FIG. 1.

Establishment of a minigenome system free of vaccinia virus. (A) Schematic diagram of the minigenome system. Plasmid pPIV2-GFP contains an hPIV2 minigenome flanked at one end by a bacteriophage T7 RNA polymerase (T7 RNAP) promoter (T7) and at the other end by a hepatitis delta virus ribozyme (Ribozyme) and T7 transcriptional terminator (T7-TΦ). T7 RNA transcripts can be synthesized under the control of the T7 promoter to generate viral negative-sense PIV2 RNA. pPIV2-GFP contains three extra G residues after the T7 RNAP promoter and prior to the PIV2 trailer sequence (Tr) in order to increase T7 RNAP transcription efficiency. The extra leader sequence (Le) is generated by cleavage with hepatitis delta virus ribozyme. The plasmids pTM1-NP, pTM1-P, and pTM1-L were used to express NP, P, and L proteins in BSR T7/5 cells, a cell line that constitutively expresses T7 RNAP. GFP gene expression can be generated from transcription of primary T7 transcript and vRNA sense genome through viral RNA replication. 5′ NP, 5′ sequence of NP gene; 3′ L, 3′ sequence of L gene. (B) GFP expression from the minigenome system. Plasmids encoding NP, P, L, and pPIV2-GFP at various combinations were transfected into BSR T7/5 cells. At 48 hpt, cells were assayed by Western blotting with anti-NP, P, L, and GFP antibodies.