Summary auto-generated
This study examined the internal ribosome entry sites (IRESs) of Rhopalosiphum padi virus (RhPV), an insect virus with two open reading frames each preceded by structurally distinct IRESs. Researchers developed an in vivo system using Spodoptera frugiperda insect cells infected with baculovirus expressing T7 RNA polymerase to test IRES activity. They found that the two RhPV IRESs (5' and intergenic) directed translation initiation with equal efficiency when tested separately. However, competition occurred between the two IRESs when present together on the same plasmid. Mutations disrupting the 3'-proximal pseudoknot of the intergenic IRES significantly reduced translation initiation both in vivo and in vitro. UV cross-linking experiments revealed that the two IRESs preferentially bound different host proteins (68 kDa for intergenic IRES; 30 and 36 kDa for 5' IRES), indicating distinct translation mechanisms. Sucrose gradient analysis confirmed that structural elements, particularly stem-loop 2, are essential for 80S ribosomal complex formation required for translation initiation.
Key findings
- RhPV 5' and intergenic IRESs direct translation with equal efficiency in insect cells, contrasting with Cricket paralysis virus where the intergenic IRES is more active
- Competition occurs between the two IRESs when present together, possibly due to ribosomal subunit availability
- The 3'-proximal pseudoknot of the intergenic IRES is essential for translation activity, confirmed by both in vivo and in vitro assays
- The two IRESs preferentially interact with different host proteins, suggesting they employ distinct translation initiation mechanisms
- Stem-loop 2 structure is required for 80S ribosomal complex formation necessary for translation initiation
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Abstract
The RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transcription and translation of plasmid templates containing the IRESs. In this system, the two RhPV IRESs directed initiation of translation from bicistronic plasmids with equal efficiency. Competition was observed between the two IRESs when they were in cis in a bicistronic plasmid. A mutation that disrupted the 3'-proximal pseudoknot of the intergenic (IG) IRES reduced translation initiation in vivo. Similarly, mutations in the RhPV IG IRES disrupted its ability to bind 80S particles in vitro. The two IRESs preferentially labelled proteins of different masses in UV cross-linking experiments, illustrating the different translation initiation mechanisms employed by the two elements.