FIG. 3.

Heterologous gene expression from TGEV replicon RNAs. (a) Cultures of BHK cells were transfected with TGEV replicon RNAs encoding GFP that contained ORF 3B and E (A) [TGEV-Rep(AvrII) transcripts] or ORF 3B, E, and M gene deletions (C). Alternatively, transcripts were treated with RNase prior to transfection into BHK cells (B and D) [TGEV-Rep(AvrII) DNA and TGEV-Rep(EcoNI) DNA only, respectively]. At ∼18 h posttransfection, GFP expression was observed by fluorescent microscopy. (b) Intracellular RNA was isolated from transfected cell cultures and used as a template for RT-PCR. Leader-containing GFP subgenomic transcripts were detected using a 5′ leader primer (TGEV-L) and 3′ primers located just downstream of the AvrII [(−)E5546] or EcoNI [M6400(−)] sites. Appropriately sized amplicons of ∼850 bp were generated, corresponding to transcripts encoding GFP. Cells transfected with TGEV-Rep(AvrII) (A) or TGEV-Rep(EcoNI) (B) RNA are shown. A 1-kb ladder is shown in both panels (lanes 1). Arrows indicate leader-containing GFP amplicons (lanes 2).