 10 segments (Seg1-Seg10) of TiLV RNA genome (A and B) and actin mRNA (A) was quantified by qRT-PCR. (A) For each pellet, the ΔΔCT value of each of the viral RNA segments was determined, using actin mRNA as a reference gene (actin CT values were 25.99, 25.84, and 25.97 for preimmune, αProtein5, or αProtein4 pellets, respectively). For each pellet in panel B, the ΔCT value of each of the viral RNA segments was determined. The fold changes of RNA levels in pellets obtained with αProtein4 or αProtein5 antibodies (“relative Co-IP”) in panels A and B were calculated relative to the RNA levels in the preimmune pellet (which was set to 1). (C and E) Boxplots show the average (with error bars) of the co-IP of all 10 viral RNA segments (dots) in the αProtein4 or αProtein5 pellets, relative to the preimmune pellet. ***, P ≤ 0.001 (Student’s t test). (D and F) Immunoblots with αProtein4 or αProtein5 antibodies. Pellets correspond to 4% of the cell or virion extracts, while the input corresponds to 2.5 or 0.8% of the extract analyzed in panels D or F, respectively.