Molecular cloning and characterization of a Sendai virus internal deletion defective RNA

Abstract

A small defective Sendai virus RNA was selectively amplified from a virus preparation obtained after serial undiluted passages in embryonated eggs. Preliminary characterization showed that this defective RNA was a true internal deletion defective RNA, containing the 5' and 3' ends of the non-defective viral genomic RNA. Cloning of this RNA after reverse transcription and polymerase chain reaction amplification was performed in such a way that an exact copy of the defective RNA could be obtained by transcription of the plasmid with T7 RNA polymerase. Sequence analysis of the plasmid allowed further characterization of the defective RNA. It was shown potentially to encode a C-terminally truncated nucleocapsid (NP) protein of 162 amino acids. This truncated NP protein was identified in cells naturally infected with the defective virus preparation. Moreover the protein produced was shown to correspond to the protein synthesized in vitro from the T7 polymerase transcript of the cloned defective genome.