Structural and functional analysis of the roles of the HCV 5′ NCR miR122-dependent long-range association and SLVI in genome translation and replication

Cell culture and transfection

Huh 7.5 human hepatocellular carcinoma cells were maintained at 37 °C, 5% CO₂, in Dulbecco’s modified minimal essential medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 1% non-essential amino acids and 2 mM l-glutamine (DMEM-FBS; GIBCO, Life Technologies, Carlsbad, CA, USA). HeLa cells were maintained at 37 °C, 5% CO₂, in DMEM supplemented with 10% (v/v) FBS. Cells were seeded in 24-well plates at 1 × 10⁵ cells/well for translation assays, or 0.8 × 10⁵ cells/well for replicon assays, in DMEM-FBS 18 h prior to transfection.

Transfections were carried out with 500 ng RNA and two μl of Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) as per manufacturers’ instructions. In addition, for translation assays five ng of a capped and polyadenylated Renilla luciferase RNA was added as a transfection control. Transfection media was replaced at 4 h with fresh DMEM-FBS. Cell lysates were harvested for analysis at 4 h (translation assay) or 4, 21, 28 and 45 h (replicon assay). Briefly, cells were washed twice with PBS and lysed with 0.1 ml/well Glo Lysis buffer (Promega, Madison, WI, USA) for 15 min with shaking. Luciferase readings were determined with Dual-Luciferase (translation assays), or Luciferase (replicon assays), Assay System Kits (Promega, Madison, WI, USA) as per manufacturers’ instruction.

HCV cDNA plasmids, JFH-1 replicon construction and mutagenesis

A variety of numbering or naming schemes have been used to specify RNA stem-loop structures in the HCV genome. Although the most logically extendable scheme involves numbering structures according to the location of the 5′ nucleotide within the relevant genome region in an H77 reference sequence (Kuiken et al., 2006), for consistency with previous work on the HCV IRES and long-range structures between coding and non-coding regions, we use the SLI-VI scheme here. For comparison, SLVI is SL87 according to the Kuiken et al. (2006) nomenclature, due to the 5′ most nucleotide being located at nt 87 of the core-coding region.

A JFH-1 based translation-only reporter construct containing a core-extended (CE) sequence, to include SLV and VI—designated pJFH1-CEtrans—was generated via modification of pJFH1-luc-trans:ΔNS5B described previously (Tuplin et al., 2015). Overlap PCR was used to generate a DNA consisting of the 5′ end of JFH-1, including the first 174 nt of JFH-1 core, fused to the 5′ end of Firefly luciferase. An AgeI/XbaI fragment was ligated into similarly digested pJFH1-luc-trans:ΔNS5B to generate pJFH1-CEtrans.

Mutations at miR122 seed site 1 (S1) or/and SLVI were introduced into pJFH1-CEtrans using the QuikChange II site-directed mutagenesis kit as per manufacturers’ instructions (Agilent, Santa Clara, CA, USA). To disrupt miR122 binding, point mutations U₂₅A and G₂₈C were introduced into the miR122 S1 site to generate pJFH1-CEtrans-S1 (F:5′-GGCGACACACCCCCATGAATCACTC-3′ and R:5′-GAGTGATTCATGGGGGTGTGTCGCC-3′). To disrupt the SLVI structure point mutations C₄₃₆G and A₄₃₉U (F:5′-CCAGATCGTTGGGGGTATACTTGTTGC-3′ and R:5′-GCAACAAGTATACACCCCCAACGATCTGG-3′), and/or U₄₉₆A and G₄₉₉C (F:5′-CGACAAGGAAAACATCCGAGCGGTCCCAGC-3′ and R:5′-GCTGGGACCGCTCGGATGTTTTCCTTGTCG-3′), were introduced into the 5′ and/or 3′ stem of SLVI to generate pJFH1-CEtrans-L, pJFH1-CEtrans-R and pJFH1-CEtrans-L/R, respectively. Mutants pJFH1-CEtrans-S1/L, pJFH1-CEtrans-S1/R and pJFH1-CEtrans-S1/L/R, containing both the miR122 S1 and SLVI mutations were generated by site-directed mutagenesis of pJFH1-CEtrans-L, pJFH1-CEtrans-R and pJFH1-CEtrans-L/R with the pJFH1-CEtrans-S1 primers described above. All mutations were confirmed by sequence analysis.

A JFH-1 replicon, containing the extended core sequence as above, and designated pJFH1-CErep, was designed based on Con1b-luc-rep (Tuplin et al., 2015). Overlap PCR was used to replace the Con1b 5′UTR with that of JFH-1 within Con1b-rep-luc to generate pJFH1-5′UTR-Con1b-rep. A second overlap PCR generated a DNA containing the 3′ end of Firefly luciferase, the EMCV IRES and an ATG codon for NS3, and introduced into the previously described pJ6/JFH-1 (Lindenbach et al., 2005) to generate pJFH1-EMCV. An SbfI/NotI fragment from pJFH1-5′UTR-Con1b-rep containing the JFH-1 5′UTR, poliovirus IRES and Firefly luciferase, was inserted into similarly digested pJFH1-EMCV to generate pJFH1-rep. An SbfI/PmeI fragment of pJFH-1-CEtrans was inserted into similarly digested pJFH1-rep to generate the core-extended JFH-1 replicon, pJFH1-CErep. Mutations at miR122 S1 and/or SLVI were introduced through SbfI/PmeI digestion of the appropriate pJFH1-CEtrans plasmid, and ligation into pJFH1-CErep. This resulted in replicon constructs pJFH1-CErep-S1, pJFH1-CErep-L, pJFH1-CErep-R, pJFH1-CErep-L/R, pJFH1-CErep-S1/L, pJFH1-CErep-S1/R and pJFH1-CErep-S1/L/R.

miR122 duplexing and electrophoretic mobility shift assay

Native (miR122), complementary (miR122-Comp) and S1 mutant (S1-miR122) miR122 RNAs (5′-UGGAGUGUGACAAUGGUGUUUGU-3′, 5′-AAACGCCAUUAUCACACUAAAUA-3′ and 5′-GGGUGUGUGACAAUGGUGUUUGU-3′) were synthesized by Integrated DNA Technologies along with an RNA oligonucleotide corresponding to nucleotides 1–50 of the JFH-1 strain of HCV (JFH1^1–50).

For addition of miR122 to replicon assays, miR122 RNA (10 mM) was duplexed with miR122-Comp (10 mM) in a final concentration of 100 mM HEPES, five mM MgCl₂, heated at 65 °C for 5 min and cooled slowly to room temperature.

For electrophoretic mobility shift assays (EMSA) JFH1^1–50 RNA (10 pmol) was heated for 5 min at 65 °C followed by cooling to 35 °C for 1 min in a 5 μl volume containing 100 mM HEPES (pH 7.6), 100 mM KCl and five mM MgCl₂. miR122 RNAs were added at molar ratios of 0, 0.5, 1, 1.5, 2, 3 and 5 and incubated for a further 30 min at 37 °C. An equal volume of loading dye (30% glycerol, 0.5× TBE and five mM MgCl₂) was added and RNA complexes separated by non-denaturing gel electrophoresis (15% 29:1 acrylamide:bisacrylamide, 0.5× TBE and 5 mM MgCl₂) at 150 V, 4 °C, 3 h using a BioRad MiniProtean III gel system. Gels were stained with SYBR Gold (Life Technologies, Carlsbad, CA, USA) and RNA visualized using a Typhoon FLA 9500 (GE Healthcare, Chicago, IL, USA).

In vitro RNA transcription

RNA transcripts were synthesized using a HiScribe™ T7 High Yield RNA Synthesis Kit (NEB), as per manufacturers’ instructions, with one μg of DNA template linearized with BspHI (for translation templates) or terminated with a 3′ cis-acting ribozyme from an MluI linearized template (for replicon templates). Newly transcribed RNA was column-purified using a GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA).

RNA modification for SHAPE

A total of 10 pmol of translation construct-derived RNA transcripts were prepared in 10 μl of 0.5× Tris-EDTA (pH 8.0) (TE), denatured at 95 °C for 3 min and incubated on ice for 3 min prior to addition of six μl of either a five mM or 10 mM MgCl₂ folding buffer [333 mM HEPES (pH8.0), 333 mM NaCl and 16.5 mM or 33 mM MgCl₂]. Samples were allowed to refold at 37 °C for 20 min before being divided in half and incubated with either one μl of 100 mM N-methylisatoic anhydride (NMIA) dissolved in DMSO, or one μl of DMSO, at 37 °C for 45 min. For reactions in the presence of miR122 a three molar excess of miR122 was added prior to addition of folding buffer. Modified RNAs were column-purified using a GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) to remove miR122 prior to reverse transcription (RT).

5′-[³²P]-primer labelling

A total of 60 μM of primer was incubated with 10 units of T4 polynucleotide kinase (NEB), two μl of supplied 10× buffer and 10 μl γ-[³²P]-ATP (3.7 × 10⁶ Bq; PerkinElmer, Waltham, MA, USA) at 37 °C for 30 min followed by heat inactivation at 65 °C for 20 min. Radiolabelled primers were purified by separation on Sephadex G-25 Quick Spin Oligo Columns (Roche, Welwyn Garden City, UK).

Primer extension for SHAPE

N-methylisatoic anhydride- or DMSO-treated RNA in 0.5× TE (10 μl) was mixed with three μl of radiolabelled primer, denatured at 95 °C for 5 min, annealed at 35 °C for 5 min and chilled on ice for 2 min. RT mix (six μl) was added (5× First Strand Buffer, five mM DTT, 0.5 mM dNTPs; Life Technologies, Carlsbad, CA, USA) and samples incubated at 55 °C for 1 min prior to addition of one μl of SuperScript®III (Life Technologies, Carlsbad, CA, USA) and further incubation at 55 °C for 30 min. The RNA template was degraded by addition of one μl of 4M NaOH and incubation at 95 °C for 5 min before addition of 29 μl of acid stop mix (140 mM un-buffered Tris-HCl, 73% formamide, 0.43× TBE, 43 mM EDTA [pH 8.0], bromophenol blue and xylene cyanol dyes) and further incubation at 95 °C for 5 min. Dideoxynucleotide (ddNTP) sequencing markers were generated by the extension of unmodified RNA with addition of two μl of 20 mM ddNTP (TriLink BioTechnologies, San Diego, CA, USA) prior to addition of RT mix. The cDNA extension products were separated by denaturing electrophoresis (7% (19:1) acrylamide:bisacrylamide, 1× TBE, 7M urea) at 70 W for 3–5 h depending on product sizes to be analysed. Gels were visualised with a phosphorimager (Typhoon FLA 9500) and densitometry analysis carried out with ImageQuant TL 8.1 software (GE Healthcare Life Sciences). Normalised reactivities indicating exposure of nucleotides in predicted RNA structures were calculated as described previously (Tuplin et al., 2012).

Mutagenesis of a miR122 binding site and the sequences implicated in the LRA

The previously probed ‘open’ and ‘closed’ structural conformations are determined by complementarity between the 5′ nucleotides forming the miR122 binding site and the base of SLVI in the core protein-coding region, together with the presence of exogenous miR122 (Beguiristain, Robertson & Gómez, 2005; Díaz-Toledano et al., 2009). The latter, by binding to the 5′UTR sequences, inhibits the LRA and ‘opens’ the structure (Fig. 1A). This transition from a ‘closed’ to an ‘open’ structure can be predicted bioinformatically using mfold (Zuker, 2003) and bifold RNA secondary structure prediction software (Reuter & Mathews, 2010), to demonstrate that if the S1 site is occupied by miR122 the ‘open’ conformation with bound miR122 is energetically more favourable (Fig. S1). To investigate the existence and potential functions of the alternative conformations we first modified our existing JFH-1 translation reporter vector, JFH1-luc-trans:ΔNS5B (Fig. 1B), to generate a core-extended version, JFH1-CEtrans, which encompasses the first 174 nucleotides of the core-coding region, thus incorporating SLVI. A JFH-1 based sub-genomic replicon, designated JFH1-CErep, was additionally constructed to include the same core-extended sequence (Fig. 1B). For both JFH1-CEtrans and JFH1-CErep we subsequently undertook a systematic mutagenesis of either, or both, of the complementary sequences required for formation of the LRA.

First, using mfold structure prediction (Zuker, 2003), we identified two sites within SLVI at which synonymous substitutions could be introduced that should disrupt formation of the structure (Fig. 1A). Substitutions C₄₃₆G and A₄₃₉U in the 5′ stem of SLVI (designated ‘L’ mutants) and U₄₉₆A and G₄₉₉C in the 3′ stem of SLVI (designated ‘R’ mutants) independently prevented base pairing of the basal stem of SLVI. Both C₄₃₆ and A₄₃₉ are implicated in the formation of the ‘closed’ structure and consequently, ‘L’ mutants were predicted to additionally prevent the LRA due to disruption of the complementarity with the miR122 seed site 1 (S1). Conversely, ‘R’ mutants would free the 5′ sequences forming the basal stem of SLVI to contribute solely to formation of the ‘closed’ structure. However, since formation of the ‘closed’ structure would also be dependent on the S1 site being unoccupied by miR122, we also introduced substitutions into the latter (at positions U₂₅A and G₂₈C) that were predicted to prevent miR122 binding and at the same time would restore complementarity with the ‘L’ mutations in SLVI (Fig. 1C; Table 1).

To verify that binding of miR122 to S1 was abrogated in the S1 mutants we conducted EMSAs using synthetic miR122 and an RNA oligonucleotide corresponding to the first 50 nucleotides of JFH-1 (JFH1^1–50). With the addition of miR122 to unmodified JFH1^1–50 we observed the expected two complexes with reduced mobility, representative of binding of miR122 to both S1 and S2 seed sites. Saturation of both seed sites was achieved upon addition of a 3:1 molar ratio of miR122:JFH1^1–50 (Fig. 2A), while addition of an antisense miR122 RNA (miR122-Comp) showed no change in mobility (Fig. 2B). In contrast to unmodified JFH1^1–50, S1-mutated JFH1^1–50 only formed the faster migrating single complex, even at a 5:1 molar ratio, indicating that miR122 remained bound to S2 alone (Fig. 2C). Restoration of both mobility-shifted complexes was achieved upon addition of a 50–50 mix of unmodified and S1-modified miR122 (S1-miR122), the latter containing mutations complementary to those introduced in S1 mutated JFH1^1–50 (Fig. 2D). These studies confirmed that substitutions introduced to the S1 site were sufficient to disrupt miR122 binding to the S1 seed site, but that binding to the S2 seed site was unaffected, in agreement with similar mutation analysis of miR122 binding (Mortimer & Doudna, 2013).

To investigate the influence on the conformation of the 5′ end of the HCV RNA the L, R and S1 mutations predicted to influence the ‘open’ or ‘closed’ conformation were introduced individually, or in combination, into the core-extended translation and replicon reporters, JFH1-CEtrans and JFH1-CErep, respectively, and individual templates validated by sequence analysis (Table 1).

The LRA is detected in the absence, but not presence, of miR122

We have previously used SHAPE mapping to demonstrate a long-range interaction between the 3′UTR of the HCV genome and distal sequences located within the polyprotein-coding region (Tuplin et al., 2012). These interactions occurred only in cis and were acutely sensitive to point mutations within the complementary regions. We were therefore confident SHAPE analysis could provide useful insights into the study of the LRA. Three regions of the HCV RNA were analysed to provide data on RNA structure: (1) the 5′ base stem of SLVI, (2) the 3′ base stem of SLVI and, (3) nts 1–80 of the 5′UTR. Unfortunately, the presence of a highly stable stem-loop (SLI; Fig. 1A) immediately 5′ to the S1 miR122 binding site acted as a strong terminator during cDNA synthesis. Consequently, as others have previously found (Pang et al., 2012), the 5′ end of the S1 miR122 binding site (nts 1–20) proved difficult to accurately map due to excessive background signal. Scrutiny of the predicted pattern of base pairing between the miR122 binding site and miR122 also shows that it is highly similar to that between the miR122 binding site and the 5′ base stem of SLVI. Together, these issues meant that the S1 region was not informative for defining the ‘open’ or ‘closed’ conformation. Determination of the ‘closed’ structure was therefore based primarily on the structure of SLVI. In particular, nucleotides 434–435 (GG), which are predicted to be paired when involved in the LRA but unpaired in formation of the basal stem of SLVI, and the overall paired/unpaired nature of the 3′ side of the basal stem of SLVI (nucleotides 494–507), which would be predominantly unpaired upon formation of the ‘closed’ structure (Fig. 1A and Fig. S1). Preliminary experiments showed that SHAPE mapping of the 5′ regions of a variety of unmodified templates, for example, JFH1-CEtrans, JFH1-CErep or a full-length in vitro transcribed RNA, resulted in the same NMIA reactivity and resulting structural predictions (data not shown). We conclude from this that the LRA interactions are essentially local in nature and are unaffected by distal sequences in the virus genome. All subsequent SHAPE mapping was conducted using JFH1-CEtrans as template.

We first compared the structural conformations of parental JFH1-CEtrans in the absence of miR122 during the RNA folding reaction, or with a 3:1 molar excess of miR122 to saturate binding to S1 and S2, as determined from EMSAs (Fig. 2A). In the presence of miR122 the basal stem of SLVI was predominantly NMIA-unreactive, indicating that the pairing through this region was in agreement with the structure predicted bioinformatically (Fig. 3A). Indeed, the reactivity of nucleotides 427–447 and 487–507 corresponded very well with both the predicted and RNAse-probed structure of this region of SLVI (Tuplin, Evans & Simmonds, 2004). As additional validation we determined the NMIA-reactivity of SLVI sequences in JFH1-CEtrans in the presence of a locked-nucleic-acid (LNA) probe, J22. LNA J22 binds with high affinity to nts 21–37 of JFH-1 across the miR122 binding sites, allowing for the determination of the SLVI structure independently of the reversible action of miR122 (Fig. 3C). The resulting SHAPE analysis recapitulated the results observed in the presence of miR122, with little or no reactivity of sequences known to form the basal stem of SLVI. These results support the previously probed structure of SLVI indicating that, in the presence of miR122, the ‘open’ conformation predominates.

microRNA 122 is present at high levels in hepatic cells in which HCV replicates (Lagos-Quintana et al., 2002). Nevertheless, since there might be compartmentalisation—in replication complexes, for example—where miR122 is limited or absent, we went on to investigate the potential formation of the ‘closed’ structure by SHAPE in the absence of miR122 (Fig. 3B). Under these conditions we observed gross changes to the structure of the basal region of SLVI. The G₄₃₄G₄₃₅ motif—predicted to be a key interaction with the S1 site—are highly unreactive, indicating that they are base paired. At the same time, the reactivity of the 3′ sequences of the basal stem of SLVI increases. There are significant increases in exposure of nt 490–503 indicative of a more extensive opening out of the SLVI structure. We interpret this as the formation of the ‘closed’ structure in the absence of miR122, despite the inability to measure the reactivity of nucleotides within the S1 site. In contrast to the results of García-Sacristán et al. (2015), but perhaps indicative of differences between in vitro techniques, we were unable to demonstrate a magnesium-dependent preference for the formation of the ‘closed’ structure while in the presence of miR122. We investigated the structure of the basal stem of SLVI in the parental templates at an increased concentration of 10 mM MgCl₂ and determined that the ‘open’ conformation predominated, irrespective of the magnesium concentration (Fig. S2).

Together, these results are in agreement with a previous conclusion by Díaz-Toledano et al. (2009) obtained via RNase III cleavage assays, and are highly indicative of an inhibitory role for miR122 in formation of the ‘closed’ structure.

In the presence of miR122, the LRA is favoured only when both miR122 binding and the SLVI structure are disrupted

Having investigated the pairing of the basal stem of SLVI and the occurrence of the LRA in unmodified templates, we went on to study the influence of mutations introduced to prevent these interactions, or that we had previously shown prevent miR122 binding. All subsequent analyses were carried out in the presence of miR122.

We first analysed those templates predicted to preferentially form the ‘open’ conformation (Fig. 4). Modification of the S1 site in template JFH1-CEtrans-S1 (Fig. 4A), shown to abrogate miR122 binding (Fig. 2C), resulted in a NMIA-reactivity pattern almost indistinguishable from the unmodified parental template (compare Fig. 3A with Fig. 4A). Since the U₂₅A and G₂₈C changes in the S1 mutant also prevents interaction with SLVI nts A₄₃₉ and C₄₃₆, respectively, this provides further support that this pattern of NMIA reactivity represents the ‘open’ conformation. Three additional modified templates, JFH1-CEtrans-L, JFH1-CEtrans-L/R and JFH1-CEtrans-S1/R, were also predicted to block the LRA by reducing the complementarity between nucleotides in the S1 site and the basal stem of SLVI (Fig. 1C). NMIA-reactivity of these three templates verified the predicted inhibition of the LRA, as evidenced by the high reactivity of the G₄₃₄G₄₃₅ motif (Figs. 4B, 4C and 4D). Interestingly, in comparison to the parental JFH1-CEtrans, all three templates displayed increases in reactivity in other regions, such as nts 427–433 (the 5′ basal stem) or nts 491–495 (3′ basal stem), suggesting that, while preventing the LRA, the introduced mutations may also lead to generation of an altered SLVI structure (compare line graph to black bars in Figs. 4B, 4C and 4D). The mfold predictions for the structure of SLVI containing mutations C₄₃₆G and A₄₃₉U, and U₄₉₆A and G₄₉₉C, did not suggest formation of such an altered structure (data not shown) and it is not possible to deduce the precise structure from the NMIA-reactivity plots.

We next investigated the conformation of templates containing combinations of mutations that were predicted to favour the LRA and the ‘closed’ conformation: JFH1-CEtrans-R, JFH1-CEtrans-S1/L and JFH1-CEtrans-S1/L/R (Fig. 5). Unexpectedly, both JFH1-CEtrans-R and JFH1-CEtrans-S1/L/R failed to demonstrate the LRA, again as evidenced by the reactivity of the G₄₃₄G₄₃₅ motif, as well as the overall lack of reactivity in the 3′ basal stem of SLVI that would be expected (Figs. 5A and 5B). As with JFH1-CEtrans-L and JFH1-CEtrans-L/R, we observed an overall increase in reactivity of the 5′ basal stem nucleotides suggestive of a similar disruption to the SLVI structure that was not predicted in mfold calculations (compare Figs. 5A and 5B with Figs. 4B and 4C). However, these result suggest that despite significant disruption to the known SLVI structure, the ‘closed’ structure is not the favoured conformation for the RNA template.

In contrast to all other modified templates JFH1-CEtrans-S1/L generated a NMIA-reactivity plot matching that of parental JFH1-CEtrans in the absence of miR122, and is highly indicative of the formation of the ‘closed’ structure (Fig. 5C). In comparison to a template in which the ‘closed’ structure is blocked, that is, parental JFH1-CEtrans in the presence of miR122, the mean NMIA-reactivities of the JFH1-CEtrans-S1/L G₄₃₄G₄₃₅ motif were reduced from 0.38 and 1.16 to 0.26 and 0.22, respectively, highlighting a substantial change in the base paired state, especially of G₄₃₅. Similarly, the average reactivity of nts 490–507 in the 3′ basal stem was increased from 0.28 to 0.64 demonstrating the overall increase in reactivity expected when the 5′ basal stem of SLVI is bound to the miR122 S1 site in the ‘closed’ conformation.

Taken together the SHAPE analyses show that, in the presence of miR122, the ‘closed’ conformation only exists when both miR122 binding at S1 is blocked, and nucleotide complementarity between S1 and the 5′ basal stem of SLVI is restored.

Phenotypic characterisation of LRA-modified templates

Using SHAPE analyses we determined that, in the presence of miR122, the LRA resulting in the ‘closed’ conformation, is highly unlikely to form. However, if HCV translation and/or replication occur in locations or complexes in which miR122 is absent then the ‘closed’ structure is the energetically favourable conformation (Fig. 3B), and as such may influence virus translation and replication. We therefore used selected modified templates with demonstrated changes in conformation, to investigate the effects of the LRA on translation and replication.

Hepatitis C virus IRES-mediated translation is known to require the first 12–30 nt of the core protein-coding region (Reynolds et al., 1995). However, to study the effects on translation of the LRA required the additional SLV–SLVI sequences included in the core-extended translation reporter described above and utilised in SHAPE analysis (Fig. 1B). We initially compared translation from JFH1-luc-trans:ΔNS5B, containing the minimal core sequence, to the core-extended JFH1-CEtrans reporter. In agreement with previous observations (Kim et al., 2003), translation was significantly decreased (∼2.5-fold) with the inclusion of the extended core sequence (Fig. 6A). This reduction in translation is proposed to be a result of formation of the LRA (Kim et al., 2003) and that a high proportion of the JFH1-CEtrans RNA templates exist in the ‘closed’ conformation (Fig. 1A), and hence are unavailable for use by the cellular translation machinery. As we have demonstrated that the LRA occurs only in the absence of miR122 (Fig. 3), this result implies either the exclusion of miR122 from sites of translation or, an alternative role for the sequences encompassing SLV and SLVI domains in regulating translation. If the observed reduction is a result of the LRA, translation levels should be restored by mutations designed to disrupt the LRA, so forcing the ‘open’ conformation, and repressed again by compensatory substitutions that—although different from the parental template—restore the LRA and the ‘closed’ conformation.

We therefore compared translation from JFH1-CEtrans with selected modified templates that had shown a distinct conformation in SHAPE analysis. We selected JFH1-CEtrans-S1 as a representative of the ‘open’ conformation due to the SHAPE analysis most closely matching the SLVI structure observed for parental JFH1-CEtrans. For the ‘closed’ conformation we were limited to the study of JFH1-CEtrans-S1/L, as the only modified template in which the LRA was demonstrated. In addition, we investigated JFH1-CEtrans-L/R and JFH1-CEtrans-S1/L/R, for which the SHAPE reactivities suggested that the 5′ and 3′ stem mutations did not recapitulate wild type SLVI as expected due to increased reactivity in the 5′ basal stem, but neither were they representative of the ‘closed’ conformation (Figs. 4C and 5B). We transfected Huh 7.5 cells with 500 ng of RNA of each template, in parallel, and normalised translation levels to a Renilla luciferase transfection control RNA (Fig. 6B). As predicted for the ‘open’ conformation and blocking of the LRA, JFH1-CEtrans-S1 showed a small, but significant, increase in translation level compared to both JFH1-CEtrans and JFH1-CEtrans-S1/L (p < 0.05). In contrast, JFH1-CEtrans-S1/L, shown to preferentially form the ‘closed’ structure, did not show the predicted reduction in translation and was unchanged from parental JFH1-CEtrans. Similarly, despite small increases and decreases in translation levels of JFH1-CEtrans-L/R and JFH1-CEtrans-S1/L/R, respectively, these were not significantly different when compared to the parental template. This suggests that, even though the wild type SLVI structure is not fully restored, with reduced base pairing remaining in the basal 5′ stem, this did not impact translation.

The limited changes in phenotype observed strongly suggested that the difference observed between JFH1-luc-trans:ΔNS5B and JFH1-CEtrans is due to an as yet unidentified role for SLV and VI, and is not miR122 or LRA dependent. In the presence of miR122 both these templates exhibit the ‘open’ conformation as determined by SHAPE analysis (Figs. 3A and 4A). Due to the mutations introduced into JFH1-CEtrans-S1 only JFH1-CEtrans is capable of forming the ‘closed’ conformation which, in the absence of miR122, is the preferred conformation (Fig. 3B). As the switch between the two conformations is miR122 dependent we reasoned that, if the ‘closed’ conformation is in fact responsible for the reduction in translation of JFH1-CEtrans, we would observe a similar difference when comparing JFH1-CEtrans (‘closed’) to JFH1-CEtrans-S1 (‘open’) in cells lacking miR122. We therefore investigated translation of JFH1-luc-trans:ΔNS5B, JFH1-CEtrans, JFH1-CEtrans-S1 and JFH1-CEtrans-S1/L in HeLa cells, which are naturally lacking in miR122 (Jopling et al., 2005) (Fig. 6C). Overall we observed translation levels that were ∼10-fold lower in HeLa cells than in Huh 7.5 cells. However, the relative translation phenotypes remained the same, with a significant reduction in translation for JFH1-CEtrans, and all other templates that contained the core-extended sequence. Additionally, no significant changes were observed between JFH1-CEtrans and JFH1-CEtrans-S1, or JFH1-CEtrans-S1/L, which is also expected to form the ‘closed’ conformation in the absence of miR122. This supports our contention that formation of the LRA does not account for the change in translation phenotype between JFH1-luc-trans:ΔNS5B and JFH1-CEtrans, and conclude that—although the presence of SLVI clearly reduces translation (Figs. 6A and 6C)—this is unrelated to the LRA and any miR122 induced switch between the ‘open’ and ‘closed’ conformations.

With no observable link between translation levels and the conformations demonstrated by SHAPE analysis we went on to investigate whether there were differences in replication between our structurally modified templates. To achieve this we used a core-extended version of a JFH-1 replicon, JFH1-CErep. Huh 7.5 cells were transfected with 500 ng of template RNA, containing the same modifications as above, and replication recorded as luciferase activity over a 45 h time course. Our results show that for JFH1-CErep-L/R, where mutations were engineered solely within SLVI, replication was not significantly altered from the unmodified parental replicon, in keeping with the results for translation (Figs. 6B and 6D). In contrast, mutants JFH1-CErep-S1, JFH1-CErep-S1/L and JFH1-CErep-S1/L/R, in which miR122 binding to S1 is blocked, all showed a >10-fold, highly significant reduction in the level of replication (p < 0.01) (Fig. 6D). This is entirely consistent with our current understanding that miR122 binding to the 5′UTR of HCV is important for HCV replication (Jangra, Yi & Lemon, 2010; Jopling et al., 2005). The addition of modified S1-miR122 to the RNA transfection mix, to complement the S1 mutations in the replicon, restored replication of JFH1-CErep-S1 to parental levels, demonstrating that the observed reductions in replication are due entirely to disruption of miR122 binding and not a consequence of possible changes in conformation promoted by the LRA (Fig. 6E).

These results demonstrate that, while miR122 binding has a profound effect on the replication of the HCV genome, neither replication nor translation phenotypes are significantly influenced by modifications to SLVI that preferentially form the ‘open’ or ‘closed’ conformations of the LRA.