The genome of hepatitis C virus (HCV) contains cis-acting replication elements (CREs) comprised of RNA stem-loop structures located in both the 5 and 3 noncoding regions (5 and 3 NCRs) and in the NS5B coding sequence. Through the application of several algorithmically independent bioinformatic methods to detect phylogenetically conserved, thermodynamically favored RNA secondary structures, we demonstrate a longrange interaction between sequences in the previously described CRE (5BSL3.2, now SL9266) with a previously predicted unpaired sequence located 3 to SL9033, approximately 200 nucleotides upstream. Extensive reverse genetic analysis both supports this prediction and demonstrates a functional requirement in genome replication. By mutagenesis of the Con-1 replicon, we show that disruption of this alternative pairing inhibited replication, a phenotype that could be restored to wild-type levels through the introduction of compensating mutations in the upstream region. Substitution of the CRE with the analogous region of different genotypes of HCV produced replicons with phenotypes consistent with the hypothesis that both local and long-range interactions are critical for a fundamental aspect of genome replication. This report further extends the known interactions of the SL9266 CRE, which has also been shown to form a "kissing loop" interaction with the 3 NCR (P. Friebe, J. Boudet, J. P. Simorre, and R. Bartenschlager, J. Virol. 79:380-392, 2005), and suggests that cooperative long-range binding with both 5 and 3 sequences stabilizes the CRE at the core of a complex pseudoknot. Alternatively, if the long-range interactions were mutually exclusive, the SL9266 CRE may function as a molecular switch controlling a critical aspect of HCV genome replication.Hepatitis C virus (HCV), a flavivirus in the genus Hepacivirus, possesses a positive (mRNA)-sense genome of approximately 9.6 kb encoding a single polyprotein. This polyprotein is cleaved co-and posttranslationally to generate proteins that form the enveloped virus particle and those that replicate the genome. Polyprotein translation is initiated within a highly structured internal ribosome entry site (IRES) occupying much of the 5Ј noncoding region (5Ј NCR). The 5Ј NCR also contains sequences required for genome replication (9,19,26), and like functionally analogous regions in the 3Ј NCR, these form defined stem-loop structures that operate in cis and are known or suspected to recruit cellular or viral proteins (5, 10, 30). In addition to these cis-acting replication elements (CREs) in the noncoding extremes of the genome, there is evidence that additional RNA structures exist within the coding regions. The latter structure is of two types, phylogenetically conserved well-defined structures occupying the 5Ј and 3Ј regions of the sense strand of the coding region of HCV (23,31,32,36) and a less well-characterized but much more extensive set of RNA secondary structures, collectively designated genome-scale ordered RNA structure (GORS), that spans the entire coding ...

The RNA structure and long-range interactions of the SL9266 cis-acting replication element located within the NS5B coding region of hepatitis C virus (HCV) were determined using selective 2′-hydroxyl acylation analysed by primer extension. Marked differences were found in the long-range interactions of SL9266 when the two widely used genotype 2a JFH-1 (HCVcc) and genotype 1b Con1b sub-genomic replicon systems were compared. In both genomes, there was evidence for interaction of the sub-terminal bulge loop of SL9266 and sequences around nucleotide 9110, though the replication phenotype of genomes bearing mutations that disrupted this interaction was fundamentally different. In contrast, a ‘kissing loop’ interaction between the terminal loop of SL9266 and sequences in the 3′-untranslated X-tail was only detectable in JFH-1-based genomes. In the latter, where both long-range interactions are present, they were independent, implying that SL9266 forms the core of an extended pseudoknot. The presence of the ‘kissing loop’ interaction inhibited the formation of SL9571 in the 3′-X-tail, an RNA structure implicated in genome replication. We propose that, SL9266 may contribute a switch function that modulates the mutually incompatible translation and replication events that must occur for replication of the positive-strand RNA genome of HCV.