Recovery of Infectious SV5 from Cloned DNA and Expression of a Foreign Gene

Abstract

A complete cDNA clone of the genome (15,246 nucleotides) of the paramyxovirus SV5 was constructed from cDNAs such that an anti-genome RNA could be transcribed by T7 RNA polymerase and the correct 3′ end generated by cleavage using hepatitis delta virus ribozyme. The plasmid encoding the antigenome sequence was transfected into cells previously infected with recombinant vaccinia virus that expressed T7 RNA polymerase, together with helper plasmids that expressed the viral replication proteins, NP, P, and L, under the control of the T7 polymerase promoter. Rescue of the RNA genome from DNA was demonstrated by recovering SV5 with the tag restriction sites introduced into the DNA clone, using RT-PCR of the genome RNA and nucleotide sequencing. Rescue of SV5 from DNA did not require expression of the viral V protein as a helper plasmid, suggesting that V protein is not essential for initial replication. The infectious cDNA of SV5 was also manipulated to express green fluorescent protein (GFP) under the control of SV5 transcriptional start and stop signals introduced between the HN and L genes. The amount of GFP that was expressed varied depending on the nature of the newly introduced transcription signals.