Stabilized recombinant suppressors of RNA silencing: Functional effects of linking monomers of Carnation Italian Ringspot virus p19

Eukaryotes have evolved complex cellular responses to double-stranded RNA including the RNA silencing pathway. Tombusviruses have adapted a mechanism to evade RNA silencing that involves a 19 kDa dimeric protein (p19) that is a suppressor of RNA silencing. In order to develop stabilized p19 proteins, linked versions of p19 from the Carnation Italian Ringspot virus (CIRV) were constructed that joined the C-terminus of one subunit to the N-terminus of the second subunit. Like the native CIRV p19, these linked p19 proteins were able to bind to double-stranded siRNAs with nanomolar affinity and discriminate siRNA according to length. In addition, the interdomain linker improved both the stability and binding properties of the p19 dimer. The observed binding properties support the idea that the semi-rigid cross-link favors the folded, binding-competent state of p19. The cross-linked recombinant CIRV-p19s represent novel stabilized suppressors of RNA silencing and may be useful in future biophysical, immunological and cell biology studies.