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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).

RNase L is an antiviral endoribonuclease that cleaves viral mRNAs after single-stranded UA and UU dinucleotides. Poliovirus (PV) mRNA is surprisingly resistant to cleavage by RNase L due to an RNA structure in the 3C Pro open reading frame (ORF). The RNA structure associated with the inhibition of RNase L is phylogenetically conserved in group C enteroviruses, including PV type 1 (PV1), PV2, PV3, coxsackie A virus 11 (CAV11), CAV13, CAV17, CAV20, CAV21, and CAV24. The RNA structure is not present in other human enteroviruses (group A, B, or D enteroviruses). Coxsackievirus B3 mRNA and hepatitis C virus mRNA were fully sensitive to cleavage by RNase L. HeLa cells expressing either wild-type RNase L or a dominant-negative mutant RNase L were used to examine the effects of RNase L on PV replication. PV replication was not inhibited by RNase L activity, but rRNA cleavage characteristic of RNase L activity was detected late during the course of PV infection, after assembly of intracellular virus. Rather than inhibiting PV replication, RNase L activity was associated with larger plaques and better cell-to-cell spread. Mutations in the RNA structure associated with the inhibition of RNase L did not affect the magnitude of PV replication in HeLa cells expressing RNase L, consistent with the absence of observed RNase L activity until after virus assembly. Thus, PV carries an RNA structure in the 3C protease ORF that potently inhibits the endonuclease activity of RNase L, but this RNA structure does not prevent RNase L activity late during the course of infection, as virus assembly nears completion.

Ribonuclease L (RNase L) cleaves RNA predominantly at single-stranded UA and UU dinucleotides. Intriguingly, hepatitis C virus (HCV) RNAs have a paucity of UA and UU dinucleotides, and relatively interferon (IFN)-resistant strains have fewer UA and UU dinucleotides than do more IFN-sensitive strains. In this study, we found that contextual features of UA and UU dinucleotides dramatically affected the efficiency of RNase L cleavage in HCV RNA. HCV genotype la RNA was cleaved by RNase L into fragments 200-1000 bases in length, consistent with 10-50 RNase L cleavage sites within the 9650-base long viral RNA. Using primer extension, we found that HCV RNA structures with multiple single-stranded UA and UU dinucleotides were cleaved most efficiently by RNase L. UA and UU dinucleotides with 3' proximal C or G residues were cleaved infrequently, whereas UA and UU dinucleotides within dsRNA structures were not cleaved. 5'-GUAC-3' and 5'-CUUC-3' were particularly unfavorable contexts for cleavage by RNase L. More than 60% of the UA and UU dinucleotides in HCV la RNA were not cleaved by RNase L because of these contextual features. The 10-30 most efficiently cleaved sites were responsible for approximately 50%-85% of all RNase L cleavage events. Our data indicate that a relatively small number of the UA and UU dinucleotides in HCV RNA mediate the overall sensitivity of HCV RNA to cleavage by RNase L.

Ribonuclease L (RNase L) is an antiviral endoribonuclease that cleaves hepatitis C virus (HCV) RNA at single-stranded UA and UU dinucleotides throughout the open reading frame (ORF). To determine whether RNase L exerts evolutionary pressure on HCV we examined the frequencies of UA and UU dinucleotides in 162 RNA sequences from the Los Alamos National Labs HCV Database (http://hcv.lanl.gov). Considering the base composition of the HCV ORFs, both UA and UU dinucleotides were less frequent than predicted in each of 162 HCV RNAs. UA dinucleotides were significantly less frequent than predicted at each of the three codon positions while UU dinucleotides were less frequent than predicted predominantly at the wobble position of codons. UA and UU dinucleotides were among the least abundant dinucleotides in HCV RNA ORFs. Furthermore, HCV genotype 1 RNAs have a lower frequency of UA and UU dinucleotides than genotype 2 and 3 RNAs, perhaps contributing to increased resistance of HCV genotype 1 infections to interferon therapy. In vitro, RNase L cleaved both HCV genotype 1 and 2 RNAs efficiently. Thus, RNase L can cleave HCV RNAs efficiently and variably reduced frequencies of UA and UU dinucleotides in HCV RNA ORFs are consistent with the selective pressure of RNase L.

Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-alpha2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 2'-5' oligoadenylate synthetase/ ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA.