1 Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
2 Division of Hepatology and Gene Therapy, Clínica Universitaria/Department of Medicine, Fundación para la Investigación Medica Aplicada (FIMA), University of Navarra, Pamplona, Spain
Correspondence
Hans C. van Leeuwen
H.C.van_Leeuwen{at}lumc.nl
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ABSTRACT |
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INTRODUCTION |
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HCV, which is a distinct member of the family Flaviviridae, has a positive-stranded RNA genome of 9600 nt that encodes a single long open reading frame of
3000 aa. This polyprotein is co-translationally and post-translationally cleaved into ten structural and non-structural proteins (Bartenschlager & Lohmann, 2000
). Flanking the open reading frame at each end are a 5' and a 3' non-translated region (NTR), approximately 342 and 225 nt in length, which contain signals required for replication (Friebe et al., 2001
; Friebe & Bartenschlager, 2002
). Translation of the HCV open reading frame is mediated via the 5' NTR that includes a structural RNA element identified as an internal ribosomal entry site (IRES) (Tsukiyama-Kohara et al., 1992
; Wang et al., 1993
). The whole 5' NTR folds into a complex RNA structure containing four distinct domains, with domains II, III and part of domain IV (up to the AUG) forming the IRES (Fig. 1a
). Through a direct interaction with this IRES element, the ribosomal 40S subunit is recruited to the vicinity of the start codon, directly placing the AUG in the ribosomal P site, thereby eliminating the requirement for a 5' cap structure and ribosomal scanning (Hellen & Sarnow, 2001
).
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In this work, we studied the 5' NTR sequence heterogeneity from serum of an HCV genotype 1b (HCV1b)-infected human containing extremely high virus titres. Several minor and major variants were found in its sequence. One major variant, which had lost base pairing in stemloop IIIa of the HCV IRES, and therefore altered the predicted secondary RNA structure fold, was tested in colony formation of subgenomic replicon HCV RNAs. We find that under selection pressure there is a strong constraint on the HCV IRES to conserve its structure and function.
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METHODS |
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HCV 5' NTR amplification.
The 5' NTR was identified by cRACE (Maruyama et al., 1995). In short, first strand cDNA was obtained from the isolated HCV RNA using a 5'-phosphorylated oligonucleotide AS2648, 5'-ACCAGGCAGCACAGAAGAACAC-3' and Thermoscript reverse transcriptase (Invitrogen). This cDNA starts at position 2648 and runs to the 5' end of the genome. After removal of the template RNA with 0·5 M NaOH, the cDNA was circularized by T4 RNA ligase (Invitrogen). This circularized single-stranded DNA was subsequently used as a template for amplification using primers S2293, 5'-TGCAATTGGACTCGAGGAGAGAGCG-3', and H179, 5'-ACTCGGCTAGCAGTC-3'. The resulting 600 bp band was cloned into the pCR2.1-TOPO vector (Invitrogen). These clones, which included the junctional region between the 5' end and the primer used for cDNA synthesis, were then sequenced in both directions by using an ABI Prism 310 genetic analyser (Applied Biosystems) and the Dye Dideoxy terminator sequencing kit (Applied Biosystems).
Plasmid constructions.
The replicon vector used to test IRES mutations was pFK-I389neo/NS3-5/5.1 [pFK5.1, see Krieger et al. (2001)]. Mutant IRES replicon constructs were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Because additional unwanted mutations could be introduced during the QuickChange procedure, a large NruISpeI fragment from the original pFK5.1 construct was placed back into the mutated replicon. The region corresponding to nt 1269 containing the mutated IRES was sequenced.
Dual luciferase reporter constructs containing mutated IRESs A53G and C168G were generated by a fusion PCR-based strategy from pDualLuc-HCVwt/C that has been described previously (Reusken et al., 2003
). For pDualLuc-A53G we used primers (A) 5'-ACTCACCGGTTCCGCAGACCACTATG-3' and (B) 5'-CCATAGATCACTCCCCTGTGAGGGAC-3' on replicon pFK5.1 A53G. The resulting PCR product was used as primer together with (C) 5'-AAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGG-3' on pDualLuc-HCVwt/
C. This fusion PCR product was cloned into pDualLuc-HCVwt/
C using EcoRV and AgeI. For pDualLuc-C168G we used primers (D) 5'-TATCAGGCAGTSWCCACAAGGCCTTTCGCG-3' and (E) 5'-GGTCTGCGGAACCGGTGAGTACAGCGG-3' on replicon pFK5.1 C168G. The resulting PCR product was used as primer together with (F) 5'-ATTCATGCATACGCGTGCCAGCCCCCGATTGGGG-3' on pDualLuc-HCVwt/
C. This fusion PCR product was cloned into pDualLuc-HCVwt/
C using NsiI and StuI. All fragments generated by PCR were sequenced upon cloning. pDualLuc-A53G+C168G was generated by exchanging the 1681 bp AgeIApaI fragment from pDualLuc-A53G with the corresponding fragment from pDualLuc-C168G.
Cell culture.
Huh-7 hepatocellular carcinoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % heat-inactivated FCS, 2 mM L-glutamine and 1 % non-essential amino acids. For cell lines carrying HCV replicons, 500 µg G418 ml1 was added (Geneticin; Life Technologies).
Transfection of cultured cells.
Electroporation and selection of G418-resistant cell lines were done as described by Lohmann et al. (1999).
Luciferase assays.
Tissue culture expression studies were performed as described previously (Reusken et al., 2003). In short, Huh-7 cells were infected with recombinant vaccinia virus, expressing T7 RNA polymerase, vTF7-3 (Fuerst et al., 1986
), at an m.o.i. of 10. After 1 h incubation at 37 °C, cells were washed in PBS and transfected with plasmid using LipofectACE reagent OptiMEM (Life Technologies). Cells were washed with PBS 7 h post-transfection, and lysed in passive lysis buffer (Dual-luciferase Reporter Assay; Promega). Cells were stored at 80 °C and thawed before reporter activities were measured with the dual-luciferase reporter assay using a luminometer (TD-20/20; Turner BioSystems). The experiments were performed in triplicate.
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RESULTS |
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In the secondary RNA structure fold, the alteration of cytidine 168, located in stemloop IIIa, to a guanidine (see Fig. 1a) results in a loss of pairing with base G159. Because stemloop IIIa, as part of the IIIabc four-way junction, is a functionally important element (Kieft et al., 2002
), which is directly contacted by both the 40S ribosomal subunit and the eukaryotic initiation factor 3 (eIF3) (Kieft et al., 2001
), we subjected this major variant to further analysis.
Effect of stemloop IIIa mutations on translation
To establish whether the major quasispecies mutations had an effect on translational activity we used the bicistronic dual-luciferase reporter system (Collier et al., 1998), which provides a way to correct for possible differences in transfection efficiencies (Reusken et al., 2003
). This dual luciferase reporter construct contains an upstream cap-dependent firefly luciferase gene (Fluc), serving as a control for transfection efficiency, and downstream an internal Renilla luciferase gene (Rluc), which is driven by the HCV IRES. A T7 promoter drives cytoplasmic transcription of this bicistronic mRNA. Huh-7 hepatocellular carcinoma cells, infected with recombinant vaccinia virus expressing T7 RNA polymerase, were transfected with dual luciferase plasmids containing the consensus IRES or the IRES quasispecies variants. Cells were lysed 7 h post-transfection and both luciferase reporter activities measured (see Methods). IRES relative translation efficiency was calculated as the ratio of the two luciferase activities (Rluc/Fluc). Relative activities of the IRES variants were compared to that of the consensus IRES, which was set at 1. The results, which represent values obtained from three independent experiments and luminometric measurements done in duplicate, are summarized in Fig. 2
. The consensus IRES was the most efficient at initiating translation. The variant containing the A53G and C168G mutations was less efficient showing approximately 70 % activity levels. To analyse the contribution of the individual nucleotide changes to this reduced activity, we introduced the A53G and C168G mutations separately into the consensus IRES. Introducing A53G had very little effect on translation efficiency but replacing position C168 with a G resulted in an almost 20 % reduction in IRES activity (Fig. 2a
). This shows that although the combination of the two mutants had the highest effect on IRES activity, the C168G mutation contributed most to the reduction in translation. When considering that stemloop IIIa is a major determinant of eIF3 and 40S ribosomal subunit binding (Hellen & Sarnow, 2001
; Kieft et al., 2001
), this decrease of
20 % in translation activity seems small for a mutation that is predicted to disrupt the stemloop (see also Laporte et al., 2000
, 2003
).
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Because sequences upstream of the IRES are essential for RNA replication, we first engineered selectable replicons in which the con11b 5' NTR was replaced with the consensus 5' NTR of our EU strain, effectively replacing nt 11 and 12 of stemloop I (see Fig. 1b). We observed that both replicons were equally efficient in G418-resistant colony formation (data not shown), and therefore used the EU strain consensus IRES in further experiments. Subsequently, the IRES quasispecies variants were engineered in this replicon. Fig. 2(b)
shows the effect of these variants in Huh-7 cells using the selectable replicon system. Unexpectedly, the double mutation rendered the replicon non-viable as no colonies were obtained. To analyse why this variant failed to generate stable colonies, we cloned the separate mutations, A53G and C168G, in the replicon. When the individual mutants were assayed in the replicon system, colonies were obtained (Fig. 2b
). Compared with the consensus 5' NTR, the efficiency of colony formation was
5-fold lower for the A53G mutation and
50- to 100-fold lower for the C168G mutation (see Fig. 2b
). This shows that although each mutation did yield G418-resistant colonies, the additive effect of both mutations is apparently deleterious in the A53GC168G mutant. When the selection pressure was lowered to 250 µg ml1 G418, a concentration that increases the efficiency of colony formation (Lohmann et al., 1999
), eight resistant colonies were found for the double mutation. However, none of them survived the initial trypsinization after picking from the plate. The replicon system might be more stringent in selecting replication-competent RNA molecules, compared to circulating virus genomes (Bartenschlager et al., 2003
), explaining the inability of the naturally occurring variant A53GC168G to form replicon colonies.
Revertants in stemloop IIIa enhance replication
The lower efficiency of colony formation of the C168G mutant could be due to lower levels of replication or because early on during selection vital changes need to be generated within the replicon in order to enhance replication to sufficient levels. To examine the latter possibility, two independent G418-resistant colonies, isolated after electroporation of the C168G replicon, were expanded, RNA was isolated, and replicon RNA was amplified by RT-PCR and cloned as described above. Ten cloned variants from each G418-resistant colony were sequenced. In both colonies the point mutation C168G was found to be present. None of the cloned variants had reverted to the parental sequence but one or two other mutations accompanied most 5' NTR variants. Interestingly, in both colonies the nucleotide originally opposite mutation G168, i.e. guanosine 159, had mutated either to a cytidine in one clone (G159C, frequency 4/10) or to a uridine in the other clone (G159U, frequency 6/10). Both alterations restore the predicted base pairing in stemloop IIIa (see Fig. 3a). To assess whether these base changes represent mutations that restore efficient replication, we cloned and tested these revertant base pairs, 159C.G168 and 159U.G168, in the replicon. The number of colonies obtained after selection with G418 clearly increased for both revertants compared to the original C168G mutant (Fig. 3b
). Levels for the 159U.G168 revertant were five to 10 times higher, while almost wild-type levels were reached for the 159C.G168 revertant (Fig. 3b
). This shows that stemloop IIIa mutations that restore the base pairing between positions 168 and 159 enhance replication and that the poor replication activity observed for the stemloop IIIa C168G mutant is attributable to disruption of the base pairing.
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DISCUSSION |
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In addition to the differences in stemloop I, a major variant was found in stemloop IIIa, located at position 168, which is predicted to alter the secondary RNA structure due to loss of base-pairing with nucleotide 159 (see Fig. 1a). This mutation, although resulting in only a small reduction in translation activity (Fig. 2a
), showed a dramatic reduction in replication (Fig. 2b
). Mutations that disrupt the IIIabc four-way junction result in a pronounced loss of translation initiation activity (Kieft et al., 1999
, 2002
). The minor reduction in IRES activity observed for the C168G variant therefore suggests that the cruciform structure for IIIabc is still intact. Stemloop IIIa is conserved between pestiviruses and HCV (Fletcher & Jackson, 2002
) and is involved in direct interaction with both eIF3 and 40S ribosomal subunits (Kieft et al., 2001
). NMR and crystallography have been used to solve detailed structures of HCV IRES stemloops IIId, IIIe and the IIIabc junction (Lukavsky et al., 2000
; Kieft et al., 2002
; Collier et al., 2002
). In the crystal of the IIIabc junction, part of loop IIIa, containing the base paired region 159161, was missing due to disorder (Kieft et al., 2002
), thereby making structural analysis of the C168G mutation impossible.
Interestingly, when RNA was recovered from colonies originating from replicons containing the stemloop IIIa C168G variant, several molecules in the population were found to carry an additional mutation that restored base pairing with the mutant G168 position (Fig. 3a). When these clones were inserted back in the original replicon, higher levels of colony formation were observed (Fig. 3b
), confirming that they are true revertants and base pairing of stemloop IIIa is required for optimal replication. The observation that the compensatory mutations failed to rescue replication to wild-type levels suggests that sequence-specific contacts with the initiation complex at this position might be essential. In addition, as colony formation by the 159C.G168 revertant reached higher levels than the 159U.G168 revertant, stability of the base pairing appears important [C.G=2·4 kcal mol1 (10·0 kJ mol1) versus U.G=1·4 kcal mol1 (5·9 kJ mol1)].
These emerging revertants could be due to their existence prior to selection in the replicon system, i.e. generated during in vitro transcription by the T7 polymerase, or by mutation from the template during RNA replication. Since the two selected colonies arose from the same in vitro transcription reaction but show different solutions for restoration of the base pairing (either G.C or G.U base pair), we consider the latter mechanism more likely.
Owing to the error-prone nature of the viral polymerase, mutations are expected to occur randomly distributed over the 5' NTR region. Clearly, only mutations compatible with replication and translation can be propagated. Whether the stemloop II/IIIa mutations we observed confer a survival advantage or disadvantage remains unclear. We suggest that, although the virus adaptation led to a low replication, it could well be that the genetic defect reflects genetic flexibility.
The con1 replicon we used contains the non-structural proteins of a genotype 1b strain different to our strain from which we obtained IRES quasispecies variants. It is possible that the 5' NTR variants found are tailored to the virus coding region and interactions may be suboptimal. Further work with subgenomic replicons containing the non-structural genes of our isolate should help to clarify this point.
The unique structure of the HCV IRES makes it an attractive target for the development of antivirus agents directed against this RNA element such as antisense oligonucleotides, transacting ribozymes and siRNAs (Kurreck, 2003). Since stemloop IIIa is highly conserved and involved in both replication and translation, it could present a good candidate for antivirus targeting. This work, however, shows that revertants can arise in the IRES during selective pressure of the subgenomic replicon system. In vivo, drug-resistant variants in HCV-infected patients have been reported in the virus RdRp during ribavirin monotherapy, where a single amino acid change conferred increased resistance (Young et al., 2003
). Strategies directed against HCV RNA elements should therefore target multiple regions in order to reduce the probability of emerging escape mutants.
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ACKNOWLEDGEMENTS |
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Received 19 December 2003;
accepted 23 February 2004.