GBV-B is a recently discovered, incompletely characterized virus belonging to the family Flaviviridae
and phylogenetically closely related to HCV (10
). Like HCV, it is hepatotropic and capable of causing acute hepatic injury in infected primates. Although recovered from an experimentally infected tamarin, only a single example of this virus has yet been identified, and its natural host species remains uncertain. The close relationship of GBV-B to HCV, a major cause of chronic liver disease in humans, makes it a particularly interesting virus to study, especially in the absence of good experimental systems for HCV. In contrast to the more distantly related flaviviruses GB virus A and GB virus C (otherwise known as hepatitis G virus), the 5′NTR of GBV-B has significant structural homology to HCV and its genome encodes a readily identifiable capsid protein (6
). The experiments described here show that GBV-B, like HCV and the pestiviruses, translates its genome by means of an efficient IRES element located within its 5′NTR.
Our results indicate that the GBV-B IRES has a number of structural and functional features in common with the HCV IRES, but they also demonstrate some impressive differences between these IRES elements. The experiments depicted in Fig. and demonstrate that the IRES spans domains II and III of the GBV-B 5′NTR structure and thus occupies a position that is analogous to the position of the HCV IRES within the 5′NTR of that virus. Placed within the same reporter sequence context, these two flaviviral sequences have approximately equal translational activities (data not shown). The GBV-B and HCV structures are remarkably similar, despite the fact that there is very little conservation of primary nucleotide sequence between these viruses (5
The most impressive difference between the two predicted structures is the inclusion of a large, approximately 97-nt insertion within domain II of the GBV-B 5′NTR. Computer modeling suggests the inserted sequence forms two extended stem-loop structures (Fig. a, stem-loops IIb and IIc) that are absent from the IRESes of HCV and the pestiviruses (6
). Although the deletion of these two stem-loops is likely to result in a structure with greater superficial similarity to the structure of the HCV IRES, we demonstrated that the retention of the two stem-loops is essential for optimal GBV-B IRES activity (Fig. ). The deletion of these stem-loops from the GBV-B sequence resulted in less impairment of translation in reticulocyte lysates than in BT7-H cells, but the effect was evident in both systems. These results are of interest with respect to a possible evolutionary relationship between the GBV-B and HCV IRES elements. It is intriguing to speculate that the GBV-B structure may be more closely related to the structure of a common ancestral IRES. Evolutionary modifications to the HCV structure appear to have allowed it to overcome the deletion of stem-loops IIb and IIc and to evolve toward a smaller, more compact sequence with approximately equivalent internal ribosome entry activity.
The analysis of compensatory mutations that were created within the extended helix at the base of domain II of the GBV-B structure (Fig. ) provides support for the secondary structure model we had proposed previously (6
). Substitutions of the nucleotide sequence between positions 231 and 236 resulted in a strong decrease in translation. The translational activity of this mutant was significantly restored by introduction of the complementary changes into the sequence between nt 63 and 68 (Fig. ). This observation confirms the presence of the predicted helical segment at the base of domain II (Fig. a). There was less reduction in translation that was observed with the disruption of the helix due to substitutions within its 5′ strand, between nt 63 and 68, than in its 3′ strand, between nt 231 and 236 (Fig. , compare the translational activity of IIL with that of IIR). This indicates that retention of the primary nucleotide sequence is more important in the 3′ segment of this helix than in the 5′ segment. This may reflect stochastic alternative base pairing that allows the partial retention of IRES activity with the 5′ mutations, but it is interesting that we have observed a similar effect with analogous mutations of domain II of the HCV IRES (5
). Thus, this appears to be a general feature of the domain II structures of the flavivirus IRESes.
In general, mutations in domain III of the GBV-B IRES had much more profound inhibitory effects on translation than mutations in domain II (for example, compare Fig. with Fig. and ). We identified several base-paired helical segments in domain III within which the primary nucleotide sequence was critically important and could not be replaced with alternative base-pair arrangements. In contrast to the helix at the base of domain II (Fig. ), replacing the stem element of stem-loop IIIe with complementary base-paired sequences led to nearly complete loss of translation (Fig. ). This hairpin structure is unique in that its primary nucleotide sequence is conserved in both its stem and loop segments between the IRES elements of GBV-B, HCV, and the pestiviruses (9
). Similarly, we found that the nearby nonpaired A253
residues could not be replaced either singly or as a pair by uridine residues and that base pairs flanking the internal bulge created by these unpaired adenosines could not be replaced by alternative base pairs (Fig. ). These results are consistent with a recent report by Pestova et al. (11
), who found that a deletion of 4 nt that included an adenosine homologous to the A253
in GBV-B resulted in a loss of ribosome binding by a pestivirus IRES. Together, these data indicate a strict requirement for conservation of the primary nucleotide sequence in this part of the IRES. This strict conservation for sequence is likely to reflect a stringent requirement for an RNA structure that is conducive to interactions with the 40S ribosome subunit or a protein translation factor (11
). An alternative interpretation of the results shown in Fig. might be that the model structure is incorrect in this region of the IRES and that the compensatory mutations we created in the GBV-B sequence do not involve actual base pairs. This is unlikely to be the case, however, because there is very strong experimental evidence for the existence of the pseudoknot in these structures and substantial natural sequence covariation in stem-loop IIId, structures that flank this region (Fig. ) (9
A similar explanation is likely for the strong inhibition of translation that we observed following the modification of the broadly conserved A271
residues in the GBV-B IRES (Fig. , mutant IIIa-ΔAA). Eucaryotic translation initiation factor 3 has been shown to interact with the apical segment of the domain III structures of both HCV and pestivirus IRES elements (2
). Since these two adenosines are located just upstream of stem-loop IIIa (Fig. a), it is possible that these substitutions interfered with an interaction between the GBV IRES and eucaryotic initiation factor 3. This initiation factor is essential for translation of the HCV polyprotein (11
All of the foregoing results show that the GBV-B IRES has a number of features in common with the IRES elements of HCV and the pestiviruses. Because there has been considerable controversy about the 3′ limits of the IRES in HCV and the pestiviruses, we examined this aspect of the GBV-B IRES in detail. Reynolds et al. (13
) reported that approximately 32 nt of HCV polyprotein-coding sequence must be present downstream of the HCV 5′NTR to ensure optimal translation and have suggested an important role for the primary nucleotide sequence of this segment in the process of internal ribosome entry. However, the HCV IRES does function efficiently when the initiator AUG is fused directly to some reporter protein sequences (Fig. ) (27
), and other results suggest that the constraints on downstream sequence are more related to a requirement for unstructured RNA rather than a requirement for specific sequence (6
). The results of the experiments depicted in Fig. and strongly support this latter hypothesis.
Taken together, our results indicate that the 3′ border of the GBV-B IRES element is located at or 5′ of the AUG codon (strictly speaking, between stem-loop IIIe and the AUG codon, or between nt 403 and 446) (Fig. and ). The inclusion of 14, 38, or 63 nt of GBV-B polyprotein-coding sequences between the AUG codon and the CAT reporter coding sequence in the +14, +38, and +63 constructs significantly reduced IRES activity compared to the wt construct (Fig. ). The reduction in translation upon inclusion of the 5′ 14 nt of the GBV-B capsid protein coding sequence was due to the inclusion of the complete stem-loop IV sequence in these transcripts. This stem-loop, which contains the AUG initiator codon within its loop sequence (Fig. a), thus appears to be inhibitory to initiation of translation. As with the analogous stem-loop in the HCV sequence (6
), we found that mutations predicted to enhance the stability of this stem-loop substantially reduced the activity of the upstream IRES (Fig. ).
However, RNA transcripts with a large 5′-terminal deletion and containing similar stabilizing mutations in the stem-loop IV sequence translated as well as transcripts with the wt stem-loop IV (Fig. ). Thus, this structure is inhibitory only for ribosomes that are entering internally on the RNA and not for ribosomes that are scanning toward it from a more 5′ point of contact. These data provide strong evidence for the absence of any scanning in the normal internal initiation of translation on GBV-B RNAs and, as for the RNAs of HCV and the pestiviruses, suggest that the 40S subunit makes an important primary contact at the site of the initiator AUG codon (6
). The presence of stem-loop IV in both the GBV-B and HCV sequences (Fig. ) remains puzzling. Its location and its suppressive effect on cap-independent translation suggest that stem-loop IV may plan an important role in regulating translation (6
). However, no data have yet confirmed this hypothesis. The presence of this stem-loop in GBV-B and HCV, but not the pestiviruses which have been increasingly used as surrogates for the study of HCV (6
), highlights the potential importance of further characterizing the GBV-B virus and its genome.