Key Points
- Determine the sequence organization and identify conserved structural features of defective-interfering RNAs generated during Sindbis virus replication.
- Cloned and sequenced a representative cDNA derived from a population of Sindbis virus defective-interfering (DI) RNAs.
- Conducted blot hybridization using cloned restriction fragments to probe for conserved genomic segments across multiple independently generated DI RNA populations in chicken embryo fibroblasts.
- The sequenced DI RNA clone contained standard viral sequences at its 3' end (50 nucleotides) and derived 75% of its sequence from repeated 5'-terminal regions, while its 5' terminus was nearly identical to host cellular tRNAAsp.
- Hybridization revealed that a conserved 51-nucleotide 5'-proximal sequence was maintained and host tRNAAsp sequences were common across DI populations, whereas the subgenomic 26S mRNA junction region was deleted.
Structured PICO
PPopulationSindbis virus defective-interfering (DI) RNA populations derived by passaging in cultured cells (chicken embryo fibroblasts)
IInterventionIsolation, sequencing, and blot hybridization of cDNA clones derived from Sindbis DI RNA
CComparatorStandard Sindbis virus RNA
OOutcomeSequence organization and conserved regions of DI RNA species
Sindbis virus defective-interfering RNAs share common structural features including conserved 3' ends, 5'-terminal standard RNA repeats, and a 5' end derived from cellular tRNAAsp.