Summary auto-generated
This study identifies a novel cis-acting element for RNA replication within the stem-loop II (SLII) region of poliovirus's internal ribosome entry site (IRES). Researchers created six poliovirus mutants with deletions in the SLII region. Two non-viable mutants, SLII-2 and SLII-3, were characterized in detail. Both mutants showed defective translation initiation, preventing viral protein synthesis. Additionally, SLII-2 was defective in viral RNA replication, while SLII-3 maintained RNA replication capacity. These findings demonstrate that the SLII region contains two distinct but overlapping functions: it serves as part of the IRES for translation initiation and contains separate cis-elements required for RNA synthesis. UV cross-linking assays revealed that different host cellular proteins bind to the SLII region in wild-type versus mutant RNA, suggesting that altered protein-RNA interactions account for the mutant phenotypes. The work expands understanding of poliovirus genome replication by showing that elements required for RNA synthesis extend beyond the previously characterized 5'-proximal 100 nucleotides into the structured IRES region.
Key findings
- SLII-2 is defective in both viral protein synthesis and RNA replication; SLII-3 is defective only in protein synthesis, identifying separate functions within SLII
- The SLII region contains overlapping cis-acting elements for IRES-dependent translation and RNA replication
- Wild-type and mutant SLII RNAs interact differently with host cell factors, with a 92 kDa protein binding wild-type RNA and a 70 kDa protein binding primarily to SLII-2 mutant RNA
- Poliovirus RNA replication signals extend beyond the previously identified 5'-proximal 100 nucleotides into the structured IRES domain
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Abstract
Several mutants of the Mahoney strain of poliovirus type 1 have been generated by introducing mutations into the stem-loop II (SLII) structure within the internal ribosomal entry site (IRES). Four of these mutants (SLII-1, -4, -5 and -6 mutants) have been characterized previously and are host-range mutants that replicate well in human HeLa cells but not in mouse cells. Two deletion mutants, SLII-2 and SLII-3, were non-viable, even in HeLa cells. It is now reported that SLII-2 was defective in genome RNA synthesis and viral protein synthesis, while SLII-3 was defective only in viral protein synthesis. These results indicate that the SLII region contains a cis-element for RNA replication as well as for IRES-dependent translation and that these two functions lie at the same sites within the SLII region. The host cellular factors that interacted with wild-type SLII and mutant SLII-2 and SLII-3 RNAs were different, suggesting that different host-factor binding regulates expression of mutant phenotypes.