The 3′-Terminal Hexamer Sequence of Classical swine fever virus RNA Plays a Role in Negatively Regulating the IRES-Mediated Translation (original) (raw)
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Biochemical and Biophysical Research Communications, 1998
The 5 nontranslated region (NTR) of hepatitis C virus (HCV) consists of 341 nucleotides (nt). This region comprises the majority of the internal ribosome entry site (IRES) which controls the efficiency of viral translation. Previous studies of the 3 boundary of the HCV IRES yielded conflicting data regarding the involvement of viral coding sequences in IRES activity. We therefore studied the functional significance of the 5 proximal coding sequences of the HCV core gene on IRES activity. We constructed monocistronic and bicistronic DNAs that contained either a chloramphenicol acetyl transferase (CAT) gene or a luciferase (Luc) gene as the reporter. Results from both in vitro and in vivo experiments indicated that the optimal IRES ranged within nt 1-371. Further mutational analyses of sequences surrounding the initiation codon revealed that primary sequences downstream of the AUG initiator rather than the secondary structure are important in regulating optimal IRES function. We are also able to demonstrate that a non-AUG codon could be used to initiate the synthesis of a reporter protein, albeit with lower efficiency. These findings bear important implications for the HCV IRES secondary structures.
RNA, 2001
Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 59 viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 59 end of the CAT sequence, or the insertion of a small stable hairpin structure (DG 5-18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 59 proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.
Journal of Virology
Bicistronic RNAs containing the 373-nucleotide-long 5 nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5 end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5 NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5 border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3 end of the CSFV 5 NTR in IRESmediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5 NTR, ribosomal scanning for the initiation codon is limited to a small region.