Article Figures & Data
Figures
- Fig. 1.
Predicted models of the secondary structures of the 5′NTRs of GBV-B (a) and HCV (b). (a) Structure proposed for the GBV-B 5′NTR by Honda et al. (5, 6). Major predicted structural domains are labeled I to IV, while individual stem-loops are labeled Ia and are analogous to similarly labeled structures in the HCV 5′NTR. Base-pair interactions involving the loop sequence of stem-loop IIIf that are predicted to result in a putative RNA pseudoknot are drawn as solid lines. Lightly shaded boxes represent AUG triplets located within the 5′NTR, while the solid black box represents the polyprotein translation initiation site. The open box indicates a helical segment at the base of domain II, and open circles represent individual nucleotides that were subjected to site-directed mutagenesis in the studies described here. Unpaired bases within domain II that are conserved in domain II of the HCV structure are shown in boldface type. The 5′ and 3′ limits of the GBV-B IRES, as determined in this study, are indicated by the arrows. (B) Structure proposed for the HCV 5′NTR (3, 5, 6). There are numerous similarities with the GBV-B structure but no predicted stem-loop structures analogous to the Ib, IIb, and IIc stem-loops of GBV-B.
- Fig. 2.
Demonstration of the GBV-B IRES and mapping of its 5′ limit. (a) Diagram showing the organization of the transcriptional unit in plasmid used to characterize the IRES. T7 and TΦ represent the T7 RNA polymerase promoter and terminator sequences. The locations of specific deletion mutations created within the 5′NTR of GBV-B in derivatives of p-wt are shown, with the nucleotides deleted listed at the right. (b) SDS-PAGE of products of in vitro translation reactions programmed with the indicated RNA transcripts. CAT/rLuc is a numeric ratio calculated from the PhosphorImager analysis of the gel, relative to an arbitrary value of 100% for p-wt transcripts. (c) Relative translational activity (CAT/rLuc) of the indicated mutants following their transfection as DNA into BT7-H cells; a value of 100% was arbitrarily assigned to cells transfected with p-wt. The number of replicate transfection assays (N) is shown for each mutant. Error bars indicate standard deviations. The results indicate that the 5′ limit of the IRES is located between nt 61 and 88, or close to the 5′ limit of structural domain II (Fig. 1a).
- Fig. 3.
Mutational analysis of domain II of the GBV-B 5′NTR. (a) Diagram showing mutations introduced into the sequence of domain II to evaluate the IRES requirement for specific stem-loop structures in this domain. (b) Expanded view of the helix at the base of domain II (see boxed sequences in Fig. 1a). Nucleotides at which mutations were introduced by site-directed mutagenesis are indicated by open circles. (c) In vitro translation of the domain II mutants. See the legend to Fig. 2b for details. (d) Translational activity of domain II mutants in transfected BT7-H cells. See the legend to Fig. 2c for details. The results of these experiments provide confirmation of the predicted helical structure at the base of domain II and demonstrate that each of the three subsidiary stem-loops of domain II contributes to efficient IRES activity.
- Fig. 4.
The phylogenetically conserved stem-loop IIIe is critically important for translational activity of the GBV-B IRES. (a) Changes made in the nucleotide sequences of stem and loop regions of stem-loop IIIe. Altered nucleotides are encircled, with effects on the predicted structure as shown. Also shown is the translational activity of the stem-loop IIIe mutants in rabbit reticulocyte lysate (b) or transfected BT7-H cells (c). See the legend to Fig. 2 for details.
- Fig. 5.
Four phylogenetically conserved, unpaired adenosine residues are critically important for GBV-B IRES activity. Changes were made in the context of p-wt to test the effect of the conserved adenosine residues at positions 253 and 392 (a) and positions 271 and 272 (b). Nucleotides that have been changed by site-directed mutagenesis are encircled. (c and d) Translational activities of the indicated mutants in rabbit reticulocyte lysate (c) and transfected BT7-H cells (d). All of the substitutions at these sites were lethal for translation, as were substitutions involving base pairs flanking the internal loop shown in panel a.
- Fig. 6.
GBV-B IRES-mediated translation does not require polyprotein-coding sequences. (a) Bicistronic RNAs expressing CAT under the control of the GBV-B IRES with different lengths of coding sequence between the IRES and the reporter sequence. Arrowheads represent the AUG at the translation initiation site, with stem-loop IV depicted in the +14 transcript and transcripts with longer lengths of coding sequence, 38 and 63 nt. Transcripts from p-wt have 3 nt of the coding sequence, the AUG codon only. The translational activities of the transcripts shown in panel a were monitored in vitro in rabbit reticulocyte lysate (b) and in transfected BT7-H cells (c). See the legend to Fig. 2 for details.
- Fig. 7.
The stability of stem-loop IV influences translational activity of the GBV-B IRES. (a) Three different mutants were created to evaluate the effect of altering the stability of stem-loop IV. RNA structures and predicted free energies are from the MFOLD program of Michael Zucker, with folding energies of Turner, on the Washington University server (http://mfold/wustl.edu/∼folder/). Nucleotides that differ from the +14 construct sequence are encircled. The initiator AUG codon is highlighted in each transcript. (b) For the left panel, translational activities of the dicistronic transcripts depicted in panel a were assessed in vitro in rabbit reticulocyte lysates. To facilitate comparisons between the stem-loop IV mutants, the relative translational activity (amount of CAT expressed per unit of rLuc) of the +14 transcripts was arbitrarily assigned a value of 100%. The right panel shows CAT expressed in reticulocyte lysates programmed for translation with 5′-truncated, monocistronic RNAs containing the indicated stem-loop IV mutations. These truncated RNAs lack the 5′ 284 nt of the GBV-B 5′NTR and are translated by a ribosome scanning mechanism. (c) The translational activities of dicistronic transcripts depicted in panel a were assessed in transfected BT7-H cells. To facilitate comparisons between the stem-loop IV mutants, the relative translational activity (amount of CAT expressed per unit of rLuc) of the +14 transcripts was arbitrarily assigned a value of 100%.
