Nucleotide sequences responsible for generation of internally deleted sendai virus defective interfering genomes

Abstract

The deletion points of four internally deleted defective interfering (DI) RNA species (7a, 7b, 7c, and 7d) that reside in a single Sendai virus strain were defined by nucleotide sequencing. DI RNA 7a ( M r 1.24 × 10 6) retained the entire NP gene with the complete NP protein-coding sequence, except for the last two U residues of the polyadenylation signal, fused to an 1800-nucleotide sequence comprising 5′-terminal genome and adjacent L gene sequences. DI RNA 7b ( M r 0.70 × 10 6) consisted of 100 3′-terminal nucleotides fused to 1900 5′-terminal bases; the deletion point in the NP gene precedes the NP protein initiation codon. DI RNA 7c ( M r 0.55 × 10 6) retained 420 3′-terminal and 1150 5′-terminal nucleotides. The sequence just downstream of the sequenced deletion site is M gene specific, indicating that 7c arose from at least two deletion events and that it comprises NP, M, and L gene fragments. Transcription of RNA 7c could yield an mRNA encoding a fusion protein with a 14,000 M r (N-terminal NP sequence fused to out of frame M-specific amino acids). DI RNA 7d ( M r 0.92 × 10 6) retained 1027 3′-terminal nucleotides fused to 1600 bases from the 5′ terminus. It has an open reading frame for a 33,000 M r N-terminal NP protein fragment. Nucleotide sequences flanking each deletion and just downstream of the NP gene deletion site suggested that these DI genomes were generated by a copy-choice mechanism, involving polymerase jumping during replication of negative polarity virus genome templates. In this process, the termination and reinitiation of RNA synthesis would involve recognition of sequences that regulate virus genome transcription and replication.