A viral RNA competitively inhibits the antiviral endoribonuclease domain of RNase L.
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 K(i) 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).
Townsend
Hannah L
HL
Department of Microbiology, University of Colorado Denver, School of Medicine, Aurora, Colorado 80045, USA.
Jha
Babal K
BK
Han
Jian-Qiu
JQ
Maluf
N Karl
NK
Silverman
Robert H
RH
Barton
David J
DJ
D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D030015:Enterovirus C, Human / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017955:Poliovirus / Q000235:genetics; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D012367:RNA, Viral; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease