title: Cysteine residues of Carnation Italian Ringspot virus p19 suppressor of RNA silencing maintain global structural integrity and stability for siRNA binding.
abstract: Carnation Italian Ringspot virus (CIRV) has evolved a protein called p19 that acts as a suppressor of RNA silencing in the host cell and aids in viral persistence. This protein has been shown to be sensitive to cysteine alkylation resulting in a reduction in its ability to bind to short-interfering RNA (siRNA). To determine the sites within the protein that are sensitive to alkylation, we systematically tested the functional role of each cysteine residue using site-directed mutagenesis. Variants of the p19 protein were created at locations C110, C134 and C160 where the cysteines were replaced by an inert amino acid such as serine or isoleucine. The results from activity measurements of the purified mutant p19 proteins indicate that the mutants maintain the ability to bind siRNAs with nanomolar affinity, however, their stabilities, as measured by circular dichroism (CD), vary. Functional studies in the presence of the cysteine alkylating agent N-ethylmaleimide (NEM) indicated that p19's ability to bind siRNAs and act as a suppressor of RNA silencing is sensitive to alkylation at all three cysteine residues with the maximum effects occurring when C110 and C134 are both alkylated. These results suggest that the role of the cysteine amino acid conservation is likely to preserve the overall structural integrity of p19 for optimal thermostability and subsequent siRNA-binding activity. We find that p19 function is maximally compromised at high levels of thiol alkylation or in an oxidizing environment.
authors.affiliation: Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Canada.
authors.forename: Jenny
authors.initials: J
authors.lastname: Cheng
authors.forename: Roger
authors.initials: R
authors.lastname: Koukiekolo
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kieliszkiewicz
authors.forename: Selena M
authors.initials: SM
authors.lastname: Sagan
authors.forename: John Paul
authors.initials: JP
authors.lastname: Pezacki