(i) Schematic representation of constructs generated as described previously (9) to replace wild-type eIF4G with either a form that was resistant to knockdown by siRNA (eIf4GIf) or versions of eIF4G where the PABP (eIF4GIf-PABP), eIF4E (eIF4GIf-4E), eIF4A (eIF4GIf-4A), or eIF3 (eIF4GIf-eIF3) binding site was mutated (12). Ct, the fragment of eIF4GI generated in cells infected with poliovirus. (ii) HeLa cells were cotransfected with si31 (eIF4G knockdown plasmid), either the resistant plasmid 4GIf or a resistant plasmid which harbored mutations in the PABP, eIF4A, eIF3, or eIF4E binding site, and a dicistronic plasmid harboring either the EMCV or HCV IRES. Firefly and Renilla luciferase assays were performed, and the data are expressed relative to those for RNAs generated from the relevant vectors (see Materials and Methods). All assays were performed in triplicate on at least three independent occasions. Relative luciferase activity is expressed relative to those in control cells, which are set at 100%. The numbers of light units generated by these assays were between 1 × 10⁵ and 2 × 10⁶.

(A) (i) HeLa cells were transfected with si31, which reduces eIF4G levels, and optionally with an eIF4G-containing plasmid which is resistant to si31 (4GIf) or with 4GIf containing a mutated PABP binding site (Fig. 1). Western blot analysis showed that eIF4G levels decreased upon si31 transfection and increased with si31 and 4GIf transfection. Cells were also transfected with si31 and a plasmid which encodes the C-terminal domain of eIF4G (CT), and Western blot analysis was used to demonstrate expression of this protein. (ii) Schematic analysis of the bicistronic construct used (pRF). The L-, N-, and c-myc IRESs were inserted between the Renilla and firefly luciferase cistrons to create pRLsF, pRNF, and pRMF, respectively. (B) HeLa cells were cotransfected with si31 (eIF4G knockdown plasmid), either the resistant plasmid (4GIf), the C-terminal fragment of eIF4G (CT), or the resistant plasmid harboring a mutation in the PABP binding site (Fig. 1), and either the L-myc (i), N-myc (ii), or c-myc (iii) IRES. Firefly and Renilla luciferase assays were performed, and the data are expressed relative to those for RNAs generated from the relevant vectors (see Materials and Methods). All assays were performed in triplicate on at least three independent occasions. Firefly luciferase activity (gray bars) and Renilla luciferase activity (black bars) were expressed relative to those in the control cells, which were set to 100%. The numbers of light units generated by these assays were between 1 × 10⁵ and 2 × 10⁶. These data show that all three IRESs are dependent on eIF4GI for function, but the N- and c-myc IRESs are able to operate with the C-terminal domain of eIF4GI. All three IRESs appear to require PABP for maximal activity, although there is partial restoration of N- and c-myc IRES activity with plasmid 4GIf containing a mutated PABP binding site. (iv to vi) Cells were additionally transfected with monocistronic hairpin vectors (8) which contained the three IRESs. These vectors were transfected into HeLa cells in conjunction with si31 (eIF4G knockdown plasmid) and either the resistant plasmid (4GIf) or the resistant plasmid harboring a mutation in the PABP binding site. Luciferase assays were performed, and the data show that all three IRESs require PABP for activity. (vii) HeLa cells were cotransfected with the three IRES-containing plasmids and either a plasmid which expresses the NSP3 protein or a mutant version where the eIF4G binding site is deleted (34). Luciferase activity was determined and normalized to the level of the RNA control. The data show that all three IRESs are inhibited by the presence of the wild-type NSP3 protein but not by the mutated version. Black bars denote Renilla luciferase activity, and gray bars denote firefly luciferase activity. (C) (i) To determine the effect of transfection with the si31 eIF4G knockdown and mutated recovery plasmids described for panel A on the expression of endogenous c-Myc, transfected cell samples were lysed and subjected to SDS-polyacrylamide gel electrophoresis, and Western blot analysis was performed. The data show that c-Myc levels were reduced to approximately 10% of the control values in the presence of si31 and were restored to slightly above wild-type levels following cotransfection with 4GIf. In comparison, c-Myc levels were slightly reduced following transfection with the plasmid containing the mutated version of PABP. The c-Myc protein levels were restored, but not to fully wild-type levels, in the presence of the C-terminal fragment of eIF4G (CT) (ii) (32).

(A) To determine whether the N-myc IRES was similar to the L- and c-myc IRESs and used a “land and scan” mechanism, site-directed mutagenesis was used to insert out-of-frame AUG codons into the IRESs within the vector pRNF. The position of each mutated sequence is numbered from the A of ATGG or the U of UUGG. (B) The resulting constructs were transfected into HeLa cells in conjunction with pcDNA3.1/HisB/lacZ. The data are presented as percentages of firefly luciferase activity compared to the activity from the wild-type IRES. (C) The location of the ribosome entry site is shown on a secondary structure model of the N-myc IRES (see Fig. S2 in the supplemental material). It was difficult to define the ribosome entry window precisely, although it can be stated with a degree of certainty that it lies between positions −232 and −173 within a pyrimidine-rich region.

(A) (i) To test the effect of hippuristanol on cellular translation, cells were incubated with 1 mM hippuristanol for 6 h and then pulse labeled with [³⁵S]methionine. The data show a 60% reduction in total protein synthesis after this period. (ii) The IRES-containing constructs were transfected into HeLa cells, and after 24 h, either hippuristanol or DMSO was added to the medium. After 6 h, luciferase activity was determined, and the values are expressed relative to those for the DMSO-treated control. In all cases, IRES activity was reduced in the presence of hippuristanol. (iii) To determine the effect of hippuristanol on the expression of endogenous c-Myc, Western blot analysis was performed. The level of c-Myc protein was reduced to approximately 20% following exposure of cells to hippuristanol. (B) HeLa cells were transfected with si31 (−eIF4G) and with either the eIF4GI-containing plasmid which is resistant to si31 (4GIf) or 4GIf containing a mutated eIF4A binding site. Western blot analysis shows that eIF4GI levels decreased upon si31 transfection and increased with 4GIf transfection. Cell lysates were then immunoblotted to determine the effect of mutant eIF4G(−4A) expression on the levels of endogenous c-Myc protein. The data show that these levels were reduced. (C) HeLa cells were cotransfected with si31 (−eIF4G) and either the resistant plasmid (4GIf) or the resistant plasmid harboring a mutation in the eIF4A or eIF4E binding site (see Fig. S1 in the supplemental material) and then transfected with either the L-myc (i), N-myc (ii), or c-myc (iii) IRES. Luciferase assays were performed, and the data are expressed relative to those for RNAs generated from these vectors. Luciferase activity in the control cells was set to 100%, and all numbers are expressed relative to the control cells. Black bars denote Renilla luciferase activity, and gray bars denote firefly luciferase activity. All assays were performed in triplicate on at least three independent occasions. The data show that all three IRESs require eIF4A bound to eIF4G for activity and that the L-myc IRES, but not the N- and c-myc IRESs, requires eIF4E. (D) (i) HeLa cells were transfected with a plasmid which contained a short hairpin sequence (si-eIF4E) against eIF4E or a control sequence. Western blot analysis shows that it was possible to reduce the levels of eIF4E to approximately 15% of the value in the control cells. (ii) HeLa cells were transfected with the si-eIF4E plasmid and then with the IRES-containing vectors shown. Luciferase activity was determined, and values are expressed relative to those for RNAs derived from the vectors. The value in the control cells was set at 100%, and changes in activity are expressed relative to the controls. Black bars denote Renilla luciferase activity, and gray bars denote firefly luciferase activity.

(A) HeLa cells were cotransfected with si31 (−eIF4G) and either the resistant plasmid (4GIf) or the resistant plasmid harboring a mutation in the eIF3 binding site. (i) Cell lysates were then immunoblotted to determine the effects on the expression of endogenous c-Myc protein. As before, a reduction in eIF4G levels resulted in a very large reduction in c-Myc expression, which was partially relieved by transfection with the recovery plasmid which contained the mutated version of eIF3. (ii to iv) Cells were transfected with si31, 4GIf harboring a mutation in the eIF3 binding site, and the IRES-containing vectors. Luciferase activity was determined and expressed relative to the level of RNA generated from the vectors. The value obtained in the control cells was set to 100%, and all values are expressed relative to the appropriate control, e.g., pRLsF-eIF4G activity is expressed relative to that of pRLsF. Black bars denote Renilla luciferase activity, and gray bars denote firefly luciferase activity. The L-myc IRES activity was not restored with the plasmid which harbors the eIF3 mutant, in contrast to the N- and c-myc IRESs. (B) (i) HeLa cells were transfected with a plasmid which contained a short hairpin sequence (sheIF3) against the p170 subunit of eIF3 (eIF3a) or a control sequence. Western blot analysis shows that it was possible to reduce the levels of eIF3a to approximately 20% of the value in the control cells. (ii) HeLa cells were cotransfected with sheIF3 plasmids and the bicistronic IRES-containing vectors. Luciferase activity was determined, and values are expressed relative to those for RNAs generated from the reporter vectors. Black bars denote Renilla luciferase activity, and gray bars denote firefly luciferase activity. The values obtained in the control cells were set to 100%, and all values are expressed relative to those for the appropriate controls. The data show that IRES activity was reduced in all cases.

(A) HeLa cells were transfected with the IRES-containing plasmids and then, after 24 h, exposed to 75 μM Salubrinal. (i) Western blot analysis was performed to confirm that exposure of cells to this compound causes a net increase in phosphorylation of the alpha subunit of eIF2. (ii) Cells were lysed, and luciferase activity was determined and shown as the percentage of luciferase produced from treated cells compared to the amount produced from the control cells, which was set at 100%. Black bars denote Renilla luciferase activity, and gray bars denote firefly luciferase activity. Assays were performed in triplicate on at least three independent occasions. The data show that the N-myc IRES is less sensitive to the effects of Salubrinal than the L- and c-myc IRESs. (B) The IRES-containing plasmids pRLsF, pRNF, and pRMF were cotransfected into HeLa cells with pcDNA3.1eIF2αS51D or -A (which harbors a constitutively active or dominant-negative version of eIF2α) or pcDNA3.1. (i) Western blot analysis was performed to show that these proteins were expressed. (ii) The data are presented as percentages of luciferase produced from control cells (pcDNA3.1) relative to the amount produced from the IRESs in cells transfected with the mutant plasmids (S51D and S51A). Assays were performed in triplicate on at least three independent occasions. Black bars denote Renilla luciferase activity, and gray bars denote firefly luciferase activity.

Model for the interaction of canonical initiation factors with the three Myc family IRESs. (A) In the absence of PABP (i) or in the presence of NSP3 (ii), eIF4G is unable to form the correct conformation to allow interaction with the IRESs. (B) (i) The L-myc IRES requires the scaffold protein eIF4G, the interaction of eIF4E, eIF4A, PABP, and eIF3 with this scaffold, and trans-acting factors (X) for function. The c- and N-myc IRESs are able to function with complexes that are comprised of trans-acting factors (X) and eIF4G (ii) or the C-terminal fragment of eIF4G (CT) (iii) in the presence of PABP and eIF4A. eIF3 may be recruited directly to the IRES RNA.