Figure 7.

BTV S10 interacts with smaller segments. (A) Beads coated with a S10-specific primer were incubated sequentially with BTV-1 S10 and with 1 pmol of segments S1–S9 individually. Interaction with RRV S9 was included as a negative control. After extensive washing, the attached RNA was released by heating. The amount of interacting RNA was determined by qRT-PCR using primers specific to each segment. The copy number was correlated by minus non-specific binding detected in beads-only control. S10 and the standard deviations from three individual experiments are indicated. (B) Beads coated with (+) or without (−) BTV-1 S10 were similarly prepared and sequentially incubated with ³²P-labelled S1, S3, S6 or S8. After three washes, RNAs were heat-released and analysed on a denaturing agarose gel and Phospho-imager exposure. The black arrows indicate positive interaction. (C) The interaction between S8 and truncated S10 was measured with a similar method: beads were coated with BTV-1 S10 (S10), S10 lacking 5′ and 3′ UTRs (ΔUTRs), 3′ UTR (Δ3′UTR) or 5′ UTR (Δ5′UTR) and incubated with equal amounts of BTV-1 S8. Interacting S8 was analysed and quantified similarly. Interaction rates and standard deviation (error bars) were calculated.