Schematic of model BUNV segments used to determine the minimal sequences required for RNA replication and primer extension analysis of the replication products they generate. (A) Model BUNV segments were generated such that progressive internal nucleotide deletions were introduced into the 3′ NTR of the anti-genomic strand. The boxed numbers within the 3′ NTR denote remaining terminal nucleotides. After RNA replication within mammalian cells, these deletions were present within the corresponding 5′ NTR of the genomic strand. Abundance of genomic strands was determined by primer extension analysis using oligonucleotide P-ext, which binds within the body of all altered model segments. (B) Progressive internal deletions were also engineered into the anti-genomic 5′ NTR, and its replication ability was determined as described above.
