Sensitivity of Hepatitis C Virus RNA to the Antiviral Enzyme Ribonuclease L Is Determined by a Subset of Efficient Cleavage Sites

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 1a 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 1a RNA were not cleaved by RNase L because of these contextual features. The 10–30 most efficiently cleaved sites were responsible for ∼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.