Summary auto-generated
This study examines the structural and functional importance of single-stranded linker regions that connect the coaxial RNA helices in the coxsackievirus B3 origin of replication (oriR). The oriR consists of two main helical domains (X and Y) anchored by a kissing (K) interaction. Researchers systematically altered the length and sequence of the linker regions connecting these helices and assessed effects on viral replication. Mutations shortening the K-Y linker (9 nucleotides) and Y-X linker (5 nucleotides) resulted in severely debilitated virus growth at 36°C, though some showed temperature-sensitive phenotypes at 39°C. Lengthening the Y-X linker also impaired replication, indicating that precise spacing is critical. Point mutations in the Y-X linker sequence caused defective growth at 36°C but wild-type phenotypes at 39°C, suggesting a cold-sensitive phenotype. These findings demonstrate that the spacing between coaxial RNA helices is finely tuned for proper oriR structure and function, likely affecting both the geometry needed for protein recognition and the overall architectural integrity required for viral RNA synthesis initiation.
Key findings
- Alterations to linker length between coaxial RNA helices of coxsackievirus B3 oriR result in defective RNA replication, indicating that precise spacing is critical for function
- Point mutations in the Y-X spacer exhibit temperature-sensitive cold-sensitive phenotypes, with defective growth at 36°C but wild-type replication at 39°C
- Both shortening and lengthening of the Y-X linker impair viral growth, suggesting the spacing acts as a geometrical determinant for protein recognition
- The identity of linker nucleotides plays a functional role, possibly through sequence-specific ligand recognition required for ribonucleoprotein complex formation
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Abstract
The enterovirus oriR is composed of two helices, X and Y, anchored by a kissing (K) interaction. For proper oriR function, certain areas of these helices should be specifically oriented towards each other. It was hypothesized that the single-stranded nucleotides bridging the coaxial helices (YX and KY linkers) are important to determine this orientation. Spatial changes were introduced by altering the linker length between the helices of the coxsackievirus B3 oriR. Changing the linker lengths resulted in defective RNA replication, probably because of an altered oriR geometry. The identity of the linker residues also played a role, possibly because of sequence-specific ligand recognition. Although each point mutation altering the primary sequence of the YX spacer resulted in defective growth at 36 °C, the mutations had a wild-type phenotype at 39 °C, indicating a cold-sensitive phenotype. The results show that the intrinsic connection between oriR structure and function is fine-tuned by the spacing between the coaxial RNA helices.
A rotating version of Fig. 1(c) is available as supplementary material in JGV Online.