There is some minor controversy in the literature with respect to where the 5′ boundary of the pestivirus IRES lies and more serious controversy over the significance of domain II for IRES activity. Rijnbrand et al. (30
) found that the deletion of 26 nt from the 5′ end actually increased IRES activity by about 60%; a deletion to nt 66 reduced the activity to about two-thirds that of the full-length construct; and an internal deletion of nt 28 to 66 decreased activity still further. Consequently, they placed the 5′ boundary as lying within what was then called stem-loop B, between nt 28 and 66, whereas we would argue that all sequences up to the 5′ side of domain II do not participate directly in IRES function. In our experiments we did not observe any stimulation of IRES activity with short 5′ deletions (though admittedly our deletion series did not include any mutants with end points very near nt 28), and we found that a deletion to nt 65 retained full activity. We would argue that our position for the 5′ boundary is more consistent with the phylogenetic data: our analysis of covariances between BDV, BVDV, and CSFV 5′ UTR sequences shows strong conservation of structure downstream of the 5′ end of domain II (Fig. ), but there is much less conservation upstream of this point. Moreover, a boundary for the CSFV IRES at the 5′ residue of domain II would be absolutely consistent with the position of the 5′ boundary of the HCV IRES (29
) in the recently revised secondary structure model of the HCV 5′ UTR (16
) and is also consistent with the position of the 5′ boundary of the BVDV IRES mapped by Chon et al. (6
As for the significance of domain II in IRES function, Rijnbrand et al. (30
) found that deletions which invaded domain II were essentially inactive in transfected Hep2 cells, whereas in our hands they retain about 20% of the activity of the full-length IRES both in vitro and in transfected BHK cells. This is very similar to the results we obtained with 5′ deletions of the HCV IRES (29
). It is only when the deletions not only remove the whole of domain II but invade stem 1 of the pseudoknot that IRES activity falls to zero in our hands. In contrast, Kolupaeva et al. (20
) found that deletion of domain II of either the CSFV or HCV IRES reduced translation efficiency only mildly, to 60 to 65% of the control, although it did subtly alter the characteristics of 40S subunit binding to the IRES and was highly debilitating when combined with other mutations which on their own had only a mild phenotype.
The recent analysis by cryo-electron microscopy of the interaction of the 40S ribosomal subunit with the HCV IRES shows that the bulk of the IRES, domain III and the pseudoknot, binds to the solvent side of the 40S subunit, behind the platform (33
). Domain II largely loops away from the ribosomal subunit, with the only contact being between the top of this domain and the E-site neighborhood of the 40S subunit. Deletion of domain II has no effect on the affinity of the IRES for the 40S subunit or on the actual contact sites between the rest of the IRES and the 40S subunit, but it does abolish the conformational change in the 40S subunit which occurs when the full-length IRES binds. It has been suggested that interaction between the top of domain II and the mRNA coding region may guide the RNA into the mRNA binding cleft on the 40S subunit (33
). Alternatively, the conformational change in the 40S subunit may open up the mRNA binding cleft. Given that a domain II deletion doesn't affect the initial binding of the IRES to the ribosome, and so the initiation codon will be tethered tightly to the 40S subunit, it would seem surprising if the deletion completely abolished all IRES activity, as was reported by Rijnbrand et al. (30
). On the other hand, retention of as much as 60 to 65% of the activity of the full-length IRES, as reported by Kolupaeva et al. (20
), would also seem surprising in view of the fact that the conformational changes induced in the 40S subunit are dependent on domain II.
The importance of both base-paired stems of the pseudoknot structure for the activity of the HCV IRES has previously been demonstrated by making compensatory mutations (34
). A similar approach was used by Rijnbrand et al. (30
) to demonstrate that stem 2 of the CSFV IRES needs to be base paired, as has been confirmed here (Table ). A further development described here and not addressed by Rijnbrand et al. (30
) is the use of the same approach to demonstrate that stem 1, or at least stem 1A, is equally important (Fig. ). In comparison with HCV, in which stem 1 of the pseudoknot is nine contiguous base pairs (eight of them being G-C pairs), the equivalent stem in the pestiviruses consists of two parts (stem 1A and stem 1B) separated by a bubble. Stem 1B, which is in the equivalent position to the HCV stem 1, is five or six contiguous base pairs (depending on the species of virus), and stem 1A is seven or eight contiguous base pairs. We have shown that base pairing in stem 1A is essential for CSFV IRES activity (Fig. ), and although we have not directly examined stem 1B, topological constraints dictate that this, too, must surely be base paired as shown in Fig. . That base pairing in stem 1B is most likely to be critical for IRES activity is shown by the fact that disruption of 3 bp in this stem severely impaired IRES activity (21
), although compensatory mutations to restore base pairing were not tested. In view of this overwhelming evidence in favor of the base pairing, but not the primary sequence, of the pseudoknot being critical for IRES activity, it is very puzzling that Kieft et al. (19
) could not rescue HCV IRES activity by making compensating mutations in stem 2. The explanation for this unique negative result is not immediately self-evident, though there is one obvious difference in the technical details of these experiments. Kieft et al. (19
) assayed IRES activity using the coupled transcription-translation system. Because this system generates uncapped transcripts and so has been optimized for translation (via the scanning mechanism) of uncapped mRNA, it is usually operated at a monovalent cation concentration that is considerably lower than that which is optimal for translation of capped mRNAs or translation dependent on the HCV and pestivirus IRESs.
Whereas there is just a single U residue between stem 1 and stem 2 in the HCV pseudoknot, in pestiviruses there is a longer loop varying from five consecutive A residues in BVDV strains to 8 nt (AUAAAAAU) in other species, including CSFV. This longer loop in CSFV than in HCV may be necessary for topological reasons related to the greater length of stem 1A plus 1B of the pestivirus pseudoknot. Certainly, reducing the length of the pseudoknot loop in the CSFV IRES by 5 nt very severely decreased IRES activity (Fig. ). If our suggestion of a topological constraint is correct, this shortening would either distort the relative positions of the two stems of the pseudoknot or possibly cause the two stems to become partly unpaired.
However, our mutational analysis showed that for maximum IRES activity it is not just the length of the pseudoknot loop that is important; it also has to be A-rich. A-rich bulges like this are found in the group I intron ribozyme, where they are likewise essential for activity (5
). The IRESs of cardioviruses and aphthoviruses also have an A-rich bulge, and mutations in this bulge influence not so much the activity of the IRES as its dependency on polypyrimidine tract binding protein for activity (17
). X-ray crystallography of the group I intron ribozyme has shown that most of the residues of the A-rich bulge are involved in tertiary interactions which depend on such residues being specifically adenosines, a conclusion consistent with the influence of mutations of these residues on ribozyme activity (5
). A common type of tertiary structure interaction, found in both the group I intron and in 23S rRNA, is the insertion of these adenosine residues into the minor groove of neighboring helices (5
). It would be intriguing if the A-rich pseudoknot bulge in the CSFV IRES were involved in similar tertiary interactions. On the whole, the influence of mutations of the A residues in the CSFV IRES pseudoknot loop is much less deleterious than in the case of the group I intron A-rich bulge, which would argue against the idea that they are involved in critical tertiary interactions. However, in structure probing experiments we find that even though this A-tract is not cleaved by cobra venom nuclease, the A residues are only moderately susceptible to reaction with dimethyl sulfate, indeed rather less reactive than nearby A residues such as the two between stem 2 and the initiation codon or the A residue in the bubble between stem 1A and stem 1B (S. P. Fletcher and R. J. Jackson, unpublished results).
Finally, the lack of any effect of mutation of the very highly conserved loop IIIa sequence was surprising. However, our results closely parallel those of Kolupaeva et al. (21
), who found that mutation of the loop sequence from the wild-type AGUA to AAAA had very little effect on translation efficiency, but mutations which would have altered the length of the stem and/or the size of the loop were deleterious. In addition, these authors observed that the binding of 40S ribosomal subunits to the IRES afforded no protection of domain IIIa of either the CSFV or HCV IRESs (20
However, almost diametrically opposite results have been reported for the HCV IRES (19
). Not only were residues in the IIIa loop protected when 40S subunits bound to the HCV IRES, but modification interference experiments strongly implicated the two A residues as critical for 40S subunit binding. In addition, mutation of the loop sequence to UCAU reduced translation efficiency and 40S subunit binding affinity about 10-fold. Nevertheless, the cryo-electron microscopy analysis actually suggests that domain IIIa, together with domains IIIb and IIIc, extends away from the surface of the 40S subunit (33
), which is hard to reconcile with the mutagenesis and structure probing data published by the same group (19
Since only two positions of this tetraloop were changed in any one of the many mutants we generated, it could conceivably be argued that the sequence of the loop is necessary but has very considerable inherent redundancy, with either just the two central residues or the two flanking A residues being sufficient. Although this type of explanation would be formally consistent with all the results of the point mutations made in the IIIa loop by Kolupaeva et al. (21
), Kieft et al. (19
), and ourselves, it seems highly implausible and without precedent in any other system. Another explanation that is formally consistent with all the data is that the CSFV and HCV IRESs differ with respect to the importance of the loop IIIa sequence, but this too seems highly improbable. It thus seems possible that despite its high degree of conservation, the sequence of the loop is not important for IRES activity, even though the length of the stem and the existence of the four-way junction probably is critical. This in turn raises the question of whether there may not be signals embedded within the IRES for other functions necessary for the viral life cycle apart from internal initiation of translation. Indeed, the results of recent experiments with the newly developed HCV replicon system indicate that there are cis
-acting signals within the IRES which are critical for efficient RNA replication (10