A Hepatitis C Virus cis -Acting Replication Element Forms a Long-Range RNA-RNA Interaction with Upstream RNA Sequences in NS5B

Abstract

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 ...

Functionally conserved architecture of hepatitis C virus RNA genomes

“…3, purple boxes). Five elements correspond to regulatory motifs that have been well characterized: the IRES domains II-IV, SL9098, and CRE (13)(14)(15)(16)(17). We focused on the four uncharacterized RNA elements (Fig.…”

“…Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

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

“…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.…”

“…It has been postulated that the S-fragment is involved in genome replication and that the alternative mutually exclusive long-range interactions represent a mechanism modulating FMDV translation and replication (Serrano et al, 2006). -Herranz, 2012) and that with the 9110 region essential for virus replication (Diviney et al, 2008). The terminal-loop of SL9266 forms a 'kissing-loop' interaction with stem loop SL9571 (alternatively SL2) in the 39NCR (Friebe et al, 2005).…”

RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand

“…The hairpin-loop sequence on 5BSL3.2 and the sequence on the loop of the 39-X (39SL2) have the potential to form a pseudoknot, which is regarded as essential for viral replication. In addition, a more recent study by Diviney et al (2008) provided evidence that the long range RNA interaction between 5BSL3.2 and its approximately 200 base upstream CGGG motif is also important for viral replication.…”