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 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.
Han
Jian-Qiu
JQ
Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Wroblewski
Gerggory
G
Xu
Zan
Z
Silverman
Robert H
RH
Barton
David J
DJ
D000998:Antiviral Agents / Q000494:pharmacology*; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004722:Endoribonucleases / Q000737:chemistry; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy*; D006801:Humans; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012330:RNA, Double-Stranded / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral; D012260:Ribonucleases / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors
D000998:Antiviral Agents; D017931:DNA Primers; D009711:Nucleotides; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; D012313:RNA; D004722:Endoribonucleases; D012260:Ribonucleases; C031956:2-5A-dependent ribonuclease