A viral RNA competitively inhibits the antiviral endoribonuclease domain of RNase L

Abstract

Ribonuclease L (RNase L) is a latent endoribonuclease in an evolutionarily ancient interferon-regulated dsRNA-activated antiviral pathway. 2′–5′ oligoadenylate (2–5A), the product of dsRNA-activated oligoadenylate synthetases (OASes), binds to ankyrin repeats near the amino terminus of RNase L, initiating a series of conformational changes that result in the activation of the endoribonuclease. A phylogenetically conserved RNA structure within group C enteroviruses inhibits the endoribonuclease activity of RNase L. In this study we report the mechanism by which group C enterovirus RNA inhibits RNase L. Viral RNA did not affect 2–5A binding to RNase L. Rather, the viral RNA inhibited the endoribonuclease domain. We used purified RNase L, purified 2–5A, and an RNA substrate with a 5′ fluorophore and 3′ quencher in FRET assays to measure inhibition of RNase L activity by the viral RNA. The group C enterovirus RNA was a competitive inhibitor of the endoribonuclease with a Ki of 34 nM. Consistent with the kinetic profile of a competitive inhibitor, the viral RNA inhibited the constitutively active endoribonuclease domain of RNase L. We call this viral RNA the RNase L competitive inhibitor RNA (RNase L ciRNA).