Hepatitis C virus cis-acting replication element forms a long-range RNA-RNA interaction with upstream RNA sequences in NS5B

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Journal of Virology (Impact Factor: 4.44). 08/2008; 82(18):9008-22. DOI: 10.1128/JVI.02326-07
Source: PubMed


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 long-range 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.

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Available from: Andrew Tuplin, Mar 20, 2014
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    • "We have shown that the closed conformation stimulates HCV IRES initiated translation (Tuplin and others, unpublished results) and the open structure preferentially binds cellular protein EWSR1 upregulating genome replication (Oakland et al., 2013). It has been suggested that SL9266/PK functions in the temporal control of early translation and replication events (Diviney et al., 2008; Oakland et al., 2013). However, much remains to be understood regarding this complex mechanism of dynamic RNA–RNA and RNA–protein interactions, in particular how alternative conformations are stabilized and interact with different trans-activating factors. "
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    • "participates in viral translation and replication. Thus, the apical loop is complementary to the apical loop of the 3′SLII within the 3′X-tail (34,42–44), while the 8-nt bulge (see Figure 1A) establishes one of two possible contacts: one with the apical loop of the IIId subdomain of the IRES region (44,45), the other with the apical loop of the stem-loop SL9110 upstream of the CRE element (34,43,44,46) (Figure 1A). It has recently been reported that all these interactions are equally probable and show similar dissociation constants (Kd) (34,44). "
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    • "An unresolved aspect of this study is why mutations that disrupt the upstream interaction in Con1b and J6/JFH-1 (Figures 3a, b and 4a, b) exert markedly different phenotypes in the two replication systems (Figure 5) (22). We suspect that this may reflect differences in the overall integrity of the extended pseudoknot structure, in which the upstream interaction in Con1b is required to stabilise SL9266 in a conformation compatible with function, for example, that allows the ‘kissing loop’ interaction to form as required. "
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