A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication

Abstract

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.

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

“…Our data confirm previous hypotheses that suggested a kissing complex between 5BSL3.2 and SL2, but they do not agree with the results from NMR analysis (Friebe et al 2005) or SHAPE mapping on the HCV genotype 1b (isolate Con1) (Tuplin et al 2012). No interaction was detected by NMR assays with the same short RNA sequences that we used, and a unique secondary structure was proposed for SL2.…”

“…These interactions were characterized either in a bimolecular context with the 5BSL3.2-SL2 and 5BSL3.2-Seq9110 complexes or in a trimolecular context with the 5BSL3.2-SL2-Seq9110 ternary complex. In contrast with previous results obtained by NMR spectroscopy with the same short RNA sequences that we used in this work (Friebe et al 2005) or SHAPE mapping with longer RNAs (Tuplin et al 2012), we show that 5BSL3.2 can form a complex with SL2 through kissing-loop interactions. The internal loop of 5BSL3.2 is also able to hybridize with Seq9110.…”

“…Evidence has now accumulated suggesting that RNA-RNA interactions between structured regions of the HCV genome are crucial to regulate the life cycle of the virus (You et al 2004;Friebe et al 2005;Diviney et al 2008;You and Rice 2008;Tuplin et al 2012).…”

“…This internal loop has also been implicated in a long-range RNA-RNA interaction with stem-loop IIId of the IRES (Romero-Lopez and Berzal-Herranz 2009), but there are conflicting results about the influence of this interaction on viral translation activity. It is hypothesized that 5BSL3.2 could act as a molecular switch depending on its interaction with SL2 or Seq9110, therefore leading the viral genome toward translation, replication, or encapsidation (Tuplin et al 2012).…”

Diverse roles and interactions of RNA structures during the replication of positive-stranded RNA viruses of humans and animals

“…The terminal-loop of SL9266 forms a 'kissing-loop' interaction with stem loop SL9571 (alternatively SL2) in the 39NCR (Friebe et al, 2005). The 'kissing-loop' is a dynamic structure forming open and closed conformations, both of which are essential for efficient virus replication (Tuplin et al, 2012). We have shown that the closed conformation stimulates HCV IRES initiated translation (Tuplin and others, unpublished results) and the open structure preferentially binds cellular protein EWSR1 upregulating genome replication (Oakland et al, 2013).…”