A Method for In Situ Localization of Single-Strand and Double-Strand RNA Elements Contained in the Hepatitis C Virus Genome 3'-Untranslated Region

Abstract

The ability to resolve secondary and tertiary structures, as present in their native state within the genome of RNA viruses, is an essential first step in elucidating the replication process of these viruses. The hepatitis C virus (HCV) genome is composed of a single (+) strand RNA whose replication is controlled primarily by the 3'-untranslated region (3'UTR). The 3'UTR comprises a genotype-specific variable region (VR), a poly (U/UC) repeat and a conserved 98-base sequence called the X-tail. Although structural models of the 3'UTR from in vitro biochemical analysis have been proposed, its native structure as part of the full-length viral genome existing within the intracellular environment is uncertain. As a means to map the native double-strand (ds) and single-strand (ss) RNA structures contained in the 3'UTR of the full-length genome, we performed in situ chemical tagging in combination with site-directed RT-qPCR. Results indicate that ssRNA is predominant in the VR-poly (U/UC) repeat sequences, whereas dsRNA elements are restricted to the X-tail. In situ chemical tagging also identified a unique dsRNA element located in the VR-poly (U/ UC) repeat region comprised in part of sequences located outside the 3'UTR. The results described here constitute a first report for in situ probing and detection of secondary structures present within the 3'UTR sequence of the HCV genome. The use of intracellular RNA modifying agents to demarcate dsRNA and ssRNA, in combination with PCR to amplify trace amounts of the targeted RNA, has potential application in mapping native secondary structures within complex intracellular RNAs.