An in Vitro and in Silico Kinetic Study of a Viral RNA Silencing Suppressor.

Abstract

RNA interference (RNAi) is a conserved gene regulatory mechanism employed by higher eukaryotes to avert emergent viruses and retrotransposons. During viral infection, the RNase III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs), 21-24 nucleotides in length, and helps load them into the RNA-induced silencing complex (RISC) to guide cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressor (RSS) proteins that tightly, and presumably quantitatively, bind siRNAs to thwart RNAi-mediated degradation. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus (CIRV), as well as by Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding ((1.69 +/- 0.07)x 10^8 M-1s-1) and marked dissociation (koff = 0.062 +/- 0.002 s-1). We also observe that p19 efficiently competes with recombinant human Dicer and inhibits formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the formation of a ternary complex between siRNA, p19, and human Dicer. The assumption of an obligatory transient ternary complex intermediate correlates well with the experimentally observed efficient shuttling of an siRNA toward the p19 bound state. A simple model based on this mechanism yields a greater than 20-fold bias in dissociation equilibrium constant for the ternary complex intermediate to dissociate into the siRNA:p19 complex rather than the siRNA:Dicer complex. We explicitly model the time dependence of the RNAi complexes in response to p19 silencing suppression by monitoring the impact of exogenously introduced p19 on the expression of a messenger RNA targets. We find p19 action to be concentration dependent and able to significantly increase the peak amount of mRNA produced and extend the length of the viral replication phase. From our experimentation and mathematical modeling we can postulate fundamental principles for the optimization of p19 in conjunction with RNAi-based techniques and therapeutics.