Figure 5

Replication of artificial vRNA-EGFP reporter transcripts in MDCK cells. (A) Schematic representation of the MDCK EcoR I- BamH I fragment in pK9Pol I EB. The G residue at position 1804 in the insert was predicted from the alignment in Fig. 4 to be the transcription initiation site (TIS). Products from primer extension reactions designed to map the TIS terminated at T (1803) and C (1805). (B) The EGFP reporter plasmids pK9GFP 1–1802(T), pK9GFP 1–1803(G) and pK9GFP 1–1804(C) were constructed by replacing the human pol I promoter sequences in pHW72-EGFP [9] with bases 1–1802, 1–1803, and 1–1804, respectively, from the MDCK EcoR I-BamH I insert in pK9Pol I EB. In each reporter construct, EGFP coding sequences are flanked by the noncoding region from an influenza M segment and this transcription unit is between a murine RNA pol I terminator (tI) and the indicated sequences from the MDCK EcoR I-BamH I insert. (C) Replication of artificial vRNA-EGFP reporter transcripts in MDCK cells. DNA mixes composed of expression plasmids for PB1, PB2, PA and NP proteins plus a single EGFP reporter plasmid or pΔHW72-EGFP were combined with MDCK cells and subjected to electroporation. At 48 hrs after electroporation, GFP expression was detected by fluorescence microscopy. The plasmid pΔHW72-EGFP is a derivative of pHW72-EGFP in which the human pol I promoter has been deleted. The MDCK cells in the panel labeled pCMV EGFP were subjected to electroporation with pCMV EGFP plasmid alone and served a positive control.