Rescue of a foreign gene by Sendai virus

Abstract

A simple protocol for the rescue of a synthetic genome into a paramyxovirus has been developed. First, a synthetic Sendai virus-like RNA, containing the antisense coding region of the chloramphenicol acetyltransferase gene replacing the coding region of the Sendai virus genome, was transcribed from a cDNA. When introduced into cells that are infected with Sendai virus, this RNA construct was transcribed, replicated, and packaged into infectious virions. The addition of infected cell extract to the RNA prior to transfection markedly enhanced levels of chloramphenicol acetyltransferase expression and rescue. However, this enhancement is not due to encapsidation of the RNA into nucleocapsids as the RNA remains nuclease-sensitive. Uninfected cell extract also enhances expression and rescue efficiency, implying involvement of a cellular factor(s) with the synthetic viral-like RNA construct that allows for enhanced polymerase recognition. This system should allow for the dissection of the various cis-acting RNA signals within the paramyxovirus genome.