Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA

Abstract

Using the unique sequence organization of copy-back defective interfering (DI) RNAs of paramyxoviruses, Sendai virus (SV), and measles virus copy-back DI RNAs were PCR amplified and cloned, without having to separate them from their helper nondefective genomes. The cloning was designed so that T7 polymerase transcription of the plasmids would generate DI RNAs with the exact 5′ and 3′ ends. The SV DI clone, transcribed from the plasmid in BHK cells using T7 polymerase produced by a vaccinia virus recombinant, was encapsidated and replicated by the SV-L, P/C, and NP proteins expressed from cloned genes. Such experiments open the possibility of examining the cis-acting sequences involved in viral multiplication directly, without using indirect markers such as CAT activity.